Chemoselective Assembly and Immunological Evaluation of

Bioconjugate Chem. 2005, 16, 1149−1159

1149

Chemoselective Assembly and Immunological Evaluation of Multiepitopic Glycoconjugates Bearing Clustered Tn Antigen as Synthetic Anticancer Vaccines Saulius Grigalevicius,†,§ Sabine Chierici,† Olivier Renaudet,† Richard Lo-Man,‡ Edith De´riaud,‡ Claude Leclerc,‡ and Pascal Dumy*,† LEDSS UMR 5616 and ICMG-FR2607, Universite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France, Unite´ de Biologie des Re´gulations Immunitaires, INSERM E352, Institut Pasteur, 75724 Paris Cedex 15, France, and Department of Organic Technology, Kaunas University of Technology, Radvilenu plentas 19, LT 50254, Kaunas, Lithuania. Received January 19, 2005; Revised Manuscript Received July 1, 2005

In this paper we investigated the use of regioselectively addressable functionalized templates (RAFTs) as new scaffolds for the design of anticancer vaccine candidates. We report the synthesis of welldefined multiepitopic RAFT scaffolds and their immunological evaluation. These conjugates exhibit clustered Tn analogue as tumor-associated carbohydrate antigen (TACA, B-cell epitope) and the CD4+ helper T-cell peptide from the type 1 poliovirus. The saccharidic and peptidic epitopes were both synthesized separately and combined regioselectively to the RAFT core using a sequential oxime bond formation strategy. B- and T-antigenicity and immunogenicity of the vaccine candidates were investigated in vitro and in vivo. These studies clearly demonstrate that the saccharidic part of the conjugates is recognized by Tn-specific monoclonal antibodies. Moreover, the antibodies elicited by immunization of mice with our vaccine candidates recognize the native form of Tn epitope expressed on human tumor cells. Together with oxime ligation technique, these results suggest that the RAFT scaffold provides a promising and suitable tool for engineering potent synthetic anticancer vaccine.

INTRODUCTION

Tumor-associated carbohydrate antigens (TACAs) such as Tn, TF, and sTn are overexpressed in clusters at the malignant cell surface in breast, prostate, lung, and pancreatic cancers (1-5). While these carbohydratebased cancer antigens represent attractive targets for epithelial cancer immunotherapy (6-10), their weak immunogenicity remains critical for the design of efficient synthetic anticancer vaccines. A first generation of vaccines in which the TACAs are conjugated to an immunogenic carrier protein such as KLH (6, 11-17), BSA, or OSA (16, 18, 19) has been developed few years ago. Particularly, a mucin-derived glycopeptide-KLH conjugate containing three Tn antigens designed by S. Danishefsky has been used in clinical trials as putative synthetic vaccine against prostate cancer (16). This approach has proved to induce an immune response specifically directed against the targeted tumors. However, in most cases the low level of the desired antibodies, due to the low molecular ratio of the TACA over the carrier protein, the resulting irrelevant response against the carrier, and the undefined composition of the molecule, represent major limitations for their use in cancer therapy. An alternative class of fully synthetic vaccines based on peptide or lipopeptide cores (20-23) has been designed to circumvent some of these drawbacks. Especially, branched templates of accurate chemical definition such * To whom correspondence should be addressed. Phone: 33 (0) 4 76 63 55 45. Fax: 33 (0) 4 76 51 43 82. E-mail: [email protected]. † Universite ´ Joseph Fourier. ‡ Institut Pasteur. § Kaunas University of Technology.

as dendrimeric lysine scaffolds, namely multiple antigen peptide (MAP) (24-29) or multiple antigen glycopeptide (MAG) (25, 30), have been used as a nonimmunogenic carrier in which the B-cell antigens are radiated from the core to immunogenic T-cell helper peptides. This alternative has emerged considerable interests by presenting a high density of clustered antigens at the surface of a small peptidic scaffold as found for Tn antigen on tumors and eliciting also a strong specific antibody response (31, 32). Indeed, MAG molecule exhibiting four copies of a CD4+ T-cell epitope together with the tri-Tn glycotope presented high immunogenicity in mice (32) and in nonhuman primates (33) and afforded good protection in prophylactic and therapeutic vaccinations against the development of Tn-expressing tumor cells in mice. The humoral response was dominated by IgG antibodies supporting the notion that a T-cell dependent response was induced. Following a similar approach, we investigated in this study the use of regioselectively addressable functionalized templates (RAFTs) (34), as new scaffolds for the design of anticancer vaccines. RAFTs are topological templates which are commonly composed of a backbonecyclized decapeptide containing two proline-glycine as β-turns inducers (35) that stabilize their conformation in solution. They have found useful applications in the past as protein mimics (36-39) and more recently as vectors for neo-vasculature targeting in tumor therapy (40) or as cell surface mimics for multivalent presentation of carbohydrates (41, 42). These cyclic templates should also provide a suitable tool for the multimeric presentation of B- and T-epitopes. Indeed they offer the advantages via lysinyl side chains of displaying two independent functional faces, one dedicated to the attachment of the TACA moieties in a multivalent manner and the

10.1021/bc050010v CCC: $30.25 © 2005 American Chemical Society Published on Web 09/02/2005

1150 Bioconjugate Chem., Vol. 16, No. 5, 2005

Grigalevicius et al.

Figure 1. Strategy for the convergent assembly of the synthetic vaccine candidates 15a and 15b via an oxime-based methodology.

other to the T-cell peptide epitopes. We thus synthesized new multiepitopic neoglycoconjugates combining clustered Tn antigen analogue as TACA and T-cell epitope from the type 1 poliovirus as a CD4+ T-cell epitope (Figure 1). B- and T-antigenicity of our vaccine candidates RAFT(4GalNAc,1PV)1 15a and RAFT(4GalNAc,2PV) 15b were finally investigated in vitro by ELISA and T-cell stimulation assays. Their immunogenicity was also studied in vivo by immunization of mice. Synthetic and biological results are reported in details in this paper. EXPERIMENTAL PROCEDURES

Syntheses. Protected amino acids and Rink amide MBHA resin were purchased from France Biochem S.A. or Advanced ChemTech Europe. All chemical reagents and solvents were purchased from Sigma Aldrich, Fluka, Acros, or Carlo-Erba and were used without further purification. Analytical TLCs were performed on 0.2 mm silica 60 coated aluminum foils with F-254 indicator (Merck). Preparative column chromatographies were done using silica gel (Merck 60, 200-63 µm). Melting points were measured on an Electrothermal Series IA9100 apparatus. 1H and 13C NMR spectra were recorded on Bruker AC300 spectrometers and chemical shift (δ) are reported in parts per million (ppm). Spectra were referenced to the residual proton solvent peaks. The cyclic templates 12a,b (34, 40) and biotin sulfone (43) were prepared as previously described. Reverse phase HPLC analyses were performed on Waters equipment using C18 columns. The analytical (Nucleosil 120 Å 3 µm C18 particles, 30 × 4.6 mm2) was operated at 1.3 mL/min and the preparative column (Delta-Pak 300 Å 15 µm C18 particles, 200 × 25 mm2) at 22 mL/min with UV monitoring at 214 and 250 nm using a linear A-B gradient (buffer A: 0.09% TFA in water; buffer B: 0.09% TFA in 90% acetonitrile). Mass spectra were obtained by electron spray ionization (ES-MS) on a VG Platform II or by chemical ionization (CI-MS) on a Thermofinnigan Polaris Q in the positive mode. 1 To facilitate the reading of this manuscript, we used a simplified nomenclature for the RAFT conjugates. For instance, RAFT(4GalNAc,1PV) 15a denotes the RAFT presenting four GalNAc moieties on the upper face and one PV fragment on the other face.

