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Jul 29, 2010 - The use of KLH-conjugate 5 as an immunogen in vivo to raise antibodies that cross-reacted with antigens expressed on melanoma cells is ...
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Stable GM3 Lactone Mimetic Raises Antibodies Specific for the Antigens Expressed on Melanoma Cells Annarosa Arcangeli,*,† Lucio Toma,*,‡ Luca Contiero,§ Olivia Crociani,† Laura Legnani,‡ Carlotta Lunghi,§ Elisa Nesti,† Gloriano Moneti,| Barbara Richichi,§ and Cristina Nativi*,§,⊥ Dipartimento di Patologia e Oncologia Sperimentali, Universita’ di Firenze, Firenze, Italy, Dipartimento di Chimica Organica, Universita’ di Pavia, Pavia, Italy, Dipartimento di Chimica, Universita’ di Firenze, Firenze, Italy, Dipartimento di Farmacologia, Universita’ di Firenze, Firenze, Italy, and FiorGen, Universita’ di Firenze, Firenze, Italy. Received December 17, 2009; Revised Manuscript Received June 23, 2010

Immunotherapy of tumors and of melanoma in particular has a long history, and recently this therapeutic approach found a reliable scientific rationale. This biological therapy aims to teach the patient’s immune system to recognize the antigens expressed on tumor cells and destroy them, leaving normal cells intact. The success of this therapy highly depends on the selection of target antigens that are essential for tumors growth and progression. The overexpression of GM3 ganglioside 1 and especially the expression of its metabolite GM3 lactone 2 characterize murine and human melanomas, playing an important role in tumor progression and making such self-antigens potential targets for the immunotherapy of these neoplasms. Although more immunogenic than its precursor, GM3 lactone 2 is unsuitable to be used in immunotherapy as a melanoma-associated antigen (MAA) because it is unstable under physiological conditions. We designed and synthesized the hydrolytically stable mimetic 3, which is remarkably simpler than the native lactone 2; after conjugation of 3 to the protein carrier keyhole-limpet hemocyanin (KLH), the obtained glycoprotein 5 was used as the immunogen in ViVo to successfully elicit specific antimelanoma antibodies. In fact, no appreciable binding to GM1 was observed. Capitalizing on the stability and on the reduced structural complexity of mimetic 3, the immunostimulant 5 we report represents a new promising synthetic glycoconjugate for the immunotherapy of melanoma.

INTRODUCTION Oncogenic transformations are accompanied by biochemical and functional changes. In particular, qualitative and quantitative changes in carbohydrate composition of the glycocalix during cell differentiation and proliferation have been observed and seem to reflect the state of malignant transformation of a variety of human tumors (1-4). Although the biological significance of such changes has not been elucidated yet, it is widely accepted that they contribute to or trigger an uncontrolled cellular adhesion, making the cell potentially metastatic. In recent years, a variety of monoclonal antibodies have been developed to specifically recognize carbohydrate epitopes that result from these faulty glycosylation processes. These epitopes (5-8), known as tumor associated antigens (TAAs), have been successfully used as markers of tumor progression and include carbohydrates expressed on normal tissues but which are accumulated in high density on the surface of tumor cells (5). Gangliosides are sialic acid containing glycosphingolipids composed of a complex carbohydrate moiety linked to a hydrophobic ceramide portion. Embedded within the outer leaflet of the cell membrane, the carbohydrate chain is exposed to the extracellular matrix. GM3 ganglioside 1 (Figure 1), a glycosph* To whom correspondence should be addressed. Prof. A. Arcangeli: Tel, +39 0554598206; Fax, +39 0554598900; E-mail, annarosa.arcangeli@ unifi.it. Prof. L. Toma: Tel, +39 0382987843; Fax, +39 0382987323; E-mail, [email protected]. Prof. C. Nativi: Tel, +39 0554573540, Fax, +39 0554573570; E-mail, [email protected]. † Dipartimento di Patologia e Oncologia Sperimentali, Universita’ di Firenze. ‡ Universita’ di Pavia. § Dipartimento di Chimica, Universita’ di Firenze. | Dipartimento di Farmacologia, Universita’ di Firenze. ⊥ FiorGen, Universita’ di Firenze.

