Synthesis of Glycocluster− Tumor Antigenic Peptide Conjugates for

Jun 29, 2007 - epitope eliciting a CD8. +. T-lymphocyte response. These data suggest that synthetically designed glycocluster- tumor antigen conjugate...
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Bioconjugate Chem. 2007, 18, 1547−1554

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Synthesis of Glycocluster-Tumor Antigenic Peptide Conjugates for Dendritic Cell Targeting Oruganti Srinivas,† Pierre Larrieu,‡,# Eric Duverger,† Claire Boccaccio,§ Marie-The´re`se Bousser,† Michel Monsigny,† Jean-Franc¸ ois Fonteneau,‡ Francine Jotereau,*,‡,# and Annie-Claude Roche*,† Glycobiologie, Vectorologie et Traffic Intracellulaire, Centre de Biophysique Mole´culaire CNRS, Rue Charles-Sadron, 45071 Orle´ans, Cedex 2, France, INSERM, Unite´ 601, 44093 Nantes, France, Faculte´ des Sciences, Universite´ de Nantes, 44322 Nantes, France and IDM Research Laboratory, LUTI, Bat.F, Institut de Recherches Biome´dicales des Cordeliers, 15 rue de l’Ecole de Me´decine, 75006 Paris, France. Received January 23, 2007; Revised Manuscript Received May 14, 2007

The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes Via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-β-alanine derivatives containing either disaccharides, a dimannoside (ManR-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycoclustertumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.

INTRODUCTION Dendritic cells (DC) are antigen-presenting cells critical for the induction of adaptive immune responses (1, 2). After capturing and internalizing antigen in peripheral organs, they efficiently associate antigenic peptides derived by limited proteolysis to nascent MHC class I protein and expose these MHC class I peptide complexes on their surface, a process termed cross-presentation. Cross-presentation of tumor epitopes to cytotoxic T cell (CTL) precursors in peripheral lymphoid organs (1) contributes to the induction of cytotoxic responses to microbes and tumors (3). Several C-type lectins and lectinlike receptors, abundantly expressed on the surface of DC, bind sugars in a calcium-dependent manner using highly conserved carbohydrate-recognition domains (CRDs) (4). Among these, the mannose specific receptor (MR, CD206) binds and internalizes glycoproteins containing terminal mannose, N-acetylglucosamine, or fucose (5, 6), and the dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN, CD209) (7) binds mannose oligosaccharides (8) and fucose-containing glycans (911). The sugar specificity was determined by using oligolysinebased Lewis type oligosaccharide clusters (9) and synthetic * To whom correspondence should be addressed. (A.-C.R) Tel: +33(0)238255537, fax: +33(0)238255557, e-mail: [email protected]. (F.J.) (for immunological data) Tel: +33(0)240084720, e-mail: [email protected]. † Centre de Biophysique Mole ´ culaire CNRS. ‡ INSERM. # Universite ´ de Nantes. § Institut de Recherches Biome ´ dicales des Cordeliers.

glycoconjugates (10) and by screening an extensive glycan array (11). While DC-SIGN and MR act similarly in mediating endocytosis and receptor recycling, they may differ in routing their ligands to distinct intracellular compartments. It was reported that DC-SIGN ligand complexes are targeted to late endosomes/lysosomes (12), while MR is preferentially targeted to early endosomes (13). DC membrane lectins which mediate efficient sugar-specific endocytosis could be potentially exploited for glycotargeting tumor antigens to enhance cross-presentation. It is known that sugar-lectin interactions depend on the density of the partners (see for reviews, refs 14, 15), so that there is a stringent requirement of clustered patches of carbohydrate ligands for high binding to the protein receptors (16, 17). High affinity binding ligands for membrane lectins have therefore been produced such as neoglycoproteins, prepared by covalent attachment of sugars to a protein, (see for reviews, refs 15, 18, 19); multivalent glycosylated peptides were obtained by coupling monosaccharides derivatives to polylysine (20, 21), as well as to oligolysine (22, 23). Glycoclusters were recently synthesized in our laboratory by coupling glycosynthons to small oligolysine-based peptides (24). Glycosynthons (25, 26) were prepared by an original two-step one-pot procedure involving the oligosaccharide reducing sugar and a peptide with a glutamyl residue in the N-terminal position. We previously showed that oligolysine substituted with dimannosides or Lewis oligosaccharides were taken up by dendritic cells (9, 27). In this study, we asked whether glycoclusters, specific for either DC-SIGN or MR and able to be internalized by DC, might

10.1021/bc070026g CCC: $37.00 © 2007 American Chemical Society Published on Web 06/29/2007

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represent efficient vectors to induce the cross-presentation by DC of a tumor epitope. We designed glycoclusters containing a 25 amino acid long peptide of the Melan-A/Mart-1 protein (a melanoma tumor antigen): Melan-A 16-40 peptide, containing the A27L analogue of the HLA-A*0201 restricted Melan-A epitope (26-35) (28-30). This epitope is a good model for developing vaccine targets for melanoma patients, as it is highly expressed by about half of melanoma tumors (31). We report here the synthesis and characterization of glycocluster-Melan-A peptide conjugates containing the Melan-A (26-35) A27L epitope and either ManR-6Man (DiMan) or a mixture of Lewisa and Lewisx oligosaccharides (Lewis) chosen on the basis of their affinity for MR and DC-SIGN, or Galβ4Glc (lactose or Lac) selected as a negative control, based on the fact that the lactose glycocluster is not recognized by DC (9). The capacity of DC to internalize fluorescent-labeled Melan-A glycopeptides was checked by confocal microscopy and flow cytometry. The kinetic constants for the binding of the glycocluster-Melan-A conjugates to MR and DC-SIGN were measured by surface plasmon resonance. Interestingly, DC loaded with DiMan- and Lewis-Melan-A glycopeptides allowed activation of a specific CD8+ T cell clone. On the basis of present results, these glycopeptides are discussed as a putative new cellular vaccination approach.

