Development of a minimal saponin vaccine adjuvant based on QS-21

Journal name:
Nature Chemistry
Volume:
6,
Pages:
635–643
Year published:
DOI:
doi:10.1038/nchem.1963
Received
Accepted
Published online

Abstract

Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability and an enigmatic molecular mechanism of action. Herein we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants. Overall, these studies have yielded critical insights into saponin structure–function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

At a glance

Figures

  1. Aryl iodide saponin 6 exhibits potent adjuvant activity and low toxicity in a preclinical mouse-vaccination model.
    Figure 1: Aryl iodide saponin 6 exhibits potent adjuvant activity and low toxicity in a preclinical mouse-vaccination model.

    a, Structure of QS-21 and its four key structural domains. b, Synthesis of aryl iodide saponins 6 (SQS-0-0-5-18) and [131I]-6. i. Fluorescein isothiocyanate, Et3N, DMF, 21 °C, 2 h, 75%. ii. 4, Et3N, DMF, 21 °C, 1 h, 52%. iii. 5, Et3N, DMF, 21 °C, 1 h, 75%. iv. [131I]-NaI, Chloramine-T, MeOH, 21 °C, 1 min, >50%. c, Structure of the adjuvant-attenuated negative control saponin 8 (SQS-0-3-7-18) and synthesis of [131I]-8. v. [131I]-NaI, Chloramine-T, MeOH, 21 °C, 1 min, >50%. df, Biological evaluation of aryl iodide saponin 6 (SQS-0-0-5-18) with the three-component vaccine for anti-KLH titres (IgG) (d), anti-MUC1 titres (IgG) (e) and anti-OVA titres (IgG) (f) that indicates potent adjuvant activity comparable to those of natural and synthetic QS-21 (compare the 20 μg doses). Horizontal bars indicate median titres; statistical significance compared to no-adjuvant control, *P ≤ 0.05, **P < 0.01, ***P < 0.001. g, Toxicity assessment based on median per cent weight loss indicates the low toxicity of 6 (SQS-0-0-5-18). Error bars indicate maximum and minimum values for five mice.

  2. Radioiodinated saponin [131I]-6 and fluorescent saponin 3 localize to lymph nodes and injection site in mice.
    Figure 2: Radioiodinated saponin [131I]-6 and fluorescent saponin 3 localize to lymph nodes and injection site in mice.

    a, Biodistribution of active adjuvant [131I]-6 ([131I]-SQS-0-0-5-18) and attenuated adjuvant [131I]-8 ([131I]-SQS-0-3-7-18) with OVA antigen, which indicates the accumulation of [131I]-6, but not [131I]-8, at the injection site and lymph nodes (see Supplementary Fig. 2). Error bars indicate the standard deviation from the mean for five mice; for clarity, statistical significance is indicated graphically only for the lymph nodes and injection site: *P ≤ 0.05, liver, muscle, lymph node, skin, thyroid; **P < 0.01, blood, lungs, spleen, kidneys, bone, injection site; ***P < 0.001, heart. b, Imaging at the injection site (yellow arrows indicate ink circles) with fluorescein-labelled active adjuvant 3 (SQS-0-0-5-12) or unlabelled inactive adjuvant 2 (SQS-0-0-5-11) and Alexa-647-labelled OVA (OVA-A647), which indicates the retention of 3 and OVA-A647 at the injection site; the green crescent in the fluorescein image for Mouse 2 results from a software ghosting effect (see Supplementary Fig. 5). c, Imaging of dissected lymph nodes with active adjuvant 3 (SQS-0-0-5-12) or inactive adjuvant 2 (SQS-0-0-5-11) and OVA-A647, which indicates the increased accumulation of OVA-A647 with 3 but not with 2. Mice were injected in the left flank and the right lymph node served as the negative control within each animal.

  3. Truncated saponin 16 lacks the entire branched trisaccharide domain of QS-21 but retains potent adjuvant activity and low toxicity in a preclinical mouse-vaccination model.
    Figure 3: Truncated saponin 16 lacks the entire branched trisaccharide domain of QS-21 but retains potent adjuvant activity and low toxicity in a preclinical mouse-vaccination model.