ingolipid found in essentially all types of cells and tissues, is the major ganglioside in normal melanocytes and is overexpressed in melanoma cells with metastatic potential (9, 10). The corresponding GM3 lactone 2 (Figure 1) has also been found in melanoma as a minor component (11, 12); its formation is likely promoted by the lower pH environment of tumor cells (13) and, possibly, by a different conformation of GM3 ganglioside induced on the tumor cell surface as a result of its local high density (12). GM3 ganglioside has been widely investigated as a potential vaccine against cancer, although with scarce success since the majority of melanoma associated antigens (MAAs) are weakly immunogenic, owing to their nature and tissue distribution (14). Nevertheless, as metastatic melanoma appears to be resistant even to the most recent molecularly targeted agents, immunotherapeutic strategies continue to be used in an attempt to improve the results of the last 10 years of clinical investigations both in active and adoptive immune therapy. Indeed, vaccines prepared by using specific tumor antigens have recently lead to the commercial availability of the first tumor immunotherapeutic agents, which are usually employed in the postsurgery treatment of removed tumors, to stimulate the patient to develop the appropriate immune defenses, in order to inhibit the recruitment of the same tumor and avoid far more invasive chemo- or radiotherapeutic agents (12, 15). To date, a large repertoire of MAAs are available, most of which are extensively studied for their possible use in immunotherapy (16). Recent findings showing relevant clinical responses in metastatic melanoma patients proved the principle that, under appropriate conditions, even large tumor masses can be made to regress by immune interventions (17). Although more immunogenic than GM3 ganglioside 1, GM3 lactone 2 (Figure 1) also failed as an immunostimulant because, under physiological conditions, the amount of lactone is below

10.1021/bc900557v  2010 American Chemical Society Published on Web 07/29/2010

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Figure 1. Structure of GM3 ganglioside 1, GM3 lactone 2, thioether mimetic 3, and GM1 ganglioside.

the recognition threshold. In this context and due to the advantage represented by nonpeptidic immunostimulants (18), hydrolytically stable analogues of GM3 lactone are extremely attractive molecules to replace the native lactone as potential immunostimulants (19, 20). We recently reported (21) the totally diasteroselective synthesis of a thioether-bridged mimetic of GM3 lactone, compound 3 (Figure 1), which is stable under physiological conditions and is structurally simpler than the endogenous lactone 2. The structure of 3 presents a deoxy manno residue replacing the sialic portion, whereas the ceramidic residue is replaced by an aliphatic chain. A striking shape similarity between GM3 lactone 2 and GM3 thioether 3 was revealed; indeed, 3 presents the folded shape characteristic of the lactone structure which might be responsible for the enhanced immunogenicity of 2 compared to that of the parent GM3 ganglioside 1. However, unlike proteins, saccharidic antigens are poorly immunogenic (22); thus, the synthesis of glycosyl derivative 4 (see Scheme 3) and of the corresponding keyhole-limpet hemocyanin (KLH) (23) conjugate 5 was realized and is here described together with the computational approach that allowed the proper choice of the synthetic precursors. The use of KLH-conjugate 5 as an immunogen in ViVo to raise antibodies that cross-reacted with antigens expressed on melanoma cells is also reported.

EXPERIMENTAL PROCEDURES Computational Methods. All of the calculations were carried out using the Gaussian 03 program package through optimizations in the gas phase at the B3LYP/6-311+G(2df,p) level for the S atom and 6-311+G(d,p) level for the other atoms to correctly describe the geometries and the electronic properties of compounds that contain a sulfur atom (24, 25). Vibrational frequencies were computed at the same level of theory to define the optimized structures as minima or transition states, which present an imaginary frequency corresponding to the forming bonds. The solvent effects were considered by single-point calculations, at the same level as that above, on the gas-phase optimized geometries, using a self-consistent reaction field (SCRF) method based on the polarizable continuum model (PCM) (26-28). Representative Procedure for Glycosylation. Azido glycoside 17. To a mixture of 15 (160 mg, 0.25 mmol), bis(cy-