EXPERIMENTAL PROCEDURES Materials. Chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) unless otherwise mentioned. Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were from Richelieu Biotechnologies (Saint-Hyacinthe, QC, Canada). N-Methylpyrrolidone (NMP) was from Applied Biosytems (Foster City, CA). Triethylammonium phosphate (TEAP), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIEA), and tert-butyl methyl ether (TBME) were from Senn Chemical AG (Dieslldorf, Switzerland). Lactose and N,Ndiisopropylethlamine (DIEA) were from Janssen Chimica (Beerse, Belgium). ManR-6Man disaccharide was from Dextra laboratories (Reading, UK). A mixture of Lewisa and Lewisx oligosaccharides isolated from human milk were a kind gift from Dr. Ge´rard Strecker (Lille, France). L-Glu-βAla-OBn was purchased from CNH Technologies (Woburn, MA). Melan-A (16-40)-oligo K containing the promiscuous CD8+ (26-35) A27L analogue epitope, Ac-GHGHSYTTAEELAGIGILTVILGVLKKKK, was synthesized by NeoMPS systems (Strasbourg, France). Spectra/Por regenerated cellulose membrane (cutoff Mr 1000) was used for the dialysis. Mannosylated serum albumin and fucosylated serum albumin (Man-BSA and Fuc-BSA, respectively) were prepared as previously described (18) and contained 25 ( 3 sugar residues per molecule. Chromatography. Conjugates were purified by gel filtration on a Biogel P-4 (Bio-Rad, Oxfordshire, England) column (2 × 45 cm) stabilized and eluted with 50 mM acetic acid. The flow rate was 0.5 mL/min. Analytical reverse-phase high performance liquid chromatography (RP-HPLC) separations were performed with a Waters 2690 (Milford, MA) system linked to a Waters 996 photodiode array detector, using an Alltech C18 column (5 µm, 4.6 mm × 250 mm) at a flow rate of 1 mL/min, with detection at 254 nm (or 280 nm) at 25 °C (solvent A: 0.1% TEAP in H2O; solvent B: 0.1% TEAP in acetonitrile; gradient from 70% A/30% B to 2% A/98% B in 20 min). Mass Spectrometry. MALDI-TOF mass spectra were recorded on a Biflex III instrument (Bruker, Wissembourg, France), in positive mode using R-cyano-4-hydroxycinnamic acid as a matrix. Synthesis of Glycocluster-Antigenic Peptide Conjugates. General Procedure for Coupling Glycosynthons and Melan-

Srinivas et al.

A-Oligo K (Scheme 1). The glycosynthons (glycosyl-pyroglutamyl-β-alanine derivatives) were obtained according to (25). Briefly, an oligosaccharide was allowed to react with R-glutamylβ-alanyl benzyl ester in the presence of imidazole, and the glycosylamine derivative obtained was stabilized by an in situ intramolecular acylation using BOP. Then, the hydrogenolysis of the benzyl ester afforded the expected glycosynthons in high yields. The coupling of glycosynthons to the peptide-oligo K was carried out using amide coupling reagents. A mixture of glycosynthon (0.01 mmol), 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU, 0.01 mmol), 1-hydroxybenzotriazole (HOBt, 0.01mmol), and 4 Å molecular sieve beads (25 mg) in N-methylpyrrolidone (NMP, 1 mL) was stirred for 20 min under N2 atmosphere. A solution of Melan-A (16-40)-oligo K peptide (0.0015 mmol) in N-methylpyrrolidone (200 µL) and 2 M N,N-diisopropylethylamine in N-methylpyrrolidone (20 µL) were added, and the mixture was stirred for another 6 h. The molecular sieve beads were removed, and 10 volumes of tert-butyl methyl ether (TBME) was added: the precipitated crude product was collected upon centrifugation. The pellet was dissolved in water (1 mL) and passed through a Biogel P-4 column with 50 mM acetic acid as an eluent, and the fast eluting sugar-containing fractions identified by a microscale resorcinol-H2SO4 assay (32) were pooled, dialyzed (cutoff Mr 1000), and lyophilized to obtain the glycoclusterMelan-A conjugate as a white fluffy powder (yield ≈ 55%). The glycocluster-peptide conjugates were analyzed by HPLC: Lac-Melan-A, DiMan-Melan-A, and Lewis-Melan-A have a retention times of 7.50, 7.51, and 6.48 min, respectively, as compared to 6.98 min for Melan-A. These glycoclusterpeptide conjugates were analyzed by MALDI-TOF mass spectrometry; the glycocluster-peptide derivatives appeared to be predominantly the trivalent derivatives. DiMan-Melan-A, Lac-Melan-A, and Lewis-Melan-A conjugates gave molecular ion peaks at m/z ) 4640.94, 4639.08, and 6165.24, respectively, corresponding to their [M + K]+ ions. In addition, [M + K]+ adducts at m/z ) 5147.48 and 5145.49 for DiMan-Melan-A and Lac-Melan-A, respectively, were also observed, corresponding to the tetravalent derivatives. General Procedure To Add Fluorescent Probe on Glycocluster-Melan-A Conjugates. A solution of iodoacetamidofluorescein (2 µmol in 15 µL of NMP) was added to the glycocluster-Melan-A conjugate (1 µmol) in 500 µL of carbonate buffer (pH 8.5) and stirred at room temperature for 2 h in darkness. The reaction mixture was purified by gel chromatography, Ultrogel GF05 (1.5 cm × 20 cm) column; the fluorescent glycopeptide was eluted with 10 mM acetic acid and was lyophilized. The concentration was determined by absorbance measurement at 495 and 280 nm, assuming that fluorescein 1Mcm at 488 nm is 80 000 and at 475 nm is 26 000 and tyrosine 1Mcm at 488 nm is 1400. Cells. DC-IL-13 were provided by IDM (Immuno Designed Molecules, Paris) in frozen vials; they were obtained from monocytes isolated by elutriation from peripheral blood mononuclear cells differentiated in the presence of GM-CSF and IL13 (33). These DC were used immediately upon thawing or plated and cultured in Aim-V medium supplemented with 500 units/mL GM-CSF and 50 ng/mL IL-13 before use. For cross-presentation experiments, DC-IL-4 were used. Briefly, healthy HLA-A*0201 donor PBMC was obtained by Ficoll separation. Monocytes and lymphocytes were isolated by elutriation (Aventi J-20, Beckman Coulter) and frozen. DC (DCIL-4) were generated by culturing the monocyte-enriched fraction in six-well plates using 3 × 106 cells/well in 3 mL of RPMI containing 1% nonautologous plasma, 100 IU/mL GMCSF (Abys), and 300 U/mL IL-4 (AbCys). The cytokines were