3,4,6-Tri-O-acetyl-2-azido-2-deoxy-D-galactopyranosyl Fluoride (4). The glycosylfluoride 4 was prepared from 3 after azidonitration of the commercial 3,4,6-triO-acetyl galactal 1 (44) then denitration reaction (45) using the following procedure. Under inert gas, the compound 3 (4.36 g, 13.2 mmol) was dissolved in dry THF (116 mL) and the solution was cooled at -30 °C. Diethylaminosulfur trifluoride (2.08 mL, 15.8 mmol) was then added, and the solution was stirred at room temperature during 30 min. After addition of methanol (10 mL) at -30 °C, the solution was concentrated and taken up with dichloromethane. The organic layer was washed successively with saturated solution of NaHCO3 then water, dried over sodium sulfate, and evaporated to get the compound 4 (4.30 g, 97%) as a colorless oil; 1H NMR (300 MHz, CDCl3) δ 5.71 (dd, J1R,2R ) 2.6 Hz, J1R,F ) 52.2 Hz, H-1R), 5.48 (dd, J4R,5R ) 1.2 Hz, J3R,4R ) 3.2 Hz, H-4R), 5.36-5.34 (m, H-3R), 5.32 (td, J4β,5β ) 1.1 Hz, J3β,4β ) 3.3 Hz, H-4β), 5.09 (dd, J1β,2β ) 7.5 Hz, J1β,F ) 51.8 Hz, H-1β), 4.83 (ddd, J3β,F ) 0.8 Hz, J3β,4β ) 3.3 Hz, J2β,3β ) 10.9 Hz, H-3β), 4.36 (td, J4R,5R ) 1.2 Hz, J5R,6R ) 6.4 Hz, H-5R), 4.17-4.06 (m, H-6R,β), 3.96 (bt, J5β,6β ) 6.3 Hz, H-5β), 3.78 (ddd, J1β,2β ) 7.5 Hz, J2β,3β ) 10.9 Hz, J2β,F ) 12.7 Hz, H-2β), 3.78-3.68 (m, H-2R), 2.13-2.01 (6s, 6OCOCH3); 13C NMR (75 MHz, CDCl3) δ 170.7 (CdO), 170.3 (CdO), 170.2 (CdO), 170.0 (CdO), 169.6 (CdO), 108.3 (d, JC-1β,F ) 217 Hz, C-1β), 106.2 (d, JC-1R,F ) 224 Hz, C-1R), 71.6 (C-5β), 71.0 (d, JC-3β,F ) 11 Hz, C-3β), 69.5 (C-5R), 68.5 (C-3R), 67.2 (C-4R), 66.3 (C-4β), 61.8 (C-6R), 61.5 (C-6β), 61.2 (d, JC-2β,F ) 22 Hz, C-2β), 57.8 (d, JC-2R,F ) 24 Hz, C-2R), 21.0 (OCOCH3), 20.9 (OCOCH3), 20.8 (OCOCH3). This product was used without further purification. O-(3,4,6-Tri-O-acetyl-2-azido-2-deoxy-D-galactopyranosyl)-N-oxyphthalimide (5). To a stirring solution in dichloromethane (200 mL) containing compound 4 (4.30 g, 13.0 mmol), N-hydroxyphthalimide (2.12 g, 13.0 mmol), and triethylamine (1.81 mL, 13.0 mmol) was added BF3‚Et2O (3.52 mL, 65.0 mmol). The reaction was stirred at room temperature until completion (reaction monitored by thin-layer chromatography). After addition of dichloromethane (200 mL), the organic layer was washed several times with 10% aqueous sodium hydrogenocarbonate, dried under sodium sulfate, and evaporated. The alpha and beta anomers were finally separated by silica gel chromatography (dichloromethane/ethyl