clopentadienyl)hafnium(IV) dichloride (Cp2HfCl2) (170 mg, 0.45 mmol), AgOTf (250 mg, 0.97 mmol), and 6-azido-1-hexanol (70 mg, 0.5 mmol) cooled to -40 °C, dry CH2Cl2 (30 mL) was added. After 40 min at -40 °C, the solution was neutralized with NEt3 and filtered through a pad of Celite, and the filtrate was washed with a saturated solution of NaHCO3 (1 × 50 mL), then with brine (1 × 50 mL). The organic phase was dried over Na2SO4 and concentrated to dryness to give 200 mg of crude product. The crude product was purified by flash column chromatography on silica gel (petroleum ether/EtOAc 1:1) to give 20 (150 mg, 80%), as a glassy solid. 1H NMR (400 MHz, CDCl3): δ 5.37 (d, 1H, Jd-e ) 2.4 Hz), 5.32 (bs, 1H), 5.26-5.21 (m, 1H), 5.12-5.09 (m,1H), 4.97 (s, 1H), 4.50-4.46 (m, 1H), 4.25-4.16 (m, 2H), 4.18-4.14 (X part of an AXY system, 1H, JXA ) 6.4 Hz, JXY ) 11.6 Hz), 4.11-4.06 (Y part of an AXY system, 1H, JYA ) 7.6 Hz, JYX ) 11.6 Hz), 3.96 (ad, 1H, J5-6 ) 10.0 Hz), 3.75-3.69 (m, 1H), 3.53-3.47 (m 1H), 3.24 (t, 2H, J ) 7.2 Hz), 3.02-2.98 (A part of an AB system, 1H, JAB ) 13.0 Hz), 2.97-2.94 (B part of an AB system, 1H, JBA ) 13.0 Hz), 2.15 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.99-1.98 (m 2H), 1.95 (s, 3H), 1.62-1.56 (m, 4H), 1.43-1.36 (m, 4H). 13C NMR (50 MHz, CDCl3): δ 170.8, 170.2, 170.1, 169.6, 169.5 (2C), 137.4, 109.0, 95.7, 93.0, 68.6, 68.0, 67.4, 67.3, 66.4, 64.4 (2C), 62.0, 61.7, 51.3, 33.8, 33.6, 29.3, 28.7, 26.4, 25.7, 20.7 (3C), 20.6 (3C). [R]D23 +22.31 (c 1.09, CH2Cl2). Elem. Anal. for C32H45N3O16S: Calcd. C 50.59, H 5.97, N 5.53. Found: C 50.62, H 6.01, N 5.42; ESI-MS 782.2 [M + Na]+, 798.2 [M + K]+. To a stirred solution of 20 (120 mg, 0.158 mmol) in CH3OH (3.2 mL), MeONa (2 mg, 0.032 mmol) was added. The mixture was stirred at RT for 2.5 h, then the pH adjusted to neutrality with HCl (10% in MeOH). Evaporation of the solvent under vacuum gave a crude product which was purified by flash chromatography on silica gel (EtOAc/CH3OH 5:1) to give 17 (72 mg, 90%) as a glassy solid. 1 H NMR (400 MHz, CD3OD): δ 4.97 (s, 1H), 4.17-4.12 (m, 1H), 4.09-4.06 (m, 1H), 4.02 (dd, 1H, J4-3 ) 3.2 Hz, J4-5 ) 1.2 Hz), 3.94 (dd, 1H, J5-6 ) 9.6 Hz, J5-4 ) 1.2 Hz), 3.84-3.79 (A part of an ABX2 system, 1H, JA-B ) 10.0 Hz, JA-X ) 6.4 Hz), 3.77-3.62 (m, 6H), 3.52-3.47 (B part of an ABX2 system, 1H, JB-A ) 10.0 Hz, JB-X ) 6.4 Hz), 3.31-3.28 (m, 2H), 3.05-3.01 (A part of an AB system, 1H, JAB ) 13.0 Hz), 2.99-2.96 (B part of an AB system, 1H, JBA ) 13.0 Hz),