Melan-A−Glycopeptide for Dendritic Cell Targeting

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Scheme 1. a Panel A: Oligosaccharides Used To Prepare Glycosynthons. Panel B: Preparation of a Typical Glycosynthon: A Reducing Oligosaccharide Is Allowed To React with a Dipeptide Having a Glutamyl Moiety in the N-Terminal Position and with R ) NHCH2CH2CO2Bn. The Glycosylamine Derivative Is Stabilized by Formation of a Pyroglutamyl Ring, and Then the Benzyl Group Is Removed by Catalytic Reduction, Leading to the Desired Glycosynthon. Panel C: Preparation of an Antigenic Glycopeptide: A Peptide (including the CD 8+ Melan-A epitope and an oligolysine tail) Is Substituted with Glycosynthons

a Abbreviations: NMP, N-methylpyrrolidone; BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; Pd/C, palladium on charcoal; CD 8+ epitope, ELAGIGILTV; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; MS, molecular sieves; Lactose, Galβ4Glc; Lewis a, Galβ3(FucR4)GlcNAcβ3Galβ4Glc; Lewis x, Galβ4(FucR3)GlcNAcβ3Galβ4Glc; DiMannose, ManR6Man.

added to the cultures at day 0 and 2. On day 5, DC were harvested and used in antigen cross-presentation experiments. M77-84 is a HLA-A2 restricted Melan-A (26-35) specific CD8+ T cell clone obtained from tumor-infiltrating lymphocytes (TIL) of a melanoma patient (34). Cell Reagents. A soluble fragment of the human MR consisting of the eight C-type CRDs (MMR1-8) and the extracellular domain of DC-SIGN were kindly provided respectively by Maureen Taylor and Kurt Drickamer (London, UK). Flow Cytometry Analysis of Endocytosis. Freshly thawed DC-IL-13 (106 cells in 100 µL of Aim-V medium) were incubated for 2 h at 37 °C in the presence of 10 µM fluoresceinlabeled glycopeptides. After being washed in PBS, DC were further incubated for 30 min at 4 °C in the absence or in the presence of 50 µM monensin. Monensin is a proton/sodium ionophore which transports monovalent cations (Na+ and H+) across membranes and, thereby, equilibrates the external and internal pH of organelles in intact cells. Therefore, monensin

neutralizes intracellular acidic compartments, allowing the recovery of fluorescein fluorescence quenched in acidic environment; a higher fluorescence intensity of the monensin-treated cells compared to that of untreated cells demonstrate that the conjugate had reached acidic compartments upon endocytosis (35). Cell-associated fluorescence was assessed using a BDLSR flow cytometer, and the data were analyzed with the Cell Quest software (BD Biosciences). Confocal Microscopy Analysis. DC-IL-13 plated on cover slip after thawing and overnight incubation in complete medium were incubated for 2 h at 37 °C with 1 µM fluorescein-labeled glycopeptides, washed, and either fixed immediately 20 min in PBS containing 4% formaldehyde at room temperature or further incubated 2 h in medium before fixation. Coverslips were mounted on slides in a PBS/glycerol mixture (1:1 v/v) containing 1% 1,4-diazabicyclo[2.2.2]octane (DABCO) as an antifading agent and fixed 20 min in PBS containing 4% formaldehyde at room temperature. Cell fluorescence was analyzed with a confocal imaging system MRC-1024 (Bio-Rad) equipped with

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a Nikon Optiphot epifluorescence microcope and a ×60 planapo objective (numerical aperture 1.4). The krypton/argon laser was tuned to produce a 488 nm beam. Images were recorded with a Kalman filter average of 5-10 images. Surface Plasmon Resonance. Surface plasmon analyses (see for a review, ref 36) were performed on a BIAcore 2000 biosensor system (BIAcore Inc., Uppsala, Sweden). The carboxymethyl dextran CM-5 research grade sensor chip and ethanolamine-HCl solution (pH 8.5) were obtained from BIAcore Inc.; the immobilization reagents (N-ethyl-N′-(dimethylaminopropyl)carbodiimide, (EDC) and N-hydroxysuccinimide, (NHS) were purchased from Sigma. All solutions were filtered using a 0.22 µm of polytetrafluoroethylene membrane syringe filter and degassed prior use. Extracellular soluble fragments (MMR1-8) of the human MR (37-39) and of the human DCSIGN (8) were immobilized on a CM5 sensor chip. The fragment of MR (MMR1-8) contains all the domains necessary for Ca2+-dependent binding of mannose, N-acetylglucosamine, and fucose-terminated glycoconjugates and binds such ligands as the whole receptor does. These soluble extracellular fragments containing the CRDs of human MMR1-8 (45 µg/mL) and DCSIGN (20 µg/mL) in 10 mM sodium acetate at pH 4.5 were covalently immobilized using a standard amine coupling procedure according to the immobilization wizard. In a typical procedure, the carboxymethyl dextran surface was activated using freshly mixed 100 mM NHS and 400 mM EDC solutions, followed by lectin coupling (immobilization time of 8 min and a constant flow rate of 20 µL/min). The remaining activated groups were blocked with 1 M ethanolamine at pH 8.5, and efficient fixation was indicated by a large increase in the refractive index, in both cases, about 5000 RU (relative units, RU). The control flow cell was also activated by EDC-NHS and then blocked by using 1 M ethanolamine. The biosensor experiments were carried out in the running buffer (pH 7.4, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 150 mM, NaCl, 17 mM NaN3, 0.005% P20 BIAcore surfactant). For binding studies, the temperature was kept at 25 °C and the flow rate was 20 µL/min for both the association and dissociation steps during 5 and 10 min, respectively. The binding capacity of the immobilized lectin fragments was ascertained by using ManBSA and Fuc-BSA neoglycoproteins; then, binding experiments with glycocluster-antigenic peptide conjugates at concentrations from 0.1 µM to 5 µM were carried out. The glycocluster-peptide conjugate solutions were prepared in the running buffer containing 1 mg/mL BSA (99% purity). Regeneration was carried out using two consecutive pulses of 30 µL of the running buffer containing 1 M NaCl and 0.3 M methyl R-mannopyranoside and 1 mg/mL BSA. Dendritic Cell Cross-Presentation to MelanA/MART-1 Specific T Cells. Immature DC (DC-IL-4) were incubated at 37 °C with various concentrations of Melan-A-glycopeptide. Maturation stimuli, 100 µg/mL poly I/C (Sigma) and 10 ng/ mL TNF-R (Abcys) were added 3 h later. After an 18 h incubation at 37 °C, DC were washed and 105 loaded DC were co-incubated with 105 M77-84 cells in the presence of 10 µg/ mL Brefeldin A (Sigma). After 6 h at 37 °C, cells were fixed and intracellular IFN-γ was stained as described (40). Briefly, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and then stained with phycoerythrin (PE)-conjugated monoclonal antibody (mAb) specific for IFN-γ (Pharmingen BD). Antibody dilutions and washes were performed with PBS containing 1 mg/mL BSA and 1 mg/mL saponin at room temperature. After staining, cells were suspended in PBS and analyzed on a FACScan (BD Biosciences) with a gate set on T cells (on the FSC-SSC dot plot).