Multiepitopic Glycoconjugates as Anticancer Vaccines

acetate, 9/1) to give pure compound 5 (2.35 g, 38%) after crystallization from diethyl ether/pentane; mp 105-107 °C; 1H NMR (300 MHz, CDCl3) δ 7.85-7.75 (m, 4 H, Har.), 5.57 (bd, 2 H, J1,2 ) J3,4 ) 3.4 Hz, H-1, H-4), 5.47 (dd, 1 H, J1,2 ) 3.4 Hz, J2,3 ) 11.3 Hz, H-3), 5.17 (bt, 1 H, J5,6a ) 6.4 Hz, H-5), 4.23 (dd, 1 H, J5,6a ) 6.4 Hz, J6a,6b ) 11.3 Hz, H-6a), 3.97-3.92 (m, 2 H, H-2, H-6b), 2.14, 2.06, 2.02 (3s, 9 H, 3OCOCH3); 13C NMR (75 MHz, CDCl3) δ 170.8 (CdOAc), 170.3 (CdOAc), 169.9 (CdOAc), 163.3 (CdOPht), 135.2 (CHar.), 129.1 (Car.), 124.2 (CHar.), 103.5 (C-1),69.2 (C-5), 68.2 (C-4), 67.7 (C-3), 61.6 (C-6), 57.0 (C-2), 21.1 (OCOCH3); MS-ES (positive mode): calcd for C20H19N4O10Na: 498.09 [M + Na]+, found: 497.96. The corresponding beta anomer (3.09 g; 50%) was also obtained; mp 78-80 °C; 1H NMR (300 MHz, CDCl3) δ ) 7.92-7.78 (m, 4 H, Har.), 5.37 (dd, 1 H, J4,5 ) 1.1 Hz, J3,4 ) 3.3 Hz, H-4), 5.03 (d, 1 H, J1,2 ) 8.4 Hz, H-1), 4.89 (dd, 1 H, J3,4 ) 3.3 Hz, J2,3 ) 10.7 Hz, H-3), 4.20-4.13 (m, 2 H, H-6), 4.00 (dd, 1 H, J1,2 ) 8.4 Hz, J2,3 ) 10.7 Hz, H-2), 3.91 (td, 1 H, J4,5 ) 1.1 Hz, J5,6 ) 6.9 Hz, H-5), 2.18, 2.09, 1.97 (3s, 9 H, 3OCOCH3); 13C NMR (75 MHz, CDCl3) δ ) 170.6 (Cd OAc), 170.4 (CdOAc), 170.0 (CdOAc), 163.0 (CdOPht), 135.2 (CHar.), 129.1 (Car.), 124.3 (CHar.), 106.2 (C-1), 71.8 (C-5), 71.4 (C-3), 66.1 (C-4), 61.1 (C-6), 59.5 (C-2), 21.0 (OCOCH3), 20.9 (OCOCH3), 20.9 (OCOCH3). O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-r-D-galactopyranosyl)-N-oxyphthalimide (6). To compound 5 (1.68 g, 3.5 mmol) dissolved in a solution of methanol/ acetic anhydride (9/1, 40 mL) was added Pd/C 10% (0.38 g, 0.3 mmol). The mixture was stirred at room temperature under an atmosphere of hydrogen for 1 h. The catalyst was then removed by filtration under Celite and washed with methanol. After evaporation of the solvent under reduced pressure, the residue was finally purified by silica gel chromatography (dichloromethane/ethyl acetate, 4/1 then pure ethyl acetate) to afford N-acetylated derivative 6 (0.92 g, 53%) as a white powder after precipitation from dichloromethane/diethyl ether; mp 148-150 °C; 1H NMR (300 MHz, CDCl3) δ 7.86-7.77 (m, 4 H, Har.), 6.07 (d, 1 H, J2,NH ) 9.6 Hz, NH), 5.54 (d, 1 H, J3,4 ) 3.0 Hz, H-4), 5.38 (d, 1 H, J1,2 ) 3.4 Hz, H-1), 5.34 (dd, 1 H, J3,4 ) 3.0 Hz, J2,3 ) 11.3 Hz, H-3), 5.08 (bt, 1 H, J5,6 ) 6.4 Hz, H-5), 4.80 (ddd, 1 H, J1,2 ) 3.4 Hz, J2,NH ) 9.6 Hz, J2,3 ) 11.3 Hz, H-2), 4.30 (dd, 1 H, J5,6 ) 6.4 Hz, J6a,6b ) 11.3 Hz, H-6a), 4.00 (dd, 1 H, J5,6 ) 6.4 Hz, J6a,6b ) 11.3 Hz, H-6b), 2.18, 2.12, 2.09, 2.04 (4s, 12 H, 3OCOCH3, NHCOCH3); 13C NMR (75 MHz, CDCl3) δ 171.2 (CdO), 135.2 (CHar.), 129.1 (Car.), 124.2 (CHar.), 105.4 (C-1), 71.5, 69.5, 67.7, 61.9 (C-3, C-4, C-5, C-6), 47.7 (C-2), 21.1 (OCOCH3). O-r-D-Galactopyranosyloxyamine (7). The compound 6 (0.86 g, 1.7 mmol) was dissolved in a solution of ethanol/methylhydrazine (1/1, 10 mL) and stirred at room-temperature overnight. After evaporation of the mixture, the residue was purified by flash silica gel chromatography (dichloromethane/ethanol, 7/3 then pure ethanol) to obtain the pure aminooxylated product 7 (0.37 g, 90%) as a white powder; 1H NMR (300 MHz, D2O) δ 4.99 (d, 1 H, J1,2 ) 4.1 Hz, H-1), 4.24 (dd, 1 H, J1,2 ) 4.1 Hz, J2,3 ) 11.3 Hz, H-2), 4.03-3.99 (m, 2 H, H-4, H-5), 3.89-3.76 (m, 3 H, H-3, H-6), 2.08 (s, 3 H, HNCOCH3); 13C NMR (75 MHz, D O) δ 175.0 (HNCOCH ), 101.0 (C2 3 1), 71.4, 68.8 (C-4, C-5), 68.0 (C-3), 61.5 (C-6), 49.6 (C-2), 22.3 (HNCOCH3); MS-CI (positive mode): calcd for C8H17N2O6 237.10 [M + H]+, found: 237.1. OHCOC-KLFAVWKITYKDT-NH2 (11). The protected peptide sequence, precursor of peptide 10, H-S(tBu)K(Boc)LFAVW(Boc)K(Boc)IT(tBu)Y(tBu)K(Boc)D(tBu)T(tBu)-NH2, was built up by Fmoc/tBu strategy from 200

