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FIG. 1.Synthesis of disialoctoside-KLH conjugates. Disialolactoside is the carbohydrate epitope of GD3. This enzymatically synthesized GD3 tetrasaccharide was chemically modified on the N-acyl substitution of both sialic acids by base treatment to remove N-acetyl groups and followed by N-acylation using propionyl anhydride, butyryl anhydride, and benzoyl chloride, respectively. The azido group was quantitatively reduced to amine by catalytic hydrogenation, which was then coupled to a maleimide-containing spacer. The final conjugates were formed by reaction of sulfhydryl-KLH with the maleimide group in GD3 tetrasaccharide derivatives.
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FIG. 2. Expression of BuGD3 in vitro on SK-MEL-28 cells. a, cells were incubated with 1, 3, and 5 mg/ml of ManNBu in standard medium for 1–3 days. The expression of GD3Bu was monitored by flow cytometry using homologous antisera raised by conjugate vaccines. At the lower concentration of 1 mg/ml, GD3Bu expression was detected after 1 day, and prolonged incubation and higher precursor concentrations did not increase the expression of GD3Bu on SK-MEL-28 cells. b, expression of GD3Bu on the cell surface after addition of ManNBu at 1 mg/ml was similar at days 1–3, 6, and 10. c, at day 10 ManNBu was removed from the medium, and at day 15 GD3Bu was still detected on most SK-MEL-28 cells by flow cytometric analysis.
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FIG. 3. Cross-reactivity of GD3 analog antisera to Bu-SK-MEL-28 cells and SK-MEL-28 cells. a, Bu-SK-MEL-28 cells were obtained by incubation of SK-MEL-28 cells with 1 mg/ml Man-NBu for 1 day. GD3Bu antiserum strongly reacts with Bu-SK-MEL-28 cells. b, only GD3Ac antiserum bound to surface GD3 of SK-MEL-28 cells. No cross-reactivity was observed between other antisera and untreated SK-MEL-28 cells.
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FIG. 4. MS analysis of GD3Bu expressed on the Bu-SK-MEL-28 cells. GD3 extracted from SK-MEL-28 cells with and without the addition of ManNBu in growth medium were subjected to capillary electrophoresis-MS analysis. Using the fragment ion scan technique (m/z 290 for NeuAc and 318 for NeuBu), a double negative charged ion (m/z 777) was found as a major peak from untreated SK-MEL-28 cells, whereas m/z 805 was the major one from ManNBu-treated SK-MEL-28 cells. a, MS-MS spectrum from m/z 777 confirms the chemical structure of cell surface GD3. b, a similar fragmentation pattern from MS-MS analysis on m/z 805 was obtained. BuGD3 was expressed on Bu-SK-MEL-28 cells.
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FIG. 5. Specificity of mAbs. mAbs 1 (a) and 2A (b) bound to both Bu-SK-MEL-28 and Pr-SK-MEL-28 cells but not Bz-SK-MEL-28 and SK-MEL-28 cells as determined by flow cytometry. c, binding of mAbs (2A and 1) to modified GD3 tetrasaccharide-BSA conjugates as determined by ELISA suggests the mAbs were more specific to GD3Bu epitope.
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FIG. 6. CDC activity in the presence of rabbit complement is mediated by mAb 2A (a) and GD3Bu antiserum (b). Without the addition of ManNBu to the growth medium neither mAb kills SK-MEL-28 cells.
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FIG. 7. Expression of GD3Bu in vivo. Nude mice (CgFoxNude) were subcutaneously injected with 1 x 107 SK-MEL-28 cells in the rear right flank. When the tumors were about 10 mm in width, mice were injected daily with ManNBu (5 mg/mouse, intraperitoneal) in RPMI medium for 2 weeks (5 days/week). Tumors were dissected, and tumor cells were analyzed by flow cytometry using mAbs R24 and 2A. The expressions of both GD3 and GD3Bu on cell surface were detected.
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