Srinivas et al.

RESULTS Design and Synthesis of Glycocluster-Melan-A Conjugates. The Melan-A peptide stands for Melan-A (16-40) A27L with the sequence: Ac-G16HGHSYTTAEE26LAGIGILTV35ILGVL40KKKK, (Ac, for acetyl) containing a variant of the HLA-A2-restricted CD8+ epitope (26-35) ELAGIGILTV instead of EAAGIGILTV, ended by a tetralysine tail. The ELAGIGILTV, A27L analogue binds to HLA-A2 better than the natural peptide because of an altered anchor residue (29, 30). The synthesis of the glycocluster-antigenic peptide conjugates involved the synthesis of glycosynthons containing Galβ4Glc (lactose, Lac), ManR-6Man (DiMan), or Lewis oligosaccharides (a mixture of Lewisa and Lewisx oligosaccharides). The coupling of glycosynthons: Galβ-4Glc-βGlp-Ala-OH, ManR6Man-βGlp-Ala-OH, and Lewis-βGlp-Ala-OH to Melan-A was carried out using amide coupling reagents HBTU/HOBT, to afford Lac-Melan-A, DiMan-Melan-A, and Lewis-MelanA, respectively (Scheme 1). After purification on a Biogel column, the glycopeptides were analyzed by MALDI-TOF mass spectroscopy, showing a high proportion of trivalent conjugates and a small proportion of tetravalent derivatives. Uptake of Fluorescent-Labeled Melan-A Glycopeptides by Dendritic Cells (DC-IL-13). Dendritic cells were incubated 2 h at 37 °C with fluorescent glycopeptides and analyzed by confocal microscopy. As expected, DiMan-Melan-A and Lewis-Melan-A were efficiently internalized and were found in numerous vesicles (Figure 1A) after 2 h pulse chase (lower panel) as well as immediately upon incubation (upper panel); conversely, Lac-Melan-A was not internalized by DC (data not shown) in agreement with our previous data (9), showing that DC are devoid of galactose or lactose-binding proteins. The cell-associated fluorescence was analyzed by flow cytometry. Monensin treatment was used to increase the fluorescence intensity of the endocytosed material (Figure 1B). The enhancement of cell fluorescence intensity, 1.5-fold upon addition of monensin, is proof that upon endocytosis, the fluorescein-labeled glycopeptide was contained in acidic compartments. Surface Plasmon Resonance Analysis. The extracellular moieties of DC-SIGN and MR (MMR1-8) were immobilized on chips, leading to ∆RU values of 5200 and 5700 for DCSIGN and MR, respectively. In preliminary experiments, we confirmed the binding activity of the immobilized lectins by evaluating their capacity to bind Man-BSA and Fuc-BSA neoglycoproteins. The binding profiles and the kinetic constants were in good agreement with our earlier results (9), indicating that the immobilized lectins did retain their binding activity (data not shown). Solutions of DiMan-Melan-A, Lewis-Melan-A, and LacMelan-A were injected over a period of 5 min, followed by a dissociation time of 10 min with a glycopeptide-free buffer. It is noteworthy to mention that upon the injection of glycocluster-Melan-A conjugates on the SPR chips, the regeneration of the lectin-immobilized surface with methyl-R-mannoside solutions was inefficient. To circumvent this problem, the glycocluster-Melan-A conjugates were injected in the presence of 1 mg/mL BSA; the regeneration solution contained 1 mg/mL BSA and 1 M NaCl in addition to methyl-R-mannoside. The presence of BSA efficiently prevents nonspecific hydrophobic interactions derived from the presence of a large number of aliphatic amino acid residues in the antigenic Melan-A peptide. Both DiMan-Melan-A and Lewis-Melan-A conjugates, injected onto the flow cell containing immobilized DC-SIGN, elicited a clear concentration-dependent response indicative of their binding; this is illustrated in Figure 2 for DiMan-MelanA. During the binding of Lewis-Melan-A to MMR1-8, the slope (kbind)of the association curve was quite small and the

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Melan-A−Glycopeptide for Dendritic Cell Targeting