1434 Bioconjugate Chem., Vol. 21, No. 8, 2010 Chart 1. Structure of Heterodiene 6 and Dienophiles 7 and 9

1.94-1.92 (m, 2H), 1.64-1.57 (m, 4H), 1.46-1.41 (m, 4H). 13 C NMR (50 MHz, CD3OD): δ 141.6, 106.4, 96.3, 92.3, 72.6, 70.9, 68.5, 67.8, 66.5, 66.3, 64.6, 62.8, 61.1, 51.1, 36.3, 33.4, 29.2, 28.6, 26.3, 25.6. [R]D23 +51.64 (c 0.25, CH3OH). ESIMS 530.3 [M + Na]+. Immunization and Fusion. Two female, 8 week old BALB/c mice (Ce.S.A.L., Firenze) were immunized with intraperitoneal (mouse A) and intravenous (mouse B) injections with antigen-KLH conjugate solution four times, twice a week. In the first immunization, the antigen was injected as a 1:1 (v/v) emulsion in Freund’s complete adjuvant (mouse A) to a final volume of 0.2 mL and as a 1:1 (v/v) solution in sterile PBS (mouse B) to a total volume of 0.2 mL. The second and third immunizations were performed in the same way, except that an incomplete Freund’s adjuvant was used in mouse A. The final immunization was given 3 days prior to cell fusion, through intravenous injection in both mice A and B. On the day of fusion, the mice were euthanized by cervical dislocation, spleens were harvested, and 1 × 108 cells were collected and used for each fusion. Spleen cells were fused with a hypoxanthine-aminopterin-thymidine (HAT) sensitive mouse myeloma cell line, NS0, by the polyethylene glycol (PEG) method (29). NS0 cells were maintained in DMEM with 10% Fetalclone I (HyClone) and split 1:2 the day before fusion. NS0 cells (1 × 108) were mixed in serum-free DMEM, centrifuged, and, after removing the supernatant, were placed in a water bath at 37 °C. Then, 1 mL of prewarmed PEG (Sigma-Aldrich) was added dropwise. At the end of PEG addition, prewarmed serum-free medium was added, and the tube was centrifuged to remove the supernatant. The fusion product was resuspended in DMEM containing 20% Fetalclone I and 1× HAT, and the ensuing cell suspension was plated in 24 well plates; cells were incubated at 37 °C in a CO2 incubator. ELISA Procedures. Hybridoma supernatants were screened for binding to the GM3 ganglioside by enzyme-linked immunosorbent assay (ELISA). The ELISA 96 well plate (Corning) was first treated with 100 µL of GM3 (30 µg/mL in 100% ethanol) overnight at room temperature. To minimize nonspecific adsorption, 300 µL of PBS containing 0.05% Tween (TPBS) and 3% BSA were added for 1 h and washed three times with TPBS. Hybridoma culture supernatants (100 µL/well) were

Arcangeli et al. Table 1. Relative Reaction and Activation Energies for the Cycloaddition Reaction of 6A to 7A and 9A Giving, Respectively, the Diastereoisomeric Cycloadducts 8-S/8-R and 10-S/10-R q

compound

∆∆E (kcal/mol)

∆∆E (kcal/mol)

8-S 8-R 10-S 10-R

3.77 0.00 0.00 5.07

0.85 0.00 0.00 10.85

added to the coated plate and incubated at room temperature for 2 h. Subsequently, the plate was washed and incubated with secondary antibody (peroxidase-labeled antimouse, 1:500 in TPBS) for 1 h. The plate was washed three times with TPBS, then 100 µL of tetramethylbenzidine (TMB, Sigma) was added to each well. The plate was further incubated for 5 min, and hence, the reaction was stopped with 0.5 M HCl. Absorbance was measured using a microplate reader (ELX800, Bio-Tek Instruments, Inc.) at 450 nm.

DISCUSSION Molecular Modeling. The entirely diasteroselective synthesis of mimetics 3 and 4 is based on an inverse electron demand [4 + 2] cycloaddition between the R-thiono-β-keto-δ-lactone 6 (Chart 1) and an appropriate exoenitol obtained from a commercially available monosaccharide. Considering the configuration of the stereogenic centers of the structure of the sialic acid moiety of 2 (ring A, Figure 1), we envisaged exoenitol 7, easily obtainable from D-galactose, as an electron rich dienophile featuring the correct orientation of the substituents at C4-C8, although the native acetamido group at C5 is replaced by an oxygenated function. The synthetic scheme may be effective if the spiranic stereocenter rising from cycloaddition would be formed with the correct stereochemistry, i.e., with the same S configuration as that of GM3 lactone 2. DFT calculations (24, 25) were performed on the simplified structure 7A, in which the isopropylidene methyl groups of 7 (Chart 1) were replaced by hydrogen atoms, and 6A, in which the benzyl and the pyvaloyloxymethyl groups of 6 (Chart 1) were both replaced by methyl groups. These simplifications do not alter the basis structural elements of cycloaddends but allow reasonable computational times. In Scheme 1, diene 6A, exoenitol 7A, and the corresponding diastereoisomeric cycloadducts 8-S and 8-R are depicted. The reaction was investigated both from the thermodynamic and the kinetic points of view. In particular, the relative stability of 8-S and 8-R and that of the corresponding transition states (TSs) was studied (30) and is reported in Table 1. It is easily appreciated that the energetically preferred diasteroisomer 8-R corresponded to the incorrect R configuration of the spiro

Scheme 1. Structures of Model Exoenitols 7A and 9A, Model Diene 6A, and of the Corresponding Cycloadducts 8-S and 8-R, and 10-S and 10-R

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Figure 4. Structure of model compounds 11A and 11B.

