Synthesis of GD3 Tetrasaccharide-KLH Glycoconjugates— Chemical syntheses of GD3 have been reported previously (44–47). However, large scale synthesis of the GD3 tetrasaccharide is still difficult to achieve. Recently, a bi-functional sialyltransferase from Campylobacter jejuni, which transfers sialic acid to both 3-O-Gal and 8-O-NeuAc, was cloned and expressed (35). We used this enzyme successfully for the synthesis of disialolactoside (GD3 tetrasaccharide) on a 200-mg scale. The main by-product was sialolactoside (GM3 trisaccharide), which was separated from disialolactoside by a Bio-Gel P-6 column eluted with 0.03 M NH4HCO3. Modified disialolactosides in which the N-acetyl groups of sialic acids were replaced by respective N-propionyl, N-butyryl, and N-benzoyl groups were derived in two steps, namely removal of N-Ac by base treatment and quantitative re-N-acylation with either propionyl and butyryl anhydride or benzoyl chloride. Consequently, four disialolactosides, GD3Ac, GD3Pr, GD3Bu, and GD3Bz tetrasaccharides, were obtained with a β-linked 3-azidopropyl spacer. The spectroscopic data of GD3Ac tetrasaccharide are listed in Table I. Catalytic reduction of the azido groups generated their respective amines quantitatively, which were subsequently converted to maleimide-containing disialolactosides as indicated by a singlet resonance at δH 6.8 ppm in their 1H NMR spectra. After reaction of the latter with thiolated KLH, four glycoconjugates, GD3Ac-KLH, GD3Pr-KLH, GD3Bu-KLH, and GD3Bz-KLH, were obtained (see Fig. 1). The ratios of disialolactoside to KLH were between 300 and 460 based on sialic acid analysis (39) and protein assay (see Table II). By using the same procedures, four respective BSA conjugates were also prepared with 5–11 disialolactosides attached to each BSA molecule.
Immunizations—BALB/c mice were immunized and boosted three times weekly with GD3Ac-KLH, GD3Pr-KLH, GD3Bu-KLH, and GD3Bz-KLH conjugates. Ten days after the last boost (day 31), whole serum was analyzed by ELISA using GD3Ac-BSA, GD3Pr-BSA, GD3Bu-BSA, and GD3Bz-BSA as coating antigens. The antibody levels, both for IgG and IgM, were determined (see Table III). All four conjugates gave high titers of antibodies. In addition, subtyping analysis revealed that a majority of the specific antibodies was IgG rather than IgM. The GD3Bu-KLH conjugate was the most immunogenic, followed by GD3Pr-KLH, GD3Ac-KLH, and GD3Bz-KLH. The extension of the N-acyl chain seemed to correlate with the increased immunogenicity, except that the N-benzoylated derivative was the least immunogenic. Since the BSA conjugates used in ELISA shared the same linkage structure with the KLH conjugates, there was a possibility that antibodies to the linkage structure might also be raised, which could contribute in part to the total antibody titers. Therefore, we also investigated whether these antisera recognize GD3Ac, GD3Pr, GD3Bu, and GD3Bz expressed on the SK-MEL-28 cell surface as well as their cross-reactivity with native cell surface GD3.
Biochemical Engineering of SK-MEL-28 Melanoma Cells in Vitro—SK-MEL-28 and G361 cells were cultured for 1–3, 6, or 10 days in standard medium supplemented with three different concentrations (1, 3, and 5 mg/ml) of ManNAc, ManNPr, Man-NBu, and ManNBz. The expression of modified GD3 was evaluated by flow cytometric analysis using diluted sera raised against the GD3 tetrasaccharide-KLH conjugates. SK-MEL-28 cells cultured in the presence of the precursors exhibited no detectable reactivity with pre-immunization sera from mice. When grown in standard medium without precursors, SK-MEL-28 cells exhibited only background fluorescence staining after treatment with dilutions of anti-GD3Pr (Bu and Bz) sera (1:100). However, cells cultured in the presence of ManNPr, ManNBu, or ManNBz exhibited a marked increase in the immunoreactivity with their respective anti-GD3Pr, anti-GD3Bu, and anti-GD3Bz sera (Fig. 2a). No increase in reactivity to the anti-GD3Ac serum was observed when ManNAc was added to the medium. Precursors at a concentration of 1 mg/ml resulted in the expression of modified GD3 within 24 h, and increased precursor concentration (3 and 5 mg/ml) did not advance further expression (Fig. 2a). In addition, incubations of SK-MEL-28 cells with precursors (ManNBu and ManNBz) for 1–3, 6, or even 10 days did not significantly change the expression of GD3Bu and GD3Bz (Fig. 2b). After the removal of precursors from the growth medium, the modified GD3 was slowly replaced by GD3, yet the cells were still found to express GD3Bu after 5 days without ManNBu (Fig. 2c). Similar results were also observed with G361 cells (data not shown).
Cross-reactivity between the above antisera and the cell surface GD3Bu and GD3Ac of SK-MEL-28 cells was also analyzed. Cell surface GD3Bu and anti-GD3Pr sera were cross-reactive as well as GD3Bu and anti-GD3Bz sera (Fig. 3a), but no cross-reactivity was observed between the cell surface GD3Ac and anti-GD3Pr, anti-GD3Bu, or anti-GD3Bz sera (Fig. 3b). In these experiments mAb R24, a murine IgG3 antibody specific for GD3 with the terminal N-Ac disialyl residues (48), was used to assess the expression of surface GD3 on SK-MEL-28 cells incubated with or without precursors. The surface GD3 expression was significantly suppressed and replaced by modified GD3 when the cells were treated with precursors (Fig. 3).