Figure 2. Surface plasmon resonance analysis of the concentrationdependent binding of DiMan-Melan-A conjugate with the immobilized extracellular domain of DC-SIGN. Solutions of DiMan-Melan-A (0.1 µM to 5 µM concentration range) were prepared in the running buffer containing 1 mg/mL BSA (pH 7.4) and were injected over a period of 5 min, with a dissociation interval of 5 min. Table 1. SPR Derived Kinetic Constants for the Binding of Glycocluster-Peptide Conjugates to Immobilized DC-SIGN and MR Lectins Ka (L mol-1) DiMan-Melan-A Lewis-Melan-A Lac-Melan-A DiMan-Melan-A Lewis-Melan-A Lac-Melan-A

DC-SIGN Extracellular 6.2 × 106 5.3 × 107 NBa MMR1-8 2.5 × 107 b NBa

kdiss (s-1) 8.8 × 10-4 6.8 × 10-5 NBa 6.7 × 10-4 b NBa

a NB: No binding observed. b The kinetic constants could not be calculated.

Figure 1. A. Confocal microscopy analysis of glycopeptide uptake by dendritic cells. DC plated on glass coverslips were incubated 2 h at 37 °C with F-DiMan-Melan-A or F-Lewis-Melan-A and fixed before mounting (upper panels); alternatively, DC were incubated 2 h at 37 °C with the glycosylated antigens and further chased 2 h without them before fixation (lower panels). B. Flow cytometry analysis of the uptake of F-glycopeptides by dendritic cells. DC were incubated for 2 h at 37 °C in the presence of either F-DiMan-Melan-A or F-Lewis-MelanA, or F-Lac-Melan-A. Fluorescence intensity was measured by flow cytometry following postincubation in the absence (open bars) or in the presence (closed bars) of monensin.

RU signal linearly increased along with the duration of the Lewis-Melan-A injection (data not shown). In a SPR binding study, the slope of the curve corresponding to the association phase depends on (kbind), the experimental kinetic constant with a value of:

kbind ) kass g0 + kdiss where kbind ) kassg0 + kdisskass stands forthe kinetic association constant, kdiss for the kinetic dissociation constant, and g0 for the free ligand (i.e., the free glycopeptide) concentration. The dissociation profiles from immobilized DC-SIGN were characteristic for each of the two conjugates: that of LewisMelan-A was more than 10 times slower (kdiss ) 6.8 × 10-5 s-1) compared to that of DiMan-Melan-A (kdiss ) 8.8 × 10-4 s-1). In the binding to immobilized DC-SIGN, the calculated equilibrium association constants Ka (Ka ) kass/kdiss) were 6.2 × 106 and 5.3 × 107 L mol-1 for DiMan-Melan-A and LewisMelan-A, respectively (Table 1). It was previously shown from inhibition experiments that the mannose-based glycoclusters have a high affinity for MR (9).

This was confirmed with the binding of DiMan-Melan-A (Ka ) 2.5 × 107 L mol-1) to immobilized MR. Lewis-Melan-A showed a poor association with MR and a very slow dissociation precluding the determination of the kinetic constants. LacMelan-A did not give any detectable response, in agreement with the failure of lactoside-based glycoconjugates to bind DC lectins (Table 1). Glycocluster-Melan-A Conjugate Uptake by DC Induces Melan-A Epitope Presentation. In order to evaluate the capacity of glycocluster-Melan-A conjugates to serve as agents for antigen vectorization into DC, DC were incubated with the various glycocluster Melan-A conjugates and the presentation of the Melan-A (27-35) A27L peptide on DC was analyzed by measuring the activation of a specific T cell clone by intracellular IFN-γ labeling. As shown in Figure 3, DC incubated with DiMan-Melan-A and Lewis-Melan-A, but not with Lac-Melan-A conjugates, induced a clone response. The level of this response, shown by the fraction of IFN-γ labeled lymphocytes, was correlated with the glycocluster dose. When DC were incubated at 4 °C with DiMan-Melan-A and LewisMelan-A peptide, the clone activation was negligible (data not shown).

DISCUSSION DC-SIGN or MR-specific antibody-antigen conjugates (4346) have been used for the induction of tumor antigen crosspresentation by DC. Glycosylated antigen targeting to macrophages or DC lectin receptors was already used, but only to induce antigen presentation by MHC class II molecules. Indeed, glycoside-clustered tetanus toxoid (among other antigens) conjugates were shown to induce MR dependent antigen

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Figure 3. Response of Melan-A (26-35) specific CD8+ T cell clone upon glycocluster-Melan-A conjugate uptake by DC. HLA-A*0201+ DC were incubated 18 h with various doses of DiMan-Melan-A (2), Lewis-Melan-A (9) or Lac-Melan-A (b). Poly I/C and TNF-R were added 3 h after the beginning of the incubation. DC were then washed and cultured with HLA-A*0201 restricted Melan-A (26-35) specific CD8+ T cell clone, in the presence of brefeldin-A to inhibit secretion. After 6 h, cells were fixed and intracytoplasmic staining of IFN-γ was performed and analyzed by flow cytometry.