Bioconjugate Chem., Vol. 16, No. 5, 2005 1151

mg of Rink amide MBHA resin (substituted at ca. 0.61 mmol/g). Coupling steps were performed using 2 equiv of NR-Fmoc-protected amino acid activated in situ with 2 equiv of PyBOP and 4 equiv of DIPEA in DMF (5 mL) for 40 min. Removal steps of NR-Fmoc protecting groups were achieved by treatment with a piperidine/DMF solution (1/4, 5 mL) three times during 10 min. Finally, the peptide was recovered upon acid cleavage by a solution of TFA/TIS/H2O/p-cresol 89/1/5/5 (5 mL x 30 min, three times). The cleavage solutions were concentrated under vacuum and the peptide H-SKLFAVWKITYKDTNH2 10 was obtained as TFA salt (114 mg, 41%) after precipitation from ether and RP-HPLC purification (0 to 50% B in 30 min); tR ) 8.3 min (5 to 100% B in 15 min, 214 and 250 nm); ES-MS calcd for C82H127N19O20 (1699.0): m/z 850.3 (M + 2H)2+, 567.3 (M + 3H)3+. The serine oxidation of the peptide 10 (40 mg, 17.6 µmol) was carried out in a phosphate buffer 0.01 M pH 7.3 (2 × 10-3 M solution) using 1.5 equiv of NaIO4 (5.6 mg) for 40 min. The solution was purified by RP-HPLC (5 to 100% B in 30 min) to afford the aldehyde 11 (31 mg, 83%) as a white salt; tR ) 8.6 min (5 to 100% B in 15 min, 214 and 250 nm); ES-MS calcd for C81H122N18O20 (1667.9): m/z 834.6 (M + 2H)2+, 556.8 (M + 3H)3+. RAFT(4Ser,1COCH2ONH2)1 (13a). Removal of Boc moieties from RAFT(4Boc,1Alloc) 12a (360 mg, 0.240 mmol) was carried out in a solution containing 50% TFA in DCM (15 mL) for 1h at room temperature. The crude was concentrated, triturated and washed with diethyl ether to yield RAFT(4NH2,1Alloc) as a white TFA salt (305 mg, 82%). This compound (295 mg, 0.19 mmol) was dissolved in 20 mL of DMF, BocSer(tBu)OH (198 mg, 0.76 mmol), and PyBOP (394 mg, 0.76 mmol) were added and the pH was adjusted to 8-9 by adding DIPEA (198 µL, 1.14 mmol). The solution was stirred 2 h at room temperature, the solvent was removed, and diethyl ether was added to precipitate the RAFT(4BocSer(tBu),1Alloc)used without further purification (275 mg, 70%); tR ) 14.8 min (5 to 100% B in 15 min, 214 nm); ES-MS calcd for C99H173N19O28 (2077.6): m/z 1039.7 (M + 2H)2+, 693.3 (M + 3H)3+. Alloc group was removed from the crude (0.13 mmol) dissolved in 20 mL of dry DCM/THF (10/1) under argon by adding PhSiH3 (583 µL, 3.25 mmol) and then Pd(PPh3)4 (55 mg, 0.03 mmol) for 1 h. The solution was filtrated on C18 silica gel, the filtrate was evaporated and precipitated in ether to get after purification (5 to 100% B in 30 min) the RAFT(4BocSer(tBu),1NH2) (231 mg, 84%); tR ) 13.4 min (5 to 100% B in 15 min, 214 nm); ES-MS calcd for C95H169N19O26 (1993.5): m/z 1993.9 (M + H)+, 997.5 (M + 2H)2+. To a solution of RAFT(4BocSer(tBu),1NH2) (60 mg, 28 µmol) in 10 mL of DMF, BocNHOCH2CO2Su (10 mg, 34 µmol) was added and DIPEA (12 µL, 68 µmol) to adjust the pH at 8-9. The reaction was stirred for 45 min at room temperature and then concentrated under reduce pressure. The crude RAFT(4BocSer(tBu),1COCH2ONHBoc) was obtained (49 mg, 81%) as a white powder after precipitation and washing with diethyl ether; tR ) 14.6 min (5 to 100% B in 15 min, 214 nm). Removal of Boc and tBu moieties were achieved by treatment with 10 mL of a solution of TFA/TIS/H2O 95/2.5/2.5 for 2 h. Evaporation of solvent and precipitation in diethyl ether afforded the template RAFT(4Ser,1COCH2ONH2) 13a (40 mg, 87%); tR ) 5.6 min (5 to 100% B in 15 min, 214 nm); ES-MS calcd for C61H108N20O20 (1441.6): m/z 721.3 (M + 2H)2+, 481.2 (M + 3H)3+, 361.2 (M + 4H)4+. RAFT(4Ser,2COCH2ONH2) (13b). The same procedure applied from 12b (100 mg, 0.06 mmol) allowed to get the intermediate RAFT(4BocSer(tBu),2NH2) (60 mg,


0.03 mmol) in 43% overall yield. tR ) 12.5 min (5 to 100% B in 15 min, 214 nm). The RAFT(4BocSer(tBu),2NH2) gave, after coupling of BocNHOCH2CO2Su and Boc and tBu removals, the peptide 13b (40 mg, 0.018 mmol) in 68% yield. tR ) 5.5 min (5 to 100% B in 15 min, 214 nm); ES-MS calcd for C66H118N22O22 (1571.8): m/z 786.4 (M + 2H)2+, 524.7 (M + 3H)3+, 393.2 (M + 4H)4+. RAFT(4Ser,1PV) (14a). RAFT(4Ser,1COCH2ONH2) 13a (16 mg, 7.9 µmol) and PV aldehyde 11 (16.8 mg, 7.9 µmol) were ligated in 5 mL of sodium acetate buffer 0.1 M pH 4. After 24 h under stirring the mixture was purified by RP-HPLC (5 to 40% B in 30 min) and the RAFT(4Ser,1PV) 14a was obtained as a white powder (25 mg, 78%); tR ) 10.9 min (5 to 60% B in 15 min); ES-MS calcd for C142H228N38O39 (3091.6): m/z 1546.5 (M + 2H)2+, 1031.3 (M + 3H)3+, 773.8 (M + 4H)4+, 619.3 (M + 5H)5+. RAFT(4Ser,2PV) (14b). The same procedure was applied from RAFT(4Ser,2COCH2ONH2) 13b (12 mg, 5.3 µmol) and an excess of PV aldehyde 11 (34 mg, 16 µmol). The excess of 11 was trapped by adding benzyloxyamine and the mixture was purified by RP-HPLC (0 to 50% B in 40 min) to yield 14b (24 mg, 72%); tR ) 14.4 min (0 to 50% B in 15 min, 214 and 250 nm). ES-MS calcd for C228H358N58O60 (4871.7): m/z 1624.4 (M + 3H)3+, 1218.8 (M + 4H)4+, 975.3 (M + 5H)5+, 812.9 (M + 6H)6+, 696.9 (M + 7H)7+, 609.9 (M + 8H)8+, 542.3 (M + 9H)8+. RAFT(4GalNAc,1PV) (15a). The RAFT(4Ser,1PV) 14a (10.5 mg, 3.4 µmol) was oxidized using a polymer supported periodate (2.5 µmol/mg, 19.75 µmol) in phosphate buffer (0.01 M, pH 7.3) for 5 h. The mixture was filtered to remove the polymer-resin and purified to yield RAFT(4COCHO,1PV) (8.7 mg, 86%) (5 to 40% B in 30 min). tR ) 11.4 min (5 to 60% B in 15 min, 214 and 250 nm). RAFT(4COCHO,1PV) (8.7 mg, 2.9 µmol) and RGalNAc-ONH2 sugar 7 (6.4 mg, 27 µmol) were stirred in sodium acetate buffer (0.1 M, pH 4) for 36 h and the solution was purified by RP-HPLC (15 to 45% B in 45 min) to afford the RAFT(4GalNAc,1PV) 15a (7.4 mg, 66%); tR ) 11.3 min (15 to 45% B in 15 min, 214 and 250 nm); ES-MS calcd for C170H264N42O59 (3840.2): m/z 1920.6 (M + 2H)2+, 1280.7 (M + 3H)3+, 970.3 (M + K+3H)4+, 776.5 (M + K + 4H)5+. RAFT(4GalNAc,2PV) (15b). The RAFT(4Ser,2PV) 14b (15 mg, 2.4 µmol) was oxidized using the same procedure to afford after purification (5 to 100% B in 30 min) RAFT(4COCHO,2PV) (11.8 mg, 87%); tR ) 9.1 min (5 to 100% B, 15 min, 214 and 250 nm). RAFT(4COCHO,2PV) (10 mg, 1.76 µmol) and 8 equiv of RGalNAc-ONH2 7 (3.3 mg, 14 µmol) were stirred in 1 mL of sodium acetate buffer (0.1 M, pH 4) for 40 days. The mixture was purified by RP-HPLC (15 to 45% B in 30 min) and 15b (7.2 mg, 63%) was obtained as a white lyophilisat; tR ) 13.7 min (15 to 45% B in 15 min, 214 and 250 nm); ES-MS calcd for C256H394N62O80 (5620.4): m/z 1405.4 (M + 4H)4+, 1124.7 (M + 5H)5+, 937.5 (M + 6H)6+. RAFT(4Ser,1Biotin) (16). The biotin sulfone (10.6 mg, 38 µmol) was coupled on the template RAFT(4BocSer(tBu),1NH2) (40 mg, 19 µmol) using PyBOP as activating agent (20 mg, 38 µmol), and DIPEA (20 µL, 114 µmol) in 6 mL of DMF. After 3 h under stirring, the solvent was concentrated and the crude product precipitated from diethyl ether. It was triturated and washed several times in ether to afford the intermediate RAFT(4BocSer(tBu),1Biotin) (30 mg, 70%) after purification (5 to 100% B in 30 min); tR ) 13.3 min (5 to 100% B in 15 min, 214 nm); ES-MS calcd for C105H183N21O30S (2251.8): m/z 1126.9 (M + 2H)2+, 751.3 (M + 3H)3+. Removal of Boc and tBu protecting groups were achieved


