Design and synthesis of potent Quillaja saponin vaccine adjuvants

Article abstract of DOI:10.1021/ja9082842

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.

Full text of DOI:10.1021/ja9082842

Published on Web 01/20/2010
Design and Synthesis of Potent Quillaja Saponin Vaccine
Adjuvants
Michelle M. Adams, Payal Damani, Nicholas R. Perl, Annie Won, Feng Hong,
Philip O. Livingston,* Govind Ragupathi,* and David Y. Gin*
Molecular Pharmacology and Chemistry Program, and the Melanoma and Sarcoma SerVice,
Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York AVenue,
New York, New York 10065
Received September 29, 2009; E-mail: gind@mskcc.org; ragupatg@mskcc.org; livingsp@mskcc.org
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.
Introduction
minor isomer, QS-21-Xyl (2, ∼35% abundance), terminates in
a ꢀ-D-xylose substituent.7
The development of vaccines to combat cancer and vaccine-
resistant infectious diseases has relied significantly on subunit
antigen constructs. While defined molecular antigens offer
advantages in terms of safety and precision in immune response
targeting, they are typically less immunogenic. Often, these
vaccine formulations require an adjuvant, a substance that
potentiates immune response.1,2 The critical roles of vaccine
adjuvants lie in their ability to: (1) enable the use of otherwise
impotent antigens; (2) extend the benefits of vaccination to poor
responders (e.g., older or immune-compromised patients); and
(3) effect dose-sparing of rare and expensive antigens in short
supply (e.g., during an epidemic). Given that the majority of
FDA-approved subunit vaccines rely on an adjuvant component,
the challenge to discover novel immunopotentiators remains at
the fore. QS-21 (Chart 1), a purified saponin fraction from the
bark extracts of Quillaja saponaria (QS),3,4 is a promising
adjuvant in numerous prophylactic and therapeutic vaccines.5
This saponin fraction comprises two principal isomers that share
a triterpene, a branched trisaccharide, and a glycosylated
pseudodimeric acyl chain.6 The two isomeric forms differ in
the constitution of the terminal sugar within the linear tetrasac-
charide segment, wherein the major isomer, QS-21-Api (1,
∼65% abundance), incorporates a ꢀ-D-apiose residue, and the
Numerous clinical trials have been conducted with QS-21
adjuvant in vaccines against infectious diseases (malaria,8,9
HIV,10,11 hepatitis,12 tuberculosis13) and cancer (melanoma,14,15
(7) Cleland, J. L.; Kensil, C. R.; Lim, A.; Jacobsen, N. E.; Basa, L.;
Spellman, M.; Wheeler, D. A.; Wu, J. Y.; Powell, M. F. J. Pharm.
Sci. 1996, 85, 22–28.
(8) Kester, K. E.; McKinney, D. A.; Tornieporth, N.; Ockenhouse, C. F.;
Heppner, D. G.; Hall, T.; Wellde, B. T.; White, K.; Sun, P.; Schwenk,
R.; Krzych, U.; Delchambre, M.; Voss, G.; Dubois, M. C.; Gasser,
R. A.; Dowler, M. G.; O’Brien, M.; Wittes, J.; Wirtz, R.; Cohen, J.;
Ballou, W. R.; Rts, S. M. V. E. G. Vaccine 2007, 25, 5359–5366.
(9) Abdulla, S.; Oberholzer, R.; Juma, O.; Kubhoja, S.; Machera, F.;
Membi, C.; Omari, S.; Urassa, A.; Mshinda, H.; Jumanne, A.; Salim,
N.; Shomari, M.; Aebi, T.; Schellenberg, D. M.; Carter, T.; Villafana,
T.; Demoitie, M. A.; Dubois, M. C.; Leach, A.; Lievens, M.;
Vekemans, J.; Cohen, J.; Ballou, W. R.; Tanner, M. New Engl. J. Med.
2008, 359, 2533–2544.
(10) Evans, T. G.; McElrath, M. J.; Matthews, T.; Montefiori, D.; Weinhold,
K.; Wolff, M.; Keefer, M. C.; Kallas, E. G.; Corey, L.; Gorse, G. J.;
Belshe, R.; Graham, B. S.; Spearman, P. W.; Schwartz, D.; Mulligan,
M. J.; Goepfert, P.; Fast, P.; Berman, P.; Powell, M.; Francis, D.;
Grp, N. A. V. E. Vaccine 2001, 19, 2080–2091.
(11) Kennedy, J. S.; Co, M.; Green, S.; Longtine, K.; Longtine, J.; O’Neill,
M. A.; Adams, J. P.; Rothman, A. L.; Yu, Q.; Johnson-Leva, R.; Pal,
R.; Wang, S. X.; Lu, S.; Markham, P. Vaccine 2008, 26, 4420–4424.
(12) Vandepapeliere, P.; Horsmans, Y.; Moris, P.; Van Mechelen, M.;
Janssens, M.; Koutsoukos, M.; Van Belle, P.; Clement, F.; Hanon,
E.; Wettendorff, M.; Garcona, N.; Leroux-Roels, G. Vaccine 2008,
26, 1375–1386.
(1) Jiang, Z. H.; Koganty, R. R. Curr. Med. Chem. 2003, 10, 1423–1439.
(2) Kwissa, M.; Kasturi, S. P.; Pulendran, B. Exp. ReV. Vaccines 2007,
6, 673–684.
(13) Garcon, N.; Chomez, P.; Van Mechelen, M. Exp. ReV. Vaccines 2007,
6, 723–739.
(3) Dalsgaard, K. Dansk Tids. Farm. 1970, 44, 327–331.
(4) Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. J. Immunol. 1991,
146, 431–437.
(14) Ragupathi, G.; Meyers, M.; Adluri, S.; Howard, L.; Musselli, C.;
Livingston, P. O. Int. J. Cancer 2000, 85, 659–666.
(5) Kensil, C. R. Crit. ReV. Ther. Drug Carrier Syst. 1996, 13, 1–55.
(6) Jacobsen, N. E.; Fairbrother, W. J.; Kensil, C. R.; Lim, A.; Wheeler,
D. A.; Powell, M. F. Carbohydr. Res. 1996, 280, 1–14.
(15) Ragupathi, G.; Livingston, P. O.; Hood, C.; Gathuru, J.; Krown, S. E.;
Chapman, P. B.; Wolchok, J. D.; Williams, L. J.; Oldrield, R. C.;
Hwu, W. J. Clin. Cancer Res. 2003, 9, 5214–5220.
9
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J. AM. CHEM. SOC. 2010, 132, 1939–1945 1939