presentation by macrophages on MHC class II molecules (47); mannosylated tetanus toxoid or proinsulin was shown to induce the uptake and targeting of these antigens to the MHC class II pathway (48). So far, there has been no report of glycotargeting of human DC lectins for the induction of tumor antigen presentation Via the MHC class I pathway, a process called cross-presentation. The aim of this study was to investigate whether glycotargeting of MR and DC-SIGN is a promising strategy to induce tumor antigen uptake by DC and to favor the MHC class I crosspresentation pathway. Our aim was to design a general route to synthesize glycocluster-tumor antigen conjugates with the flexibility of varying either the sugar moieties or the peptide epitope. In order to synthesize glycocluster-tumor antigenic peptide conjugates, two strategies were considered: (i) coupling of glycosynthons to an oligolysine template added to one of two ends of the antigenic peptide and (ii) synthesis of an oligolysine-based glycocluster (24) followed by an attachment to an antigenic peptide. The former strategy was selected, and four lysine residues were appended at the C-terminal end of the antigenic peptide Melan-A chosen for the study in order to allow attachment of up to four glycosynthons per molecule. Along this line, glycocluster-Melan-A peptide conjugates were prepared in a one-pot reaction involving the coupling of pyroglutamyl-based glycosynthons to -amino groups of lysine residues present at the C-terminal end of the peptide. This approach allowed an efficient attachment of DiMan-, Lewis-, and Lac-glycosynthons to the Melan-A 16-40 sequence and an efficient recovery of the glycocluster-Melan-A conjugates. In previous papers (9, 27), we showed that DiMan-glycoclusters and Lewis-glycoclusters were taken up by DC. In order to monitor the cell uptake of the glycocluster-Melan-A conjugates, they were substituted with a fluorescent probe. The glycocluster-Melan-A, being devoid of any cysteine residue, has two histidine resisues (H17,H19) that were fluoresceinylated by allowing iodoacetamido fluorescein to react at neutral pH; this procedure is based on the fact that iodoacetamide leads to carboxymethylation of the imidazole moiety of histidine as previously described (41, 42). Here, we report that glycosylated antigenic peptides bearing DiMan- and Lewis-oligosaccharides were efficiently taken up in slightly acidic compartments, while the lactose-based glycopeptides were not. A series of SPR experiments were carried out to evaluate the apparent binding of glycocluster-Melan-A conjugates to the DC membrane lectins, MR and DC-SIGN. Owing to the

Srinivas et al.

relatively high molecular weight of the glycocluster-Melan-A conjugates, in the present work, we were able to determine the association constants of the glycocluster-Melan-A conjugates by direct surface plasmon resonance measurements of their binding to the immobilized lectins, while in our earlier report (9), by using low molecular weight glycoclusters, the binding constants were deduced from their ability to inhibit the binding of neoglycoproteins (Man-BSA or Fuc-BSA). Here, we showed that DiMan-Melan-A and Lewis-Melan-A exhibited a high apparent affinity for DC-SIGN and MR (MMR1-8) lectins. However, their binding behavior differed. The binding constant Ka of DiMan-Melan-A to MMR1-8 was 4 fold higher than that to DC-SIGN; this difference is related to the association rate, not to the dissociation rate. In contrast, the binding constant Ka of Lewis-Melan-A to DC-SIGN was 10 times higher than that of DiMan-Melan-A; this difference is related to the dissociation rate of Lewis-Melan-A from this lectin, which is 10 times slower than that of DiMan-Melan-A. The binding of DiMan-Melan-A to DC-SIGN and MMR1-8, and that of Lewis-Melan-A to DC-SIGN, being within the 10-7 L mol-1 range clearly shows that these glycopeptides benefit from an avidity effect similar to that shown with the glycoclusters themselves (9). In the case of the interaction between LewisMelan-A and MMR1-8, no kinetic constant could be obtained, precluding any determination of the binding parameters. This result sheds light onto our previous negative results, when, by using a competitive approach, we were not able to show any interaction (9) between Lewis glycoclusters and immobilized MR. As expected, Lac-Melan-A did not exhibit any significant binding to either DC-SIGN or MMR1-8 lectins, in agreement with the fact that the lactose-based glycocluster is not a ligand for either DC-SIGN or MR. Therefore, in the present study, by direct SPR measurements with glycocluster peptide conjugates, we were able to demonstrate a binding of DiMan-Melan-A to DC-SIGN and to determine the binding parameters, while with small glycoclusters used as inhibitors of a neoglycoprotein binding, we were not able to detect interaction between DiMan glycoclusters and DC-SIGN (9). In addition, glycocluster-Melan-A conjugates were clearly shown to serve as agents for antigen vectorization into DC. The fact that DC incubated with DiMan-Melan-A and LewisMelan-A allowed activation of specific CTL clones, and that DC incubated with Lac-Melan-A did not induce such a peptide presentation, strongly suggest that glycocluster-Melan-A conjugates induce Melan-A epitope cross-presentation through a sugar-specific mediated uptake, a subsequent intracellular routing, and an epitope processing through the MHC class I presentation pathway. As stated above, the efficacy of this antigen cross-presentation, as well as its time-course and doseligand dependency, is being investigated to ascertain the putative interest of glycotargeting tumor antigens to DC lectins for the stimulation of tumor specific T cell responses in cancer patients. Having assessed the binding affinities of DiMan-Melan-A and Lewis-Melan-A to DC membrane lectins, their uptake by DC, their capacity to induce the presentation by DC of the epitope ELAGIGILTV of Melan-A (26-35) A27L on HLAA2, inducing CTL specific clones activation, we find that such glycoconjugates are promising for DC-mediated ex ViVo and/ or in ViVo cellular vaccination.

ACKNOWLEDGMENT This work was supported by grants from la Ligue Nationale Contre le Cancer (AC.R, Commite´ du Loiret and F. J Labellisation 05), Fondation pour la Recherche Me´dicale (FRM), Cance´ropoˆle Grand Ouest, and Institut National du Cancer (INCA, projet libre 2005, F. Jotereau). We are grateful to Maureen Taylor and Kurt Drickamer (Imperial College, London)

Melan-A−Glycopeptide for Dendritic Cell Targeting

for kindly providing us with fibroblasts expressing MR and DCSIGN and with MMR1-8 and DC-SIGN lectins, and for critical reading of the manuscript. We thank Prof. Ge´rard Stecker (University of Lille, France) for providing Lewis oligosaccharides, and Dr. Natacha Frison for the synthesis of fluorescent glycoclusters. We thank Margarita Salcedo (IDM, Paris) for kindly providing us with the DC-IL-13 and for critical reading of the manuscript, and C. Bure´ for her skillful help with MALDI, CBM-CNRS, Orle´ans). O.S. thanks La Ligue Nationale Contre le Cancer for the postdoctoral fellowship. ED is Assistant Professor (Universite´ d’Orle´ans), ACR is Research Director (Inserm), MM is Emeritus Professor (Universite´ d’Orle´ans).