Figure 2. Three-dimensional plots of the preferred conformations of the diastereoisomeric cycloadducts 8-S and 8-R and of the corresponding TSs.

Figure 3. Three-dimensional plots of the preferred conformations of the diastereoisomeric cycloadducts 10-S and 10-R and of the corresponding TSs. Scheme 2. Synthesis of Thioether Mimetic 3

center (see Figure 2), suggesting that exoenitol 7 is the incorrect precursor for the desired mimetic. We therefore moved to exoenitol 9 (Chart 1), easily available from D-mannose, which may react with 6 to give the tricyclic cycloadduct with S configuration at the spiro center. Indeed, when the reaction of the simplified dienophile 9A (Scheme 1) was investigated using a DFT computational approach at the same level of theory previously employed, thermodynamic and kinetic preference for the adduct 10-S over the adduct 10-R could neatly be appreciated (Scheme 1, Table 1, and Figure 3), indicating that a mimetic of 2 of the correct configuration could be obtained by the described synthetic strategy. Actually, when 6 was reacted with 9, the predicted stereochemical outcome was confirmed (see below), and the reaction afforded a cycloadduct (12, Scheme 2) with a tricyclic moiety presenting a double bond at the junction between the B and C rings. To evaluate the effects of this insaturation, the conformational properties of the model structure 11A were investigated through DFT calculations in comparison with the saturated portion of GM3 lactone 11B (Figure 4). The energies of all of the conformers were recalculated in water, to reflect the actual situation in solution. Calculations confirmed the preference for a boat-like conformation of the lactone ring of 11B (31) and showed a very similar geometry of the most stable conformation of 11A. Figure 5 shows the global minimum conformations of 11A and 11B and their good superimposition, although their A rings differ in the configuration of two stereocenters. Moreover, experimentally determined 1H NMR coupling constants of

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Arcangeli et al.

Figure 5. Three-dimensional plots of the preferred conformations of 11A and 11B and their superimposition. Scheme 3. Synthesis of the KLH-Conjugate Mimetic 5

significant vicinal hydrogen atoms in the tricyclic moiety are in agreement with the preferred geometry of 11A depicted in Figure 5 (see J5,6, J3,4, and J4,5 of compounds 17 and 20 in the Experimental Procedures section). Diasteroselective Synthesis. [4 + 2] Cycloaddition between R-thiono-β-keto-δ-lactone 6 (32) and exoenitol 9 (21, 33) was performed in CHCl3 at 40 °C in the presence of pyridine and potassium carbonate affording the desired product (Scheme 2) with complete diasteroselectivity (21). The configuration of the spiranic stereocenter of cycloadduct 12 (73% over two steps) was unambiguously assigned by X-ray crystal structure determination (21). Sequential removal of protecting groups and acetylation of free hydroxyl groups under standard conditions gave the peracetylated derivative 13. Reduction of the lactonic function with freshly prepared sodium bis(2-methoxyethoxy)aluminum (RedAl, 1.06 M toluene) afforded the hemiacetalic derivative 14 (93%). Activation of 14 as fluorinated glycoside with diethylaminosulfotrifluoride (DAST) in CH2Cl2 allowed the isolation of 15 as a pseudoalpha anomer in 87% yield which, in turn, was treated with tetradecanol under Ley’s glycosylation conditions (34) to afford 16 (42%). Deacetylation of pseudoR-glycoside 16 gave the desired thioether 3 in 77% yield (Scheme 2). The hydrolytic stability of 3 at pH ∼7 (1 month at room temperature) and at pH 5-6 (15 days, room temperature) was monitored by 1H NMR analysis. In order to use thioether 3 as an immunogen to raise polyclonal and monoclonal antibodies, considering the known low immunogenicity of saccharidic antigens (35), the ω-amino glycosyl derivative 4 (Scheme 3) was prepared from the fluoro derivative 15 by treatment with azido hexanol under Ley’s glycosylation conditions (34). The pseudo-R azido derivative 17 obtained after the removal of acetyl groups underwent a Staudinger reduction to give 4, which was reacted with bis-p nitrophenyl adipic ester 18 (36) and gave