A R T I C L E S
Adams et al.
Chart 1
breast,16,17 small cell lung,18 prostate19). Despite the promise
of QS-21, however, there are several liabilities associated with
its use as an adjuvant. First, high variability in molecular
composition of saponins is seen even among QS trees of similar
age and local environment.20 Purification of the multicomponent
QS-21 fraction entails elaborate low-yielding extraction and
HPLC protocols.21 Not surprisingly, this saponin adjuvant is
currently not available commercially. Second, potency of QS-
21 is proportional to dose, but the tolerated dose of QS-21 in
cancer patients does not exceed 150 µg, above which significant
local erythema and systemic flu-like symptoms arise. Finally,
QS-21 was reported to degrade in a matter of days on storage
in solutions of physiological pH at ambient temperature,7
wherein the labile ester group within the acyl chain (Chart 1)
of the molecule undergoes spontaneous hydrolysis to produce
byproducts with severely attenuated adjuvant activity.22,23
Importantly, the latter issue of chemical instability is a significant
factor in precluding the advancement of QS-21 alone as an
adjuvant for vaccines in developing countries, the epidemic
strongholds of malaria, HIV, and tuberculosis. Efforts to address
these impediments have largely focused on the incorporation
of additives (copolymers, lipids, etc.)13,24 to impart varying
degrees of stability to QS-21. However, such complex formula-
tions confer additional dimensions of heterogeneity to the
vaccine and introduce challenges associated with maintaining
formulation consistency for advancement to clinical evaluation.
An alternate approach to overcoming all of the problems
associated with QS-21 is to improve this adjuvant through
controlled structural modifications at the molecular level. The
extent of these efforts has been quite limited, however, as the
chemical sensitivity of the natural product presents an exceed-
ingly narrow window for its functional group derivatization.
This barrier can be overcome through chemical synthesis of
the QS-adjuvants. The successful validation of this approach is
exemplified herein by the design, synthesis, and evaluation of
novel synthetic saponins based on the QS-21 parent architecture.
These new hydrolytically stable molecular adjuvants exhibit in
vivo immunostimulating potencies rivaling or exceeding that
of naturally derived QS-21. Moreover, when compared to the
natural product, these molecular entities can be obtained in
homogeneous form with higher efficiency, and signal the
potential for modulation of toxicity by further structural
modification.
Results
Chemical Synthesis. A key observation from early stability
studies on QS-21 revealed that the removal of the acyl chain of
the natural product furnished a deacylated saponin byproduct
that was acutely compromised in its ability to stimulate antibody
and CTL responses against OVA antigen in mice.22 The
establishment of the unstable acyl chain as a critical component
for the bioactivity of QS-21 prompts the investigations herein,
which focus on the structural modification of this quadrant to
arrive at novel saponin adjuvants. In this context, simple
replacement of the unstable ester linkages within the acyl chain
with more robust amide linkages would enhance the stability
of the adjuvant with the intention of increasing potency and/or
duration of therapeutic activity per dose.
Access to these amide-stabilized structural variants would
require a novel monosaccharide moiety to serve as a structural