NOTE ADDED IN PROOF A recently published paper (Burgdorf et al. (2007) Distinct pathways of antigen uptake and intracellular routing in CD4 and CD8 T cell activation. Science 216, 612-616) clearly demonstrates that mannose receptor-mediated endocytosis of ovalbumin (OVA), used as a soluble model antigen, targets early endosomes and enables its cross-presentation to CD8+ T cells. In the conclusion they suggest that “targeting antigen to the MR may represent an avenue for improving vaccines aimed at inducing CD8+ T cells mediated immunity against viruses or tumors”.

LITERATURE CITED (1) Bevan, M. J. (1976) Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J. Exp. Med. 143, 1283-1288. (2) Mellman, I., and Steinman, R. M. (2001) Dendritic cells: specialized and regulated antigen processing machines. Cell 106, 255258. (3) Banchereau, J., and Steinman, R. M. (1998) Dendritic cells and the control of immunity. Nature 392, 245-252. (4) Figdor, C. G., van Kooyk, Y., and Adema, G. J. (2002) C-type lectin receptors on dendritic cells and Langerhans cells. Nat. ReV. Immunol. 2, 77-84. (5) Stahl, P. D., Rodman, J. S., Miller, M. J., and Schlesinger, P. H. (1978) Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc. Natl Acad. Sci., U.S.A. 75, 1399-1403. (6) Taylor, P. R., Gordon, S., and Martinez-Pomares, L. (2005) The mannose receptor: linking homeostasis and immunity through sugar recognition. Trends Immunol. 26, 104-110. (7) Geijtenbeek, T. B., Kwon, D. S., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Middel, J., Cornelissen, I. L., Nottet, H. S., KewalRamani, V. N., Littman, D. R., Figdor, C. G., and van Kooyk, Y. (2000) DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100, 587-597. (8) Mitchell, D. A., Fadden, A. J., and Drickamer, K. (2001) A novel mechanism of carbohydrate recognition by the C-type lectins DCSIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands. J. Biol. Chem. 276, 28939-28945. (9) Frison, N., Taylor, M. E., Soilleux, E., Bousser, M. T., Mayer, R., Monsigny, M., Drickamer, K., and Roche, A. C. (2003) Oligolysinebased oligosaccharide clusters: selective recognition and endocytosis by the mannose receptor and dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin. J. Biol. Chem. 278, 23922-23929. (10) Appelmelk, B. J., van Die, I., van Vliet, S. J., VandenbrouckeGrauls, C. M., Geijtenbeek, T. B., and van Kooyk, Y. (2003) Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells. J. Immunol. 170, 1635-1639. (11) Guo, Y., Feinberg, H., Conroy, E., Mitchell, D. A., Alvarez, R., Blixt, O., Taylor, M. E., Weis, W. I., and Drickamer, K. (2004) Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR. Nat. Struct. Mol. Biol. 11, 591-598. (12) Engering, A., Geijtenbeek, T. B., van Vliet, S. J., Wijers, M., van Liempt, E., Demaurex, N., Lanzavecchia, A., Fransen, J., Figdor,

Bioconjugate Chem., Vol. 18, No. 5, 2007 1553 C. G., Piguet, V., and van Kooyk, Y. (2002) The dendritic cellspecific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells. J. Immunol. 168, 2118-2126. (13) Engering, A. J., Cella, M., Fluitsma, D. M., Hoefsmit, E. C., Lanzavecchia, A., and Pieters, J. (1997) Mannose receptor mediated antigen uptake and presentation in human dendritic cells. AdV. Exp. Med. Biol. 417, 183-187. (14) Lee, Y. C., and Lee, R. T. (1995) Carbohydrate-protein interactions: basis of glycobiology. Acc. Chem. Res. 28, 321-327. (15) Monsigny, M., Mayer, R., and Roche, A. C. (2000) Sugar-lectin interactions: sugar clusters, lectin multivalency and avidity. Carbohydr. Lett. 4, 35-52. (16) Drickamer, K. (1995) Multiplicity of lectin-carbohydrate interactions. Nat. Struct. Biol. 2, 437-439. (17) Crocker, P. R., and Feizi, T. (1996) Carbohydrate recognition systems: functional triads in cell-cell interactions. Curr. Opin. Struct. Biol. 6, 679-691. (18) Monsigny, M., Roche, A. C., and Midoux, P. (1984) Uptake of neoglycoproteins via membrane lectin(s) of L1210 cells evidenced by quantitative flow cytofluorometry and drug targeting. Biol. Cell 51, 187-196. (19) Lee, Y. C., and Lee, R. T. (1994) in Neoglycoconjugates: Preparation and Application (Lee, Y. C., Lee, R. T., Eds.), pp. 2350, Academic Press, San Diego, CA. (20) Derrien, D., Midoux, P., Petit, C., Negre, E., Mayer, R., Monsigny, M., and Roche, A. C. (1989) Muramyl dipeptide bound to poly-Llysine substituted with mannose and gluconoyl residues as macrophage activators. Glycoconjugate J. 6, 241-255. (21) Midoux, P., Mendes, C., Legrand, A., Raimond, J., Mayer, R., Monsigny, M., and Roche, A. C. (1993) Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells. Nucleic Acids Res. 21, 871-878. (22) Biessen, E. A., Noorman, F., van Teijlingen, M. E., Kuiper, J., Barrett-Bergshoeff, M., Bijsterbosch, M. K., Rijken, D. C., and van Berkel, T. J. (1996) Lysine-based cluster mannosides that inhibit ligand binding to the human mannose receptor at nanomolar concentration. J. Biol. Chem. 271, 28024-28030. (23) Grandjean, C., Rommens, C., Gras-Masse, H., and Melnyk, O. (2000) One-Pot Synthesis of Antigen-Bearing, Lysine-Based Cluster Mannosides Using Two Orthogonal Chemoselective Ligation Reactions. Angew. Chem., Int. Ed. 39, 1068-1072. (24) Frison, N., Marceau, P., Roche, A. C., Monsigny, M., and Mayer, R. (2002) Oligolysine-based saccharide clusters: synthesis and specificity. Biochem. J. 368, 111-119. (25) Que´tard, C., Bourgerie, S., Normand-Sdiqui, N., Mayer, R., Strecker, G., Midoux, P., Roche, A. C., and Monsigny, M. (1998) Novel glycosynthons for glycoconjugate preparation: oligosaccharylpyroglutamylanilide derivatives. Bioconjugate Chem. 9, 268-276. (26) Monsigny, M., Quetard, C., Bourgerie, S., Delay, D., Pichon, C., Midoux, P., Mayer, R., and Roche, A. C. (1998) Glycotargeting: the preparation of glyco-amino acids and derivatives from unprotected reducing sugars. Biochimie 80, 99-108. (27) Be´douet, L., Bousser, M. T., Frison, N., Boccaccio, C., Abastado, J. P., Marceau, P., Mayer, R., Monsigny, N., and Roch, A. C. (2001) Uptake of dimannoside clusters and oligomannosides by human dendritic cells. Biosci. Rep. 21, 839-855. (28) Romero, P., Gervois, N., Schneider, J., Escobar, P., Valmori, D., Pannetier, C., Steinle, A., Wolfel, T., Lienard, D., Brichard, V., van Pel, A., Jotereau, F., and Cerottini, J. C. (1997) Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201restricted Melan-A/MART-1 antigenic peptide in melanoma. J. Immunol. 159, 2366-2374. (29) Valmori, D., Gervois, N., Rimoldi, D., Fonteneau, J. F., Bonelo, A., Lienard, D., Rivoltini, L., Jotereau, F., Cerottini, J. C., and Romero, P. (1998) Diversity of the fine specificity displayed by HLA-A*0201-restricted CTL specific for the immunodominant Melan-A/MART-1 antigenic peptide. J. Immunol. 161, 6956-6962. (30) Valmori, D., Fonteneau, J. F., Lizana, C. M., Gervois, N., Lienard, D., Rimoldi, D., Jongeneel, V., Jotereau, F., Cerottini, J. C., and Romero, P. (1998) Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J. Immunol. 160, 1750-1758. (31) Chen, Y. T., Stockert, E., Jungbluth, A., Tsang, S., Coplan, K. A., Scanlan, M. J., and Old, L. J. (1996) Serological analysis of