19 (87%, over two steps) as an activated carboxylic derivative (Scheme 3). Finally, 19 was coupled to the protein carrier KLH (23, 35). The thioether-KLH conjugate 5 was prepared by mixing 19 with KLH (phosphate buffer, room temperature, 20 h). Saccharide molecule/KLH loading was determined by the trinitrobenzensulfonic (TNBS) test (37) to be ∼27%. Biological Tests. To determine the immunogenicity of mimetic 5, BALB/c mice were immunized with the KLH conjugate. Following Galfre`’s procedure (29) for the production of monoclonal antibodies, after sequential immunizations spleen cells from immunized mice were collected and fused with a HAT sensitive mouse myeloma cell line (NS0). The ensuing hybridomas were screened by testing supernatants for binding to GM3 ganglioside 1 by an enzyme-linked immunosorbent assay (ELISA). The supernatant displaying the highest absorbance (IV1:4C5C4) was then chosen for further analyses. An ELISA test aimed at isotype determination was also performed. The IV1:4C5C4 supernatant turned out to be composed by two isotypes: IgM and IgG3, with the IgM quantitatively prevailing on the IgG3 (Figure 6). The immunoreactivity of the IV1:4C5C4 supernatant toward either GM3- or GM1-ganglioside was then screened through an ELISA test. Results (Figure 7) clearly indicated that the supernatant indeed binds GM3, whereas no appreciable binding to GM1 was observed. In order to demonstrate the potential use as an immunostimulant of a newly synthesized thioether-bridged mimetic of GM3 lactone, we described here the procedure and the results obtained from the production and the first step of characterization of monoclonal antibodies raised against the saccharidic antigen. To this purpose, three BALB-C mice were immunized by multiple injections of the conjugated KLH-antigen (38, 39) every two-three weeks. We periodically measured the antibody

Stable GM3 Lactone Mimetic

Figure 6. ELISA test performed for isotype determination. The hybridoma IV1:4C5C4 supernatant was composed of two isotypes: IgM and IgG3.

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titer in mice serum, and we proceeded to the sacrifice and splenic explants only after reaching a good polyclonal titer (1:10000). Such results were obtained after three months from the first inoculum. The serum specificity for GM3 was confirmed by ELISA, using microtiter plate wells coated with 3 µg/well of GM3 antigen in ethanol, incubated overnight at room temperature. To evaluate the cross-reactivity of the polyclonal serum for the human GM3, it was also checked by immunocytochemistry on human colon cancer cells HCT116 expressing the GM3 antigen. As we expected, the results reported below indicate a clear positivity of the cell line tested, evidenced by the brown staining. Note that there is no detectable signal at the nuclear level (Figure 8). The same protocols were performed on melanoma cells expressing or not expressing the GM3 antigen with the hybridoma population IV1:4C5C4 supernatant, obtaining a staining of good intensity and specificity (Figure 9). The specificity of the signal is demonstrated by the absence of staining in the negative cell line (WM-266-4) and by the focal staining at the plasma membrane level of GM3 expressing cells (A375). Note that the signal obtained with the hybridoma supernatant is more localized at the cell periphery with respect to polyclonal one. These latter results can encourage further cloning of the IV1: 4C5C4 hybridoma and purification of the IV1:4C5C4 supernatant to obtain a monoclonal antibody against GM3 to be used in the future for the immunotherapy of melanoma.

CONCLUSIONS

Figure 7. ELISA test to compare antibody immunoreactivity toward either GM3- or GM1-ganglioside.

Figure 8. Human HCT116 cell line: staining with polyclonal serum obtained from mice immunized with the GM3 mimetic.

In conclusion, the rational design and the diasteroselective synthesis of a novel thioether-bridged mimetic of GM3 lactone, characterized by the replacement of the sialic ring by a manno residue and the stability under physiological conditions, were reported. Conjugation to the proteic carrier KLH gave glycoconjugate 5, which was employed for the immunization of Balb/c mice. Hybridoma IV1:4C5C4, obtained after immunization, was screened for binding to GM3 by ELISA and ICC tests. The results obtained demonstrated that 5 is immunogenic and able to raise the production of antibodies that bind GM3 expressed on melanoma cells. The reduced structure complexity of the mimetic with respect to the native antigen, its stability in ViVo, as well as the notable ability of the KLH-glycoconjugate 5 to induce a specific immune response against melanoma suggest that it possesses significant advantages over the known compounds and represents a new promising synthetic glycoconjugate for the immunotherapy of melanoma.

