Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines

Abstract

The problem of immunotolerance to GM3, an important tumor-associated trisaccharide antigen, seriously hinders its usage in cancer vaccine development. To solve this problem, the keyhole limpet hemocyanin (KLH) conjugates of a series of GM3 derivatives were synthesized and screened as therapeutic cancer vaccines. First, the beta-linked anomeric azides of differently N-acylated GM3 analogues were prepared by a highly convergent procedure. Next, a pentenoyl group was linked to the reducing end of the carbohydrate antigens following selective reduction of the azido group. The linker was thereafter ozonolyzed to give an aldehyde functionality permitting the conjugation of the antigens to KLH via reductive amination. Finally, the immunological properties of the resultant glycoconjugates were studied in C57BL/6 mice by assessing the titers of specific antibodies induced by the GM3 analogues. While KLH-GM3 elicited low levels of immune response, the KLH conjugates of N-propionyl, N-butanoyl, N-iso-butanoyl, and N-phenylacetyl GM3s induced robust immune reactions with antibodies of multiple isotypes, indicating significantly improved and T-cell dependent immune responses that lead to isotype switching, affinity maturation, and the induction of immunological "memory". It was suggested that GM3PhAc-KLH is a promising vaccine candidate for glycoengineered immunotherapy of cancer with GM3 as the primary target.

Figure 1

The structure of GM3 antigen

Figure 2

Structures of N-modified GM3 and conjugate vaccines

Figure 3

Antigen-specific total antibody contents in sera analyzed by ELISA. Each line represents the antibody level in serum pooled from six mice. Anti-GM3 and anti-derivatized GM3 specific antibody levels were obtained from mice immunized with various GM3-KLH glycoconjugates. Anti-KLH specific antibody level, which was used as a positive control, was obtained from mice immunized with GM3NPr, and anti-GM3 specific antibody level of pre-immune serum was obtained from the same group as the negative control (equivalent results were obtained from the other groups). For ELISA assays, the corresponding GM3-HSA glycoconjugates were used as the capture antigens. Goat anti-mouse Kappa antibodies were used to detect antibodies bond to the capture antigens. Error bars are smaller than the symbol width.

Figure 4

Titer analysis of antigen-specific antibodies determined by ELISA assay (see Procedures). Each represents the titer in pooled serum obtained on day 35 after primary and booster immunizations (see Procedures) from six replicate animals.

Figure 5

Titer analysis of antibodies cross-reactive with the natural GM3 determined by ELISA (see Procedures). ELISA plates were coated with GM3-HSA conjugate. Each bar represents total anti-GM3 reactivity in pooled sera from six replicate animals immunized with the indicated derivatized GM3.

Scheme 1

Reagents and conditions: (a) MsOH (cat.), MeOH, reflux, 24 h; then CF₃COOMe, Et₃N, MeOH; (b) Ac₂O, pyridine, 84% (two steps); (c) NIS, TfOH, MS 4Å, MeCN, ca. 63%; (d) Lindlar catalyst, H₂, MeOH-EtOAc (1:1), 4-pentenoic anhydride, 6 h, 90%. (e) 0.5 N NaOH, 10 h; then various anhydride, rt, 83–94%.

Scheme 2

Reagents and conditions: (a) O₃, MeOH, −70 °C, 0.5 h; then Me₂S, to rt, 2 h, 85–90%; (b) KLH or HSA, NaBH₃CN, 0.1 N NaHCO₃, 37 °C, 3d.