1554 Bioconjugate Chem., Vol. 18, No. 5, 2007 Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas. Proc. Natl Acad. Sci., U.S.A. 93, 5915-5919. (32) Monsigny, M., Petit, C., and Roche, A. C. (1988) Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod. Anal. Biochem. 175, 525-530. (33) Goxe, B., Latour, N., Chokri, M., Abastado, J. P., and Salcedo, M. (2000) Simplified method to generate large quantities of dendritic cells suitable for clinical applications. Immunol. InVest. 29, 319-336. (34) Pandolfino, M. C., Viret, C., Gervois, N., Guilloux, Y., Davodeau, F., Diez, E., and Jotereau, F. (1992) Specificity, T cell receptor diversity and activation requirements of CD4+ and CD8+ clones derived from human melanoma-infiltrating lymphocytes. Eur. J. Immunol. 22, 1795-1802. (35) Midoux, P., Roche, A. C., and Monsigny, M. (1987) Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry. Cytometry 8, 327-334. (36) Duverger, E., Frison, N., Roche, A. C., and Monsigny, M. (2003) Carbohydrate-lectin interactions assessed by surface plasmon resonance. Biochimie 85, 167-179. (37) Simpson, D. Z., Hitchen, P. G., Elmhirst, E. L., and Taylor, M. E. (1999) Multiple interactions between pituitary hormones and the mannose receptor. Biochem. J. 343 Pt 2, 403-411. (38) Napper, C. E., Dyson, M. H., and Taylor, M. E. (2001) An extended conformation of the macrophage mannose receptor. J. Biol. Chem. 276, 14759-14766. (39) Taylor, M. E., and Drickamer, K. (1993) Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor. J. Biol. Chem. 268, 399-404. (40) Jung, T., Schauer, U., Heusser, C., Neumann, C., and Rieger, C. (1993) Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197-207. (41) Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Racs, J., Groff, T., and Raftery, M. A. (1969) The nature of amino acid

Srinivas et al. side chains which are critical for the activity of lysozyme. Biochemistry 8, 700-712. (42) Takahashi, K. (1976) The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide. J. Biochem. (Tokyo) 80, 1267-1275. (43) Ramakrishna, V., Treml, J. F., Vitale, L., Connolly, J. E., O’Neill, T., Smith, P. A., Jones, C. L., He, L. Z., Goldstein, J., Wallace, P. K., Keler, T., and Endres, M. J. (2004) Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs antimelanoma T cell responses via multiple HLA molecules. J. Immunol. 172, 2845-2852. (44) Keler, T., Ramakrishna, V., and Fanger, M. W. (2004) Mannose receptor-targeted vaccines. Expert Opin. Biol. Ther. 4, 1953-1962. (45) Tacken, P. J., de Vries, I. J., Gijzen, K., Joosten, B., Wu, D., Rother, R. P., Faas, S. J., Punt, C. J., Torensma, R., Adema, G. J., and Figdor, C. G. (2005) Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody. Blood 106, 1278-1285. (46) Dakappagari, N., Maruyama, T., Renshaw, M., Tacken, P., Figdor, C., Torensma, R., Wild, M. A., Wu, D., Bowdish, K., and KretzRommel, A. (2006) Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses. J. Immunol. 176, 426-440. (47) Gras-Masse, H. (2001) Chemoselective ligation and antigen vectorization. Biologicals 29, 183-188. (48) Narendran, P., Elsegood, K., Leech, N. J., Macindoe, W. M., Boons, G. J., and Dayan, C. M. (2004) Dendritic cell-based assays, but not mannosylation of antigen, improves detection of T-cell responses to proinsulin in type 1 diabetes. Immunology 111, 422-429. BC070026G