    a, Synthesis of aryl iodide saponins 16 (SQS-1-0-5-18) and [131I]-16. i. TESOTf, 2,6-lutidine, CH2Cl2, 0 °C, 1 h, 80%. ii. (1) 11, 12, BF3·OEt2, 4 Å molecular sieves, CH2Cl2, −35 °C, 30 min; (2) PhSeH, Et3N, 38 °C, 8 h, 58% (over two steps). iii. (1) HO2C(CH2)5NHBoc (14), EtOCOCl, Et3N, THF, 0 °C, 2.5 h, (acid preactivation), then add to 13, 0 °C, 1.5 h; (2) H2 (50 p.s.i.), Pd/C (Degussa), THF/EtOH (1:1), 21 °C, 24 h; (3) TFA/H2O (4:1), 0 °C, 2 h, 65% (over three steps). iv. 4, Et3N, DMF, 21 °C, 2 h, 67%. v. 5, Et3N, DMF, 21 °C, 1.5 h, 75%. vi. [131I]-NaI, Chloramine-T, MeOH, 21 °C, 1 min, 55%. bd, Biological evaluation of truncated saponin 16 with three-component vaccine for anti-KLH (IgG) (b), anti-MUC1 (IgG) (c) and anti-OVA (IgG) (d) titres that indicates potent adjuvant activity. Horizontal bars indicate median titres; statistical significance compared to no-adjuvant control, *P ≤ 0.05, **P < 0.01, ***P < 0.001. e, Toxicity assessment based on median per cent weight loss indicates the low toxicity of 16 (SQS-1-0-5-18). Error bars indicate the maximum and minimum values for five mice.

  4. Oleanolic acid derivative 18, which lacks both the C4-aldehyde substituent and the C16-alcohol in the triterpene domain of QS-21, exhibits poor adjuvant activity in a preclinical mouse-vaccination model.
    Figure 4: Oleanolic acid derivative 18, which lacks both the C4-aldehyde substituent and the C16-alcohol in the triterpene domain of QS-21, exhibits poor adjuvant activity in a preclinical mouse-vaccination model.

    ac, Biological evaluation of oleanolic acid derivative 18 (SQS-1-7-5-18) with a three-component vaccine for anti-KLH titres (IgG) (a), anti-MUC1 titres (IgG) (b) and anti-OVA titres (IgG) (c) indicates attenuated adjuvant activity. Horizontal bars indicate median titres; statistical significance compared to no-adjuvant control, *P ≤ 0.05, **P < 0.01, ***P < 0.001. d, Toxicity assessment based on median per cent weight indicates low toxicity of 18 (SQS-1-7-5-18). Error bars indicate maximum and minimum values for five mice.

  5. Caulophyllogenin derivative 19 and echinocystic acid derivative 20, which lack the C4-aldehyde substituent but retain the C16-alcohol in the triterpene domain of QS-21, exhibit potent adjuvant activity and no toxicity in a preclinical mouse-vaccination model.
    Figure 5: Caulophyllogenin derivative 19 and echinocystic acid derivative 20, which lack the C4-aldehyde substituent but retain the C16-alcohol in the triterpene domain of QS-21, exhibit potent adjuvant activity and no toxicity in a preclinical mouse-vaccination model.

    a, Structures of saponin variants 1922 with modifications at the C4-aldehyde substituent and C16-alcohol of the triterpene domain of QS-21. bf, Biological evaluation of triterpene variants 1922 with a four-component vaccine (MUC1–KLH, OVA, GD3–KLH) for anti-KLH (IgG) (b), anti-MUC1 (IgG) (c), anti-OVA (IgG) (d), anti-GD3 (IgM) (e) and anti-GD3 (IgG) (f) titres indicates that the C4-aldehyde substituent is not required for adjuvant activity (19, 20) and removal of the C16-alcohol attenuates activity (21, 22) (see Supplementary Fig. 10 for full data with both 20 and 50 μg doses). Horizontal bars indicate median titres; statistical significance compared to no-adjuvant control, *P ≤ 0.05, **P < 0.01, ***P < 0.001. g, Toxicity assessment based on median per cent weight loss indicates lack of toxicity of 1922. Error bars indicate maximum and minimum values for five mice.

  6. Adjuvant-active quillaic acid derivative 16 localizes to the injection site and lymph nodes in mice, but adjuvant-attenuated oleanolic acid derivative 18 does not.
    Figure 6: Adjuvant-active quillaic acid derivative 16 localizes to the injection site and lymph nodes in mice, but adjuvant-attenuated oleanolic acid derivative 18 does not.

    In vivo biodistribution in mice of active adjuvant [131I]-16 ([131I]-SQS-1-0-5-18) and attenuated adjuvant [131I]-18 ([131I]-SQS-1-7-5-18) at 24 hours post-injection in the presence of 20 μg OVA. Error bars indicate standard deviation from the mean for five mice; for clarity, statistical significance indicated graphically only for lymph nodes and injection site: *P ≤ 0.05, lymph nodes, injection site, skin; **P < 0.01, lungs, spleen, stomach, muscle, bone; ***P < 0.001, blood, heart.