Spectroscopic Confirmation of Modified GD3 Expression— The biochemical engineering of GD3Bu on the SK-MEL-28 cell surface in the presence of ManNBu was further confirmed by MS spectroscopic analysis. The glycolipids extracted from SK-MEL-28 cells were subjected to capillary electrophoresis, and the negatively charged glycolipids were analyzed by electrospray-MS. By using the fragment ion (m/z 290 for NeuAc and 318 for NeuBu) scan technique, GD3 was found to be a dominant ganglioside on the SK-MEL-28 cells with a molecular weight of 1556 based on the observation of a double negative charged ion at m/z 777 (Fig. 4a). The fragmentation derived from MS-MS analysis confirmed the GD3 structure and the ceramide composition (m/z 647), which were in agreement with previous reports (49, 50). When ManNBu was added to the growth medium at the concentrations of 1, 3, and 5 mg/ml, GD3Bu was spontaneously expressed, which was detected in the MS analysis as a major component (m/z 805, a double negative charged ion); an increase of m/z 28 from 777 or a 56-Da difference in molecular mass represents the difference in molecular mass between GD3Bu and GD3Ac (Fig. 4b). Unmodified GD3 and mono-N-Bu GD3 (m/z 777 or 791) were not detected in the glycolipid extraction under the above ManNBu concentrations by MS analysis (data not shown). Therefore, we assume that the modified GD3 molecules are highly expressed on the cell surface.
Production of Monoclonal Antibodies—Immunization of mice with glycoconjugated GD3Bu-KLH, fusion, and initial screening by ELISA against GD3Bu-BSA gave hybridomas 2A (IgG2a) and 1 (IgG1). After re-cloning by limiting dilution, each culture supernatant was tested by ELISA against the following glycoconjugates as antigens: GM3-BSA, GD3Ac-BSA, GD3Pr-BSA, GD3Bu-BSA, and GD3Bz-BSA. mAb 1 only reacted with GD3Bu-BSA, whereas mAb 2A bound to both GD3Pr-BSA and GD3Bu-BSA (Fig. 5c). Neither mAbs were reactive with SK-MEL-28 or Bz-SK-MEL-28 cells by flow cytometry, but both reacted with Bu-SK-MEL-28 cells and Pr-SK-MEL-28 cells (Fig. 5, a and b). To confirm further the epitope specificity of mAbs 2A and 1, ELISA inhibition was performed with GD3Bu-BSA glycoconjugate as coating antigen by using four disialolactosides as inhibitors (GD3Ac, GD3Pr, GD3Bu, and GD3Bz). Only GD3Bu disialolactoside was able to inhibit the binding of both mAbs to the antigen. This result indicates that N-butyryl group(s) are involved in the binding, but detailed structural parameters of the epitope have yet to be established. Because of its better ability to bind complement, mAb 2A (IgG2a) was selected for the further experiments.
In Vitro Anti-tumor Effects (CDC Mediated by Monoclonal Antibody 2A)—We investigated whether the mAb 2A and the anti-GD3Bu serum were capable of mediating CDC on SK-MEL-28 cells. Thus, CDC of mAb 2A and anti-GD3Bu serum on SK-MEL-28 cells with and without treatment of ManNBu (1 mg/ml) was examined in the presence of rabbit complement. mAb 2A killed only Bu-SK-MEL-28 cells that were treated with ManNBu in a dose-dependent manner in the range of 0.05–100 µg/ml (Fig. 6a). Similar results were also obtained with the anti-GD3Bu serum at various dilutions (1:400–1:1600, Fig. 6b). Neither antibody showed any killing effect on SK-MEL-28 cells without precursor treatment.
Expression of Modified GD3 on SK-MEL-28 in Vivo—After administration of ManNBu to nude mice (CgFoxNude) for 2 weeks (5 days/week, 5 mg/mouse), SK-MEL-28 cells from tumors were examined by flow cytometry, and both GD3 and GD3Bu epitopes were detected by mAbs R24 and 2A (Fig. 7). Compared with the expression of modified GD3 in vitro (Fig. 2), the incorporation of GD3Bu epitope in vivo was less effective, but nevertheless it was still sufficient to provide a specific immunotarget.
Anti-tumor Effects in Vivo—After tumors grafted on nude mice had grown to about 10 mm in diameter, mAb 2A in combination with ManNBu was able to suppress tumor growth during the 2 weeks of treatment (average V/V01.0), but tumors started to grow rapidly after treatment was withdrawn (data not shown). In the control group, tumors in mice treated with mAb 2A alone grew faster (average V/V01.4). Flow cytometry analysis of cells from dissected tumors after the treatment of mAb2A/ManNBu showed reactivity only to R24 but not mAb 2A (data not shown). This observation was in direct contrast to the prolonged expression of modified GD3 observed in SK-MEL-28 cells in vitro. Thus this mAb/precursor combination had a limited effect in controlling tumor growth, but the antibodies were obviously not effective in removing solid tumors.
Further experiments designed for early intervention were more successful. Without any treatment, all 10 nude mice after transplantation of SK-MEL-28 cells developed solid tumors within 30 days that grew to about 4–7 mm in diameter after 45 days. Whereas subcutaneous injection, 3 days after tumor cell transplantation for 2 weeks (5 times/week), of either mAb 2A or ManNBu alone provided some protection (about 30% of mice did not develop solid tumor), the best results were obtained with the combination of both, delivered by either subcutaneous or intraperitoneal routes, that resulted in 90% of mice being free of tumors after 45 days (see Table IV).