by treatment with 10 mL of TFA for 2 h. Evaporation of solvent and precipitation with diethyl ether allowed to get the RAFT(4Ser,1Biotin) 16 (24 mg, 86%) after purification (5 to 40% B in 40 min); tR ) 6.1 min (5 to 100% B in 15 min, 214 nm); ES-MS calcd for C69H119N21O22S (1626.9): m/z 814.0 (M + 2H)2+, 543.2 (M + 3H)3+. RAFT(4Glc,1Biotin) (17) and RAFT(4GalNAc,1Biotin) (18). The serine oxidation of 16 (20 mg, 9.6 µmol) was carried out in 1 h using 4.8 equiv of NaIO4 (10 mg) in 5 mL of water. The solution was directly purified by RP-HPLC (0 to 50% B in 30 min) to afford the RAFT(4COCHO,1Biotin) (12 mg, 83%); tR ) 8.9 min (0 to 50% B in 15 min, 214 nm). To a solution containing the intermediate RAFT(4COCHO,1Biotin) (6 mg, 4 µmol) in 2 mL of sodium acetate buffer (0.1 mM, pH 4) was added the βGlc-ONH2 derivative 9 (6.3 mg, 32 µmol) or the RGalNAc-ONH2 7 (7.5 mg, 32 µmol). The reactions followed by RP-HPLC were placed under stirring at room temperature for 24 h. Conjugates RAFT(4Glc,1Biotin) 17 (5.3 mg, 66%) and RAFT(4GalNAc,1Biotin) 18 (6.3 mg, 60%) were isolated after RP-HPLC purification (0 to 50% B in 30 min) as white powders; 17: tR ) 9.2 min (0 to 50% B in 15 min, 214 and 250 nm); ES-MS calcd for C89H143N21O42S (2211.3): m/z 1106.5 (M + 2H)2+; 18: tR ) 9.1 min (0 to 50% B in 15 min, 214 and 250 nm); ESMS calcd for C97H155N25O42S (2375.5): m/z 1188.8 (M + 2H)2+, 792.4 (M + 3H)3+. ELISA and Flow Cytometry. The antigenicity of the RAFT(4GalNAc,1Biotin) 18 was assessed by ELISA using two monoclonal antibodies specific for the Tn antigen, the 83D4 (IgM; kindly given by Dr. E. Osinaga, Facultad de Medicina, Montevideo, Uruguay) and the 6E11 (IgG3; produced in our lab (unpublished data)). RAFT(4Ser,1Biotin) 16 and RAFT(4Glc,1Biotin) 18 were used as control for specificity. The immunogenicity of the RAFT compounds was assessed in BALB/c mice (CER Janvier, Le Genest St Ile, France). For this purpose, mice were ip immunized with the different RAFTs containing one and two PV sequences 15a,b and 14a,b in alum. Various times after immunization sera were collected and tested for IgG antibodies by ELISA using RAFT(4GalNAc,1Biotin) 18 or RAFT(4Ser,1Biotin) 16. Serial dilutions of sera were performed and bound antibodies were revealed using goat anti-mouse IgG peroxidase conjugate (Sigma, St. Louis, MO) and O-phenyldiamine/ H2O2 substrate. Plates were read photometrically at 492 nm in an ELISA auto-reader (Dynatech, Marnes la Coquette, France). The negative control consisted of naive mouse sera diluted 100-fold. ELISA antibody titers were determined by linear regression analysis, plotting dilution versus absorbance at 492 nm. The titers were calculated to be the Log10 highest dilution which gave twice the absorbance of normal mouse sera diluted 1/100. Titers are given as the arithmetic mean ( SD of the Log10 titers. Sera were also tested at 1/50 dilution by flow cytometry for recognition of Tn-expressing human Jurkat tumor cell line. Cells were first incubated for 30 min with serial dilutions of sera at 4 °C in PBS containing 5% fetal calf serum and 0.05% sodium azide. Then, cells were incubated for 30 min with an anti-mouse IgG conjugated to phycoerythrin (Caltag, Burlingame, CA). Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), and data analysis was performed with CellQuest software (Becton Dickinson). The data are shown as histograms corresponding to the fluorescence of cells incubated with the secondary reagent alone or together with sera. T-Cell Assays. The recognition by specific T-cells of the poliovirus T-cell epitope contained in the RAFT-

Multiepitopic Glycoconjugates as Anticancer Vaccines Scheme 1. Synthesis of Aminooxylated Tn Antigen 7 (A) and Glucose 9 (B)a

a Reagents and conditions: (A) (i) NaN , CAN, CH CN, -15 3 3 °C; (ii) NaNO2, dioxane/H2O (1/1), 80 °C; (iii) DAST, THF; (iv) PhtOH, TEA, CH2Cl2, BF3‚Et2O; (v) H2, Pd/C, CH3OH/Ac2O (9/ 1); (vi) MeHNNH2; (B) phase-transfer catalysis (PTC) (70).