Compounds

23 compounds View all compounds
  1. QS-21-Api
    Compound 1a QS-21-Api
  2. QS-21-Xyl
    Compound 1b QS-21-Xyl
  3. SQS-0-0-5-11
    Compound 2 SQS-0-0-5-11
  4. SQS-0-0-5-12
    Compound 3 SQS-0-0-5-12
  5. N-Succinimidyl-4-iodobenzoate
    Compound 4 N-Succinimidyl-4-iodobenzoate
  6. N-Succinimidyl-4-trimethylstannyl-benzoate
    Compound 5 N-Succinimidyl-4-trimethylstannyl-benzoate
  7. SQS-0-0-5-18
    Compound 6 SQS-0-0-5-18
  8. [SQS-0-0-5-18]-SnMe3
    Compound 7 [SQS-0-0-5-18]-SnMe3
  9. SQS-0-3-7-18
    Compound 8 SQS-0-3-7-18
  10. [SQS-0-3-7-18]-SnMe3
    Compound 9 [SQS-0-3-7-18]-SnMe3
  11. Quillaic acid
    Compound 10 Quillaic acid
  12. TES-protected Quillaic acid
    Compound 11 TES-protected Quillaic acid
  13. (2R,3R,4S,5S,6S)-5-Azido-4-(benzyloxy)-6-((benzyloxy)methyl)-3-(((3aR,4S,6S,7S,7aR)-2,2,6-trimethyl-7-(((2S,3R,4S,5R)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yl)oxy)tetrahydro-2H-pyran-2-yl 2,2,2-trichloroacetimidate
    Compound 12 (2R,3R,4S,5S,6S)-5-Azido-4-(benzyloxy)-6-((benzyloxy)methyl)-3-(((3aR,4S,6S,7S,7aR)-2,2,6-trimethyl-7-(((2S,3R,4S,5R)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-4-yl)oxy)tetrahydro-2H-pyran-2-yl 2,2,2-trichloroacetimidate
  14. Protected quillaic acid saponin amine
    Compound 13 Protected quillaic acid saponin amine
  15. 6-((tert-Butoxycarbonyl)amino)hexanoic acid
    Compound 14 6-((tert-Butoxycarbonyl)amino)hexanoic acid
  16. Aminoacyl quillaic acid saponin
    Compound 15 Aminoacyl quillaic acid saponin
  17. SQS-1-0-5-18
    Compound 16 SQS-1-0-5-18
  18. [SQS-1-0-5-18]-SnMe3
    Compound 17 [SQS-1-0-5-18]-SnMe3
  19. SQS-1-7-5-18
    Compound 18 SQS-1-7-5-18
  20. SQS-1-11-5-18
    Compound 19 SQS-1-11-5-18
  21. SQS-1-8-5-18
    Compound 20 SQS-1-8-5-18
  22. SQS-1-9-5-18
    Compound 21 SQS-1-9-5-18
  23. SQS-1-10-5-18
    Compound 22 SQS-1-10-5-18

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Author information

Affiliations

  1. Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA

    • Alberto Fernández-Tejada,
    • Jeffrey R. Gardner,
    • Derek S. Tan &
    • David Y. Gin
  2. Pharmacology Program, Weill Cornell Graduate School of Medical Sciences, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA

    • Eric K. Chea,
    • Derek S. Tan &
    • David Y. Gin
  3. Melanoma & Immunotherapeutics Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA

    • Constantine George,
    • Philip O. Livingston &
    • Govind Ragupathi
  4. Radiochemistry & Imaging Science Service, Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA

    • NagaVaraKishore Pillarsetty &
    • Jason S. Lewis
  5. Tri-Institutional Research Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA

    • Derek S. Tan &
    • David Y. Gin
  6. Present address: Adjuvance Technologies Inc., 116 West 22nd St, Suite No. 1, New York 10011, USA.

    • Philip O. Livingston
  7. Deceased

    • David Y. Gin

Contributions

A.F-T., E.K.C., N.P., J.R.G., G.R., J.S.L., D.S.T. and D.Y.G. conceived and designed the experiments. A.F-T. and E.K.C. performed the syntheses. C.G. performed the preclinical mouse-vaccination experiments. C.G. and N.P. performed the biodistribution experiments. J.R.G. performed the fluorescence imaging experiments. A.F-T., E.K.C., N.P., J.R.G., P.O.L., G.R., J.S.L., D.S.T. and D.Y.G. analysed the data. A.F-T. and D.S.T. wrote the manuscript.

Competing financial interests

J.R.G., P.O.L., G.R. and D.Y.G are founders of Adjuvance Technologies Inc. and have financial interests in the company.

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