ACKNOWLEDGMENT We thank Professor Lucio Luzzatto (Istituto Toscano Tumori (ITT), Italy) and Professor Gabriele Mugnai (University of

Figure 9. Human melanoma cell lines expressing (A375) and not expressing (WM-266-4) GM3 antigen. Signals were obtained using the hybridoma population IV1:4C5C4 supernatant.

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Florence) for fruitful discussions. Finantial support was provided by Ministero Universita’ e Ricerca (MiUR), Italy, (PRIN 2006) and Ente Cassa di Risparmio di Firenze. Supporting Information Available: Synthesis and characterization of compounds 5 and 19 and immunocitochemistry. This material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED (1) Feizi, T. (1985) Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are oncodevelopmental antigens. Nature 314, 53. (2) Hakomori, S.-I., and Zhang, Y. (1997) Glycosphingolipid antigens and cancer therapy. Chem. Biol. 4, 97. (3) Kim, Y. J., and Varki, A. (1997) Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconjugate J. 14, 569. (4) Mannori, G., Cecconi, O., Mugnai, G., and Ruggieri, S. (1990) Role of glycolipids in the metastatic process: characteristics of neutral glycolipids in clones with different metastatic potentials isolated from a murine fibrosarcoma cell line. Int. J. Cancer 45, 984. (5) Singhal, A., and Hakomori, S. (1990) Molecular changes in carbohydrate antigens associated with cancer. BioEssay 12, 223. (6) Muramatsu, T. (1993) Carbohydrate signals in metastasis and prognosis of human carcinomas. Glycobiology 3, 291. (7) Buck, C. A., Glick, M. C., and Warren, L. (1971) Glycopeptides from the surface of control and virus-transformed cells. Science 172, 169. (8) Wickus, G. G., and Robbins, P. W. (1973) Plasma membrane proteins of normal and Rous sarcoma virus-transformed chickembryo fibroblasts. Nat. New Biol. 245, 65. (9) Harada, Y., Sakatsume, M., and Nores, G. A. (1989) Density of GM3 with normal primary structure determines mouse melanoma antigenicity; a new concept of tumor antigen. Jpn. J. Cancer Res. 80, 988. (10) Tardif, M., Coulumbe, J., and Souliers, D. (1996) Gangliosides in human uveal melanoma metastatic process. Int. J. Cancer 68, 97. (11) Nores, G. A., Dohi, T., and Taniguchi, M. (1987) Densitydependent recognition of cell surface GM3 by a certain antimelanoma antibody, and GM3 lactone as a possible immunogen: requirements for tumor-associated antigen and immunogen. J. Immunol. 139, 3171. (12) Harada, Y., Sakatsume, M., and Taniguchi, M. (1990) Induction of mouse anti-melanoma cytotoxic and suppressor T cells in vitro by an artificial antigen, GM3-lactone. Jpn. J. Cancer Res. 81, 383. (13) Kawashima, I., Kotani, M., and Ozawa, H. (1994) Generation of monoclonal antibodies specific for ganglioside lactones: Evidence of the expression of lactone on human melanoma cells. Int. J. Cancer 58, 263. (14) Pan, Y., Chefalo, P., and Nagy, N. (2005) Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines. J. Med. Chem. 48, 875. (15) Jarvis, L. M. (2006) Body, heal thyself. After false starts, cancer immunotherapies tiptoe toward commercialization. Chem. Eng. News 84, 23. (16) Parmiani, G., De Filippo, A., Novellino, L., and Castelli, U. (2007) Unique human tumour antigens: immunobiology and use in clinical trials. J. Immunol. 178, 1975. (17) Dudley, M. E., Wunderlich, J. R., and Yang, J. C. (2005) Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J. Clin. Oncol. 23, 2346. (18) Riker, A. I., Radfar, S., Liu, S. H., Wang, Y., and Khong, H. T. (2007) Immunotherapy of melanoma: a critical review of current concepts and future strategies. Expert Opin. Biol. Ther. 7, 1. (19) Wilstermann, M., Kononov, L. O., Nilsson, U., Ray, A. K., and Magnusson, G. (1995) Synthesis of ganglioside lactams