(4Ser,1PV) 13a and the RAFT(4GalNAc,1PV) 15a was analyzed using a specific T-cell hybridoma and bone marrow-derived dendritic cells as antigen-presenting cells. T-cell hybridomas (105) were cultured with dendritic cells (105) in the presence of the RAFT at several concentrations for 24 h in RPMI 1640 supplemented with 10% FCS, antibiotics, L-glutamine, and mercaptoethanol. Interleukin 2 production by hybridoma T-cells was assessed by the measure of the proliferative response of the interleukin 2-dependent CTLL cell line using [3H]thymidine. RESULTS AND DISCUSSION

Design of the Vaccine Candidates. Taking advantages of the synthetic versatility of the RAFT platform, we designed our vaccine candidates 15a and 15b so that to mimic through one domain of the template the clustering of the mucin-associated Tn antigen (Figure 1) which characterizes the surface of epithelial tumors (4, 5). We assembled regioselectively on the other domain a peptide fragment from poliovirus in order to stimulate CD4+ T-cells and activate the production of specific antibodies for the TACA by B-cells (Figure 1). We chose the peptide fragment KLFAVWKITYKDT from the type I-poliovirus protein since it previously proved to provide efficient T-cell help for the induction of antibody responses in BALB/c mice (31). One critical aspect of our approach focuses on controlling the molecular assembly processes which are essential to warrant the structure of the target molecule. While many chemical methodologies including solution and


solid-phase synthesis protocols have been reported so far, the assembly of carbohydrate-based bioconjugates remains fastidious due to numerous manipulations of orthogonal protecting groups (46-49). As an attractive synthetic alternative, we and other have demonstrated recently that chemoselective ligation via oxime or polyoxime bond formation offers a convenient strategy for the convergent assembly of bioconjugates (40-42, 50-59). Indeed, this ligation technique is highly efficient, compatible with a wide range of chemical functions and allows the ligation between unprotected fragments in minimal chemical transformations without any coupling reagent. Moreover, the oxime linkage has been shown to be stable under physiological conditions (51, 60), and its use for ligation of peptide epitopes in vaccines has been found advantageous (61-63). Such oxime-based strategy requires the synthesis of the RAFT molecule, bearing serine residues as masked glyoxylyl aldehyde functions on the upper domain and protected aminooxy groups on the other domain, and of the B/T-epitopes displaying the suitable complementary functions. For this purpose, we prepared the aminooxylated RGalNAc moiety 7 as a carbohydrate analogue of the native Tn form (RGalNAcSer/Thr) in mucin glycoproteins (1-3). Since it was previously attested that other carbohydrate-based Tn analogues present similar antigenicity as the native antigen (21, 64-66), we expected that this modification should not affect the immunological properties of our constructions. Especially, a vaccine bearing an O-aliphatic aglycone instead of natural amino acid carrier has proved to be more antigenic than the natural Tn-based conjugate (67). In the case of the PV fragment moiety, this one was functionalized by an N-terminal aldehyde spacer prior to its grafting to the RAFT template. Synthesis of the Aminooxylated Tn Analogue 7 and Glucose 9. The Tn analogue (RGalNAc-ONH2) 7 containing an aminoxy group at its anomeric position has been synthesized according to the previously published procedure (Scheme 1A) (68). Starting from the commercial triacetylated galactal 1, the azidonitration reaction described by R. U. Lemieux and R. M. Ratcliffe (44) followed by the anomeric denitration of compound 2 with sodium nitrite in aqueous dioxane at 80 °C (45) gave 3. Treatment of 3 with DAST in THF (69) provided glycosyl fluoride 4 which was glycosylated in CH2Cl2 with Nhydroxyphthalimide as precursor of the aminooxy function using BF3‚Et2O as a promoter in the presence of triethylamine. The alpha and beta anomers were easily separated by silica gel chromatography to obtain 5 in 50% yield (38% for the beta anomer). The azido group was then converted into the NHAc function by a catalytic hydrogenation in CH3OH/Ac2O to get 6. After complete acetate deprotection and aminooxy formation with methylhydrazine, we obtained the alpha aminooxylated Tn antigen 7 in 90% yield. The aminooxylated β-glucose 9 was obtained following the phase-transfer catalysis pro-

Scheme 2. Synthesis of T-Epitope Peptide 11a

a

Reagents and conditions: (i) TFA/TIS/H2O/p-cresol (89/1/5/5); (ii) NaIO4, phosphate buffer 0.01 M, pH 7.3.



Scheme 3. Synthesis of Vaccine Candidates 15a,ba

a Reagents and conditions: (i) a) 50% TFA/CH Cl ; b) BocSer(tBu)OH, PyBOP, DIPEA, DMF; c) Pd(PPh ) /PhSiH , CH Cl ; d) 2 2 3 4 3 2 2 BocNHOCH2CO2Su, DIPEA, DMF; e) TFA/TIS/H2O (95/2.5/2.5); (ii) 11, AcONa buffer 0.1 M pH 4; (iii) a) NaIO4, phosphate buffer 0.01 M, pH 7.3; b) 7, AcONa buffer 0.1 M pH 4.

cedure described by R. Roy from the activated derivative 8 (Scheme 1B) (70). Synthesis of the T-Cell PV Epitope Aldehyde 11. The minimal CD4+ T-cell epitope sequence from the poliovirus type 1, KLFAVWKITYKDT, corresponds to the 103-115 amino acid sequence that is immunogenic in BALB/c mice. We have introduced a serine residue at the N-terminal position to access to the aldehyde function after oxidation. The targeted sequence H-SKLFAVWKITYKDT-NH2 7 was built up by standard Fmoc/tBu strategy from the Rink amide MBHA resin using PyBOP as coupling reagent and diisopropylethylamine (DIPEA) as base. We obtained the fully unprotected peptide 10 in 41% overall yield after removal from the resin and reverse phase HPLC purification (Scheme 2). An adapted oxidation procedure was used for the sodium periodate oxidation of the serine residue due to the presence of tryptophane. We carried out the reaction in phosphate buffer at pH 7.3, and the peptide aldehyde 11 was obtained in 83% yield. Synthesis of the RAFT Scaffolds 13a,b. The chemoselective oxime-based strategy requires the appropriate RAFT cores 13a or 13b to combine the PV aldehyde 11 and the aminooxy sugars 7 and 9. These key intermediates 13a,b present two distinct domains, one bearing aminooxy acetyl sites to ligate the PV sequence and the other bearing four serine residues allowing the combination with the sugar moieties after an oxidation step. They have been prepared (Scheme 3) from the corresponding cyclic scaffolds 12a and 12b (34, 40) presenting Alloc and Boc orthogonal protecting groups at the lysinyl side chains. The removal of Boc groups from the four lysine residues was first achieved in acidic conditions (50% of TFA in CH2Cl2) and followed by the coupling of protected serine BocSer(tBu)OH residues using standard coupling conditions. The aminooxyacetyl moieties were incorporated on the other face as N-BocO-(carboxymethyl)hydroxylamine succinimide ester (71)

Figure 2. (A) HPLC profile (Nucleosil 100 Å 5 µm C18 particles, 250 × 4.6 mm; solvent B: 0.09% TFA in 90% acetonitrile and solvent A: 0.09% TFA; 1.3 mL/min; linear gradient 100:0 A:B to 50:50 in 15 min; detection λ ) 214 and 250 nm) of the reaction mixture 14b after 20 h (the RAFT core 13b is completely consumed after 24 h); (B) ES-MS (positive mode) of 14b after purification; (C) HPLC profile (linear gradient 85:15 A:B to 55: 45 A:B in 15 min; detection λ ) 214 and 250 nm) of the reaction mixture 15a after 36 h; (D) ES-MS (positive mode) of 15a after purification: numbers in italics correspond to the potassium adducts.

