Design and synthesis of potent Quillaja saponin vaccine adjuvants

Abstract

The success of antitumor and antiviral vaccines often requires the use of an adjuvant, a substance that significantly enhances the immune response to a coadministered antigen. Only a handful of adjuvants have both sufficient potency and acceptable toxicity for clinical investigation. One promising adjuvant is QS-21, a saponin natural product that is the immunopotentiator of choice in many cancer and infectious disease vaccine clinical trials. However, the therapeutic promise of QS-21 adjuvant is curtailed by several factors, including its scarcity, difficulty in purification to homogeneity, dose-limiting toxicity, and chemical instability. Here, we report the design, synthesis, and evaluation of chemically stable synthetic saponins. These novel, amide-modified, non-natural substances exhibit immunopotentiating effects in vivo that rival or exceed that of QS-21 in evaluations with the GD3-KLH melanoma conjugate vaccine. The highly convergent synthetic preparation of these novel saponins establishes new avenues for discovering improved molecular adjuvants for specifically tailored vaccine therapies.

Figure 1. Preclinical Immunological Evaluation of SQS-Adjuvants with GD3-KLH Conjugate Vaccine

Each adjuvant, plus a no-adjuvant negative control, was evaluated by vaccination of a group of 5 mice (C57BL/6J, female). Vaccinations took the form of weekly subcutaneous injection of antigen (10 µg) and various saponin adjuvants (10 µg for ELISA and FACS results; 20 µg for CDC results) for three weeks (days 0, 7, and 14), followed by a booster at day 65. Post-boost serological data at day 72 are presented. (A) Anti-GD3 titers (IgM) after vaccination; median values in parentheses. (B) Anti-GD3 titers (IgG) after vaccination; median values in parentheses. (C) Anti-KLH titers (IgG) after vaccination; median values in parentheses. (D) Anti-GD3 IgG subtyping as assessed by class-specific indirect ELISA; median absorbance levels >0.1 are considered positive. (E) Cell surface reactivity of anti-GD3 against SK-Mel-28 tumor cell line expressing GD3 antigen following vaccination; median values in parentheses. (F) Complement dependent cytotoxicity (CDC) activity of anti-GD3 against SK-Mel-28 tumor cell line expressing GD3 antigen.

Figure 2. Toxicity Assessment via Median Weight Loss

Initial toxicity assessment was performed by tracking median weight loss of each group of mice following the first vaccination with GD3-KLH (10 µg) antigen with various synthetic saponin adjuvants (10 µg).

Scheme 1^a

^aReagents and conditions: (a) NaN₃, Bu₄NCl, PhMe, 110 °C, 66%; (b) NaOH, MeOH, 23 °C; NaH, BnBr, DMF, 23 °C, 78%; (c) PhI(OAc)₂, BF₃•OEt₂, CH₂Cl₂, −50→−25 °C, 85%; (d) K₂CO₃, MeOH, H₂O, 23 °C, 84%; (e) TIPSCl, imidazole, DMAP, DMF, 23 °C, 59%; (f) TBAF, THF, 23 °C, 98%; (g) 8, Tf₂O, Ph₂SO, TBP, CH₂Cl₂, −55 °C; add 6, −78→23 °C, 67%; (h) TBAF, THF, 0 °C, 93%; (i) CCl₃CN, DBU, CH₂Cl₂, 0→23 °C, 95%.

Scheme 2^a

^aReagents and conditions: (a) NaOH, BnOCOCl, H₂O, 0→23 °C, >99%; (b) EtOCOCl, Et₃N, THF; CH₂N₂, 0→23 °C, 78%; (c) CF₃CO₂Ag, Et₃N, THF, −50→23 °C, 79%; (d) CDI, THF, 23 °C; 13, THF, −78 °C, 60%; (e) H₂, RuCl₂•(S)-BINAP, MeOH, 23 °C, 89%; (f) TBSOTf, 2,6-lutidine, CH₂Cl₂, −78 °C, 94%; (g) Pd/C, H₂, MeOH, 23 °C, 97%; (h) 17, EtOCOCl, Et₃N, THF, 0 °C; add 16, 23 °C, 80%; (i) TMSOTf, 2,6-lutidine, CH₂Cl₂, 0→23 °C, 76%.

Scheme 3^a

^aReagents and conditions: (a) 21, Tf₂O, Ph₂SO, TBP, CH₂Cl₂, −45 °C; add 20, −78→0 °C, 91% (1.1:1, α:β, SiO₂ separation); (b) BaOH•8H₂O, MeOH, 23 °C, 81%.

Scheme 4^a

^aReagents and conditions: (a) BF₃•OEt₂, 4Å ms, CH₂Cl₂, −78→23 °C, 82%; (b) PhSeH, Et₃N, 30 °C, 91%; (c) 19, EtOCOCl, Et₃N, THF, 0 °C; add 26, 10 °C, 93%; (c’) 22, EtOCOCl, Et₃N, THF, 0 °C; add 26, 12 °C, 89%; (c”) 23, TBP, CH₂Cl₂, 23 °C, 98%; (d) Pd/C, H₂, THF, EtOH, 23 °C; TFA, H₂O, 23 °C, RP-HPLC, 85% (for 27), 78% (for 28), 69% (for 29).

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