Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies

Abstract

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.

Keywords: antibodies; carbohydrates; immunology; synthesis; vaccines.

Figure 1

ELISA analysis of the epitope profiles of post-immune sera from mice immunized with triazole linked Qβ–GM2 conjugate 13 and thiourea-linked Qβ–GM2 17, respectively. For 13, the anti-triazole antibody level was significantly higher for than other types of antibodies, such as anti-GM2 or anti-GM3 antibodies (p <0.0001). Qβ-GM2 17 induced significantly higher anti-GM2 antibodies (p =0.002) but much lower levels of anti-triazole antibodies (p <0.0001) than did 13. Sera from each group were analyzed at 1600-fold dilution. The average of optical density value and SEM were shown. Statistics were performed by Student’s t-test.

Figure 2

Immunological evaluation of Qβ–GM2 conjugate vaccine 17. A) IgM and IgG titers of anti-GM2 antibodies tested by ELISA. Sera from mice immunized with wild-type Qβ particle were tested as a control. B) The levels of anti-GM2 IgG subclasses as determined by ELISA. Sera were tested at 1:1000 dilution. C) Binding of GM2-expressing Jurkat cells and D) MCF-7 cells with representative mouse sera diluted at 1:20. Gray filled: pre-immune sera and sera from mice immunized with Qβ only; solid line: day 35 sera from a mouse immunized with Qβ–GM2 17. E) Complement-dependent toxicity against Jurkat cells as measured by LDH assay. Sera from two mice immunized with Qβ-GM2 17 are shown (mouse 1: ■, mouse 2: ▲). Pre-immune serum was utilized as a control (●). Sera from mice immunized with Qβ gave similar results as the pre-immune sera.

Scheme 1

Retrosynthetic analysis of GM2 tetrasaccharide 1.

Scheme 2

Synthesis of GM2 tetrasaccharide 1. a) NaN₃, DMF; b) NaOMe, MeOH; c) acetone, p-TsOH, 2,2-dimethoxypropane; d) NaH, BnBr, DMF; e) TFA, CH₂Cl₂, (44 % for five steps); f) sialyl donor 3, p-TolSCl, AgOTf, −40 °C, MeCN (65 %); g) GalN donor 4, p-TolSCl, AgOTf, −78 °C, CH₂Cl₂, Et₂O (63 %); h) NaOH, THF; i) Ac₂O, TEA, MeOH; j) PMe₃, NaOH; k) Pd(OH)₂, H₂ (54 % for four steps).

Scheme 3

Synthesis of GM2–Qβ conjugates. a) NaHCO₃, H₂O; b) CuSO₄, sodium ascorbate, THPTA, PBS buffer (CuAAC conditions); c) thiophosgene, NaHCO₃, CHCl₃/H₂O; d) Na₂B₄O₇ buffer (pH 8.5).