after the removal of the Alloc groups using Pd(PPh3)4/ PhSiH3 procedure (72). Further removal of the Boc and tBu from the serine and aminooxyacetyl residues gave the appropriate templates 13a and 13b in 72% and 68% yields, respectively. Regio/chemoselective Assembly of the Vaccine Candidates 15a and 15b. We prepared vaccine candidates bearing four copies of the Tn analogues and one or two copies of the T-cell epitope sequence to determine whether we could further increase the immunogenicity of our vaccines. The ligation of the T-cell peptide fragment 11 and RGalNAc-ONH2 7 were carried out by consecutive oxime bond formation (Scheme 3). PV alde-



Scheme 4. Synthesis of Biotinylated Compounds 16-18a

a Reagents and conditions: (i) (a) 50% TFA/CH Cl ; (b) BocSer(tBu)OH, PyBOP, DIPEA, DMF; (c) Pd(PPh ) /PhSiH , CH Cl ; (d) 2 2 3 4 3 2 2 Biotin sulfone, PyBOP, DIPEA, DMF; (e) TFA; (ii) (a) NaIO4, water; (b) 7 or 9, AcONa buffer 0.1 M pH 4.

Figure 3. Antigenicity of the RAFT bearing the Tn antigen analogue. 6E11 (IgG3) and 83D4 (IgM) monoclonal antibodies specific for the tumor-associated Tn carbohydrate antigen were tested by ELISA for the recognition of RAFT(4GalNAc,1Biotin) 18, RAFT(4Glc,1Biotin) 17 or RAFT(4Ser,1Biotin) 16 directly coated on microtiter plates. Error bars refer to the standard error to the mean of triplicate measures.

hyde 11 was first assembled to the cores 13a and 13b in sodium acetate buffer following the typical chemoselective ligation procedure we previously described (40-42). The progress of the reactions was monitored by RPHPLC, and no significant difference of reactivity was noticed between the templates 13a and 13b. The completeness of the coupling reaction required up to 24 h to give cleanly mono- and bivalent compounds 14a and 14b which have been fully characterized by ES-MS (Figure 2A,B). These conjugates 14a,b bearing serine residues have been used as reference compounds in immunological assays. After liberation of the four aldehyde functions by sodium periodate oxidation of serine moieties, the aminooxylated Tn antigen 7 was incorporated through the same chemoselective coupling conditions. As we expected, the crude reaction mixtures were clean confirming the high efficiency of the chemoselective ligation method (Figure 2C,D). The conjugates 15a,b were finally isolated in almost 65% yield after semipreparative HPLC purification. Synthesis of Biotinylated Glycoconjugates. Several compounds presenting a biotin moiety instead of the PV fragment on the lower face of the core have also been synthesized for the ELISA tests. The conjugates 16, 17, and 18 (Scheme 4) bearing serine, βGlc, or RGalNAc residues, respectively, have been used as reference compounds to assess the specificity of the immune response. They were prepared from the intermediate displaying four protected serine residues and one unprotected lysine site for biotin attachment. While preliminary experiments have shown that biotins are partially oxidized during the oxidative cleavage of serines, we decided to incorporate biotin sulfone (43) to get welldefined conjugates. The biotin was directly coupled using amide linkage to get 16 after removal of Boc and tBu

Figure 4. Activation of a PV-specific T-cell hybridoma by the PV-containing RAFTs 14a and 15a in the presence of antigenpresenting cells. T-cell hybridoma were cultured with dendritic cells in the presence of the indicated RAFTs at different doses. Interleukin-2 secretion following T-cell hybridoma stimulation was assessed by the proliferation of the interleukin 2-dependent CTLL cell line using [3H]-thymidine. Error bars refer to the standard error to the mean titer of 5 individual sera.

protecting groups of serine. Oxidation of 16 and further ligation of aminooxy sugars RGalNAc-ONH2 7 and βGlcONH2 9 afforded the glycoconjugates 18 and 17 in 60 and 66% yield, respectively. B-Cell Antigenicity. We first investigated by ELISA the ability of the GalNAc moiety incorporated into the RAFT platform to be recognized by monoclonal antibodies (mAbs) specific for the Tn antigen. The two anti-Tn mAbs, an IgG3 (6E11) and an IgM (83D4), were both able to interact with the RAFT(4GalNAc,1Biotin) 18, but not with the RAFT(4Ser,1Biotin) 16 or the RAFT(4Glc,1Biotin) 17 (Figure 3). This clearly demonstrates that the recognition is directed specifically toward the carbohydrate moiety of compound 18. These results also give an accurate view of the display of TACA at the RAFT surface for antibody recognition. The recognition of RAFT(4GalNAc,1Biotin) by these antibodies indicates that the multimeric presentation in the RAFT system is able to mimic the carbohydrate part of the repeated glycosylated serine/threonine unit found on natural tumor-associated mucins. Therefore, our unnatural Tn analogues represent suitable models for humoral stimulation as other reported analogues (6467). T-Cell Antigenicity. To elicit an IgG humoral response, an efficient presentation of the T-cell epitope by MHC II molecules on the antigen-presenting cells (APC) surface is required for T-cell stimulation. Therefore, we analyzed in vitro by a T-cell stimulation assay the recognition of the PV epitope when incorporated into the



Figure 5. Immunogenicity of the RAFT displaying the Tn analogue. (A) BALB/c mice (n ) 5) were immunized on days 0, 21 and 42 with 10 µg of RAFT(4GalNAc,1PV) 15a in alum. Mice were then bled at various times after immunization. Sera from these mice were analyzed by ELISA for antibody titers against RAFT(4GalNAc,1Biotin) 18 or RAFT(4Ser,1Biotin) 16 as indicated. (B) Mice were immunized on days 0 and 21 with 10 µg of RAFT(4GalNAc,1PV) 15a, RAFT(4GalNAc,2PV) 15b, RAFT(4Ser,1PV) 14a or RAFT(4Ser,2PV) 14b. Sera collected at day 28 were analyzed for antibody titers against RAFT(4GalNAc,1Biotin) 18. Results are expressed as mean of log10 IgG antibody titers.