Arcangeli et al. corresponding to GM1-, GM2-, GM3-, and GM4-ganglioside lactones. J. Am. Chem. Soc. 117, 4742. (20) Tietze, L. F., Keim, H., Janβen, C. O., Tappertzhofen, J., and Olschimke, J. (2000) Synthesis of a novel ether-bridged GM3lactone analogue as a target for an antibody-based cancer therapy. Chem.sEur. J. 6, 2801. (21) Toma, L., Di Cola, E., Ienco, A., Legnani, L., Lunghi, C., Moneti, G., Richichi, B., Ristori, S., Dell’Atti, D., and Nativi, C. (2007) Synthesis, conformational studies, binding assessment and liposome insertion of a thioether-bridged mimetic of the antigen GM3 ganglioside lactone. ChemBioChem 8, 1646. (22) Tzianabos, A. O. (2000) Polysaccharide immunomodulators as therapeutic agents: structural aspects and biological function. Clin. Microbiol. ReV. 13, 523. (23) Danishefsky, S. J., and Allen, J. R. (2000) From the laboratory to the clinic: a retrospective on fully synthetic carbohydratebased anticancer vaccines. Angew. Chem., Int. Ed. 39, 836. (24) Becke, A. D. (1993) Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648. (25) Lee, C., Yang, W., and Parr, R. G. (1988) Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. ReV. B 37, 785. (26) Cance´s, E., Mennucci, B., and Tomasi, J. (1997) A new integral equation formalism for the polarizable continuum model: Theoretical background and applications to isotropic and anisotropic dielectrics. J. Chem. Phys. 107, 3032. (27) Cossi, M., Barone, V., Cammi, R., and Tomasi, J. (1996) Ab initio study of solvated molecules: a new implementation of the polarizable continuum model. Chem. Phys. Lett. 255, 327. (28) Barone, V., Cossi, M., and Tomasi, J. (1998) A new integral equation formalism for the polarizable continuum model: theoretical background and applications to isotropic and anisotropic dielectrics. J. Comput. Chem. 19, 404. (29) Galfre`, G., and Milstein, C. (1981) Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73 (Pt B), 3–46. (30) All the degrees of conformational freedom of the ground state structures 8-S and 8-R as well as of the corresponding TSs were taken into consideration by performing several optimizations in each case. (31) Ray, A. K., Nilsson, U., and Magnusson, G. (1992) Synthesis and conformational analysis of GM3 lactam; a hydrolytically stable analog of GM3 ganglioside lactone. J. Am. Chem. Soc. 114, 2256. (32) Bartolozzi, A., Pacciani, S., Benvenuti, C., Cacciarini, M., Liguori, F., Menichetti, S., and Nativi, C. (2003) Totally Stereoselective synthesis of 1,3-disaccharides through Diels-Alder reactions. J. Org. Chem. 68, 8529. (33) Haudrechy, A., and Sinay¨, P. (1992) Cyclization of hydroxy enol ethers: a stereocontrolled approach to 3-deoxy-D-manno-2-octulosonic acid containing disaccharides. J. Org. Chem. 57, 4142. (34) Baeschlin, D. K., Green, L. G., Hahn, M. G., Hinzen, B., Ince, S. J., and Ley, S. V. (2000) Rapid assembly of oligosaccharides: 1,2-diacetal-mediated reactivity tuning in the coupling of glycosyl fluorides. Tetrahedron: Asymmetry 11, 173. (35) Hermanson, G. T. (1996) Bioconjugate Technique, pp 421427, Academic Press, Orlando, FL. (36) Wu, X., Ling, C.-C., and Bundle, D. (2004) A new homobifunctional p-nitro phenyl ester coupling reagent for the preparation of neoglycoproteins. Org. Lett. 6, 4407. (37) Habeeb, F. S. A. (1966) Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal. Biochem. 14, 328. (38) Bitton, R. J., Guthmann, M. D., Gabri, M. R., Carnero, A. J. L., Alonso, D. F., Fainboim, L., and Gomez, D. E. (2002) Cancer vaccines: an update with special focus on ganglioside antigens (review). Oncol. Rep. 9, 267. (39) Gabri, M. R., Mazorra, Z., Rispoli, G. V., Mesa, C., Fernandez, L. E., Gomez, D. E., and Alonso, D. F. (2006) Complete antitumor protection by perioperative immunization with GM3/ VSSP vaccine in a preclinical mouse melanoma model. Clin. Cancer Res. 12, 7092. BC900557V