Departamento de Química Biológica-CIQUIBIC-CONICET and 2Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina
Received on November 22, 1999; revised on February 10, 2000; accepted on February 11, 2000.
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Abstract |
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Key words: immunogenicity/Thomsen-Friedenreich disaccharide/tumor-associated antigen
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Introduction |
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We demonstrated recently that Agaricus bisporus lectin (ABL) binds mainly to TFD where GalNAc (through its axial C-4 hydroxyl group and C-2 acetamido residue) has a more significant role in the interaction than terminal ß-linked Gal (Irazoqui et al., 1999). ABL is a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines (Yu et al., 1993
; Yu et al., 1999
), in contrast to TFD-specific lectin, peanut agglutinin (PNA), and human anti-TFD mAbs, which stimulate proliferation of these cells (Ryder et al., 1994a
, 1994b; Yu et al., 1997
). Both PNA and anti-TFD mAbs recognize primarily the terminal Gal of TFD (Lotan et al., 1975
; Dahlenborg et al., 1997
; Loris et al., 1998
).
The purpose of the present study was to construct an immunogen that gives rise to anti-TFD antibodies with a fine carbohydrate-binding specificity similar to that of ABL. Such immunogen could be an alternative carbohydrate target antigen for active specific immunotherapy of patients with epithelial tumors. Synthesis of the novel immunogen was accomplished by molecular rotation of TFD on its carrier conjugation, where it is linked to protein through C-6 of Gal, thereby exposing more GalNAc than Gal.
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Results |
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Discussion |
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Comparison of ABL binding to BSA or KLH by ELA indicates a major interaction with KLH (Figure 4). This observation is consistent with the presence of O-glycans in KLH (Wirguin et al., 1995). However, immunization of mice with KLH containing synthetic TFD C-6 linkage in addition to natural O-glycans did not raise significant anti-TFD antibody titer, confirming the poor immunogenicity of TFD. In contrast, when Bzl
TFD was C-6 attached to KLH and used as immunogen, strong immune response against TFD was obtained. This illustrates the effect of chemical modification, in this case benzyl group, on carbohydrate target immunogenicity to improve anti-TFD antibody titer. Similarly, Gu et al. (1998)
observed enhanced cellular and humoral immunity using an hydrophobized polysaccharide.
BzlTFD-KLH immunogen raises anti-ASG antibody titer, demonstrating recognition of TFD when it is
-linked through C-1 of GalNAc. Bzl
TFD-KLH also improves anti-MUC antibody titers, and shows a binding ability more similar to ABL than to PNA, since PNA does not bind to MUC (Figure 4B).
The anti-TFD antibodies were analyzed in terms of fine carbohydrate-binding specificity, using TFD-related carbohydrates as inhibitors in CELISA. High specificity of IgG and IgM antibodies to TFD was evidenced by the fact that TFD and pNPhTFD were the major inhibitors of all assayed carbohydrates. Both disaccharides showed ID50 of ~0.02 mM, similar to that reported for inhibition of ABL interaction (Irazoqui et al., 1999
). When TFD-related monosaccharides were assayed as competitors, GalNAc was found to have greater inhibitory activity than Gal on TFD-IgG and -IgM antibody interaction. These findings are contrary to those for previously described anti-TFD mAbs (Dahlenborg et al., 1997
) and PNA (Lotan et al., 1975
; Loris et al., 1998
) where Gal was more relevant than GalNAc on TFD interaction.
Our results indicate that C-2 acetamido addition improves binding of Gal, and the axial C-4 hydroxyl group in GalNAc provides a significant binding locus, since GlcNAc does not bind to the IgG antibody. However, the GalNAc ID50 obtained here (100 mM) is 4-fold higher than that described for ABL. This may reflect minor importance of the C-4 hydroxyl group of GalNAc from TFD, as evidenced by the fact that lacto-N-biose (Galß13GlcNAc) is a better inhibitor of the competitive assays of these antibodies than for ABL (Irazoqui et al., 1999). This explanation is also supported by the lack of inhibitory activity of lactose and Galß16GlcNAc on TFD-antibody interaction, in contrast to ABL. Binding of ABL is attributed to the C-4 and C-3 hydroxyl groups of reducing Glc from lactose and Galß16GlcNAc respectively, since conformational analysis shows that they occupy a position similar to C-4 hydroxyl group of GalNAc from TFD. This C-4 hydroxyl group position is less relevant for IgM anti-TFD antibody interaction, since GalNAc and GlcNAc do not differ significantly in their inhibitory activity. CELISA also indicates a lower ID50 difference between TFD and lacto-N-biose for IgM anti-TFD antibody.
In conclusion, we found that immunization of mice with BzlTFD conjugated to KLH through C-6 of Gal produces anti-TFD antibodies with enhanced binding to GalNAc over Gal, and consequently a fine carbohydrate-binding specificity more similar to that of ABL than other TFD-binding proteins. The TFD immunogen synthesized as described here could be an effective alternative carbohydrate target antigen for active specific immunotherapy of patients with epithelial tumors. Experiments to test this hypothesis are in progress.
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Materials and methods |
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Preparation of (Bzl)TFD C-6 aldehyde and its conjugation to protein
Galactose oxidase (50 U) was immobilized to cyanogen bromide-activated Sepharose 4B gel (100 mg) as described by Cuatrecasas and Anfinsen (1971). One milliliter of 2 mM carbohydrate (TFD or Bzl
TFD) in PBS was incubated with immobilized galactose oxidase for 2 h at room temperature with constant stirring. The carbohydrate C-6 aldehyde was recovered by gel separation, detected by DNPH reaction (Liggins and Furth, 1997
), and separated by HPTLC. Briefly, 50 µl carbohydrate solution was added with an equal volume of methanol and 7 mg/ml DNPH (50 µl) in methanol:HCl (80:1) for 2 h at room temperature, then added with 2.5 mM pyridine (50 µl) in methanol. The tubes were dried in nitrogen atmosphere and dissolved in 50 µl methanol for HPTLC analysis. Carbohydrates were separated on HPTLC silica gel 60 (Merck) in the running solvent chloroform-methanol-aqueous 0.2% CaCl2 (60:37:8), using a tank to obtain highly reproducible chromatograms (Nores et al., 1994
). After air-drying for 15 min, carbohydrates were visualized chemically using orcinol-sulfuric acid spray reagent, for 5 min at 120°C.
TFD and BzlTFD C-6 aldehydes were mixed with 3 mg BSA or KLH in PBS for 10 min, then added with 3 mg NaCNBH3. The reaction mixture was incubated with gentle stirring for 4 h at room temperature. The glycoconjugate was dialyzed with four changes of PBS, at 4°C. The presence of carbohydrates linked to proteins was analyzed by Western blot for BSA and enzyme-lectin assay (ELA) for KLH.
Immunization of mice
Groups of 5 mice (Rockefeller female, age 6 weeks) were intradermally injected with 100 µg TFD-KLH or BzlTFD-KLH mixed with 100 µl Freunds complete adjuvant, at four shaved sites. Three weeks later this process was repeated, but using Freunds incomplete adjuvant. Three weeks later, mice were boosted by subcutaneous injection with the same sample as the second injection. Mice were bled at day 0 and day 10 after the third immunization.
Competitive ELISA (CELISA)
The three antisera having highest anti-TFD titer were analyzed by CELISA and the results averaged. The optimal antiserum dilution showing 1.0 optical density against TFD-BSA (10 µg/ml) as target antigen was determined in preliminary experiments. All steps of CELISA were developed as ELISA, except that the optimal antiserum dilution was preincubated with several carbohydrates for 1 h at room temperature before adding to wells.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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