RAFT core. Dendritic cells were incubated with the RAFT(4GalNAc,1PV) 15a or the RAFT(4Ser,1PV) 14a in the presence of a PV-specific T-cell hybridoma. Secreted interleukin-2 (IL-2) was determined in a radioactive proliferation assay by measuring the incorporation of [3H]-thymidine by the IL-2 dependent CTLL cell line (Figure 4). IL-2 secretion by activated T-cell hybridoma following the recognition of peptide-MHC II complexes on the dendritic cell surface by T-cell receptors (TCR) correlates with the incorporation of [3H]-thymidine by CTLL cells. As shown in Figure 4, both the unglycosylated 14a and the glycosylated 15a conjugates stimulate the PV-specific T-cell hybridoma. This indicates that despite being ligated in an unnatural way the T-cell determinant is efficiently presented by APC to CD4+ T-cells. The response (IL-2 production) induced by the RAFT 15a was dramatically enhanced at significantly lower doses compared to the response obtained with the unglycosylated RAFT 14a. Indeed, T-cell stimulation was obtained with about 10 000-fold less RAFT 15a dose compared to the RAFT 14a, suggesting that the presence of the Tn antigen increases the presentation of the PV peptide by MHC class II molecules. This result further confirms previous observations on the enhanced T-cell stimulation induced by peptides bearing the Tn antigen (31, 64). Although the mechanism underlying this phenomenon is unclear, it may strongly benefit to the glycosylated RAFT 15a for stimulating immune responses. Immunogenicity. We next evaluated the immunogenicity of our RAFT constructs exhibiting one or two copies of PV peptide in BALB/c mice. The RAFT(4GalNAc,1PV) 15a was first used to immunize mice and to elicit Tnspecific IgG antibodies. Indeed, sera from immunized mice and tested by enzyme-linked immunosorbent assay against biotinylated conjugates were able to strongly recognize the RAFT(4GalNAc,1Biotin) 18 (Figure 5A). Only 0.1-1% of the antibodies raised by the RAFT(4GalNAc,1PV) 15a recognized the RAFT devoid of the GalNAc moiety (i.e. RAFT(4Ser,1Biotin) 16) demonstrating a limited reactivity against the RAFT platform. These results confirm the ability of the cyclic template as nonimmunogenic carrier for vaccine constructs which is in good agreement with a previous study (39). To determine whether we could further increase the immunogenicity of our conjugates, the antibody response induced by the RAFT(4GalNAc,2PV) 15b carrying two copies of the T-helper PV peptide was also investigated. However, the incorporation of a second copy of the T-cell epitope did not result in an increase of immunogenicity since we observed a similar antibody response when compared with 15a (Figure 5B). In addition, the immune

Figure 6. Antibodies induced following immunization with 15a or 15b recognize the native form of Tn on human Jurkat cells. Sera obtained from mice (n)5) immunized with RAFT(4GalNAc,1PV) 15a, RAFT(4GalNAc,2PV) 15b, RAFT(4Ser,1PV) 14a or RAFT(4Ser,2PV) 14b were analyzed by flow cytometry for the recognition of native Tn on Jurkat cells. FACS data are shown as histograms corresponding to the mean fluorescence intensity of Jurkat cells stained with sera or in the absence of serum and revealed PE-conjugated anti-mouse IgG antibodies. For each group, staining obtained with five individual mouse sera (empty histogram) is shown as compared to the fluorescence obtained with PE-conjugated anti-mouse IgG antibodies in the absence of serum (plain histogram).

response induced by our RAFT-based vaccine candidates results probably from the convenient presentation of multiple copies of the B-cell epitope on the RAFT surface. Indeed, a previous study using dimeric or trimeric molecules based onto a lysine core bearing both T-cell and B-cell determinants has shown that the presentation of the determinants to T-cells may be different for linear compared to branched immunogens bearing the same determinants (27). Particularly, the number of the B-cell


epitopes into a branched construct could facilitate its uptake by specific B-cells in vivo and the subsequent presentation to helper T-cells. RAFT(4GalNAc,1PV) 15a Induces Abs That Recognize Tumor Cells. To clearly determine whether the antibodies elicited by the RAFT are able to recognize the native form of Tn, we tested by flow cytometry their binding to the human Jurkat tumor cell line expressing the Tn antigen. As shown in Figure 6, sera from mice immunized with RAFT(4GalNAc,1PV) 15a or RAFT(4GalNAc,2PV) 15b were able to recognize the Jurkat cells, whereas control RAFT(4Ser,1PV) 14a and RAFT(4Ser,2PV) 14b devoid of TACA did not. These results show the ability of the RAFT constructs bearing Tn- and PV-epitopes to elicit an immune response specifically directed against the native form of Tn displayed by cancer cells. CONCLUSION

In conclusion, we investigated here the use of the RAFT platforms for the preparation of potential anticancer vaccine candidates. On the basis of previous studies, we designed RAFT molecules displaying clustered Tn antigen analogue to mimic the epithelial tumor surface and immunogenic CD4+ T-cell epitope. The synthesis of such well-defined multiepitopic glycoconjugates is based on a sequential oxime bond ligation between the carrier and the B/T-cell epitopes. The immunological evaluation of our RAFT glycoconjugates clearly demonstrate a promising potential of such scaffold as a nonimmunogenic build-in vaccine carrier. Indeed, the antibodies elicited by immunization of mice with our molecules recognize the native form of Tn epitope expressed on human tumor cells, suggesting that the RAFT display Tn antigen analogues in a convenient clustered manner. Altogether, this preliminary study opens interesting perspectives to employ the RAFT scaffold in engineering potent immunogenic conjugates as vaccine candidates. Indeed, the modularity and versatility of the molecular assembly as well as the efficiency of oximebased strategy might be further exploited to improve the immune response by combining TACAs with other immunogenic peptides and/or immunoadjuvants and to develop multicomponent therapeutic vaccines. ACKNOWLEDGMENT

We thank the Association pour la Recherche contre le Cancer (ARC), the Centre National pour la Recherche Scientifique (CNRS), the Ministe`re de la Recherche (MENRT), the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), and the Institut Universitaire de France (IUF) for supporting this work. LITERATURE CITED (1) Springer, G. F. (1984) T and Tn, general carcinoma autoantigens. Science 224, 1198-1206. (2) Itzkowitz, S. H., Yuan, M., Montgomery, C. K., Kjeldsen, T., Takahashi, H. K., Bigbee, W. L., and Kim, Y. S. (1989) Expression of Tn, sialosyl-Tn, and T antigens in human colon cancer. Cancer Res. 49, 197-204. (3) Kim, Y. S., Gum, J., and Brockhausen, I. (1996) Mucin glycoproteins in neoplasia. Glyconjugate J. 13, 693-707. (4) Springer, G. F. (1997) Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy. J. Mol. Med. 75, 594-602. (5) Springer, G. F. (1995) T and Tn pancarcinoma markers: autoantigenic adhesion molecules in pathogenesis, prebiopsy carcinoma-detection, and long-term breast carcinoma immunotherapy. Crit. Rev. Oncogenesis 6, 57-85.

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Chemoselective Assembly and Immunological Evaluation of

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