mimic to the acylated fucose sugar (Chart 1, Fuc-residue within
1 and 2). Accordingly, the principal design criterion in this
carbohydrate component is the replacement of the C4-oxygen
functionality with an appropriate nitrogen group, as exemplified
by C4-deoxy-C4-azidogalactopyranoside 6 (Scheme 1). The
synthetic sequence to prepare the fucose surrogate 6 (Scheme
1) was initiated with 3,6-di-O-benzoyl-4-O-methanesulfonyl-
D-glucal (3), derived in one step from D-glucal according to
the procedure of Piancatelli.25 This C4-mesylate 3 was a
competent electrophile for SN2 displacement with sodium azide
(16) Gilewski, T.; Ragupathi, G.; Bhuta, S.; Williams, L. J.; Musselli, C.;
Zhang, X. F.; Bencsath, K. P.; Panageas, K. S.; Chin, J.; Hudis, C. A.;
Norton, L.; Houghton, A. N.; Livingston, P. O.; Danishefsky, S. J.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3270–3275.
(17) Musselli, C.; Ragupathi, G.; Gilewski, T.; Panageas, K. S.; Spinat,
Y.; Livingston, P. O. Int. J. Cancer 2002, 97, 660–667.
(18) Krug, L. M.; Ragupathi, G.; Ng, K. K.; Hood, C.; Jennings, H. J.;
Guo, Z. W.; Kris, M. G.; Miller, V.; Pizzo, B.; Tyson, L.; Baez, V.;
Livingston, P. O. Clin. Cancer Res. 2004, 10, 916–923.
(19) Ragupathi, G.; Slovin, S. F.; Adluri, S.; Sames, D.; Kim, I. J.; Kim,
H. M.; Spassova, M.; Bornmann, W. G.; Lloyd, K. O.; Scher, H. I.;
Livingston, P. O.; Danishefsky, S. J. Angew. Chem., Int. Ed. 1999,
38, 563–566.
(20) Kamstrup, S.; San Martin, R.; Doberti, A.; Grande, H.; Dalsgaard, K.
Vaccine 2000, 18, 2244–2249.
(21) Kensil, C. A. Saponin adjuvant compositions. U.S. Pat. 6,231,859,
2001.
(22) Kensil, C. R.; Soltysik, S.; Wheeler, D. A.; Wu, J. Y. Structure/function
studies on QS-21, a unique immunological adjuvant from Quillaja
saponaria In Saponins Used in Traditional and Modern Medicine;
Waller, G. R., Yamasaki, K., Eds.; Plenum Press: New York, 1996;
pp 165-172.
(23) Liu, G.; Anderson, C.; Scaltreto, H.; Barbon, J.; Kensil, C. R. Vaccine
2002, 20, 2808–2815.
(24) Drane, D.; Gittleson, C.; Boyle, J.; Moraskovsky, E. Exp. ReV. Vaccines
2007, 6, 761–772.
(25) Squarcia, A.; Vivolo, F.; Weinig, H. G.; Passacantilli, P.; Piancatelli,
G. Tetrahedron Lett. 2002, 43, 4653–4655.
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A R T I C L E S
Scheme 1a
Scheme 2a
a Reagents 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, -50f-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, -78f23
°C, 67%; (h) TBAF, THF, 0 °C, 93%; (i) CCl3CN, DBU, CH2Cl2, 0f23
°C, 95%.
a Reagents and conditions: (a) NaOH, BnOCOCl, H2O, 0f23 °C, >99%;
(b) EtOCOCl, Et3N, THF; CH2N2, 0f23 °C, 78%; (c) CF3CO2Ag, Et3N,
THF, -50f23 °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,
0f23 °C, 76%.
to provide the corresponding azide, whose ester groups were
subsequently exchanged for benzyl ethers by Zemple´n saponi-
fication (NaOMe) and alkylation with benzyl bromide to furnish
azido-galactal 4 (51% from 3). Stereoselective 1,2-bis(acyloxy-
lation)26 of glycal 4 (PhI(OAc)2, BF3 ·OEt2) was accomplished
in 85% yield, allowing for subsequent acetate methanolysis (5,
84%). Selective protection of the hemiacetal with TIPSCl
provided the C4-azidogalactoside 6 (59%), which serves as the
fucose surrogate incorporating the C4-nitrogen functionality. It
is worth noting that the fucose mimic 6 bears an “extra” C6-
benzyloxy group as compared to the natural fucose moiety,
which incorporates only a C6-methyl group. This artifact of the
synthesis not only allows for a relatively short synthetic
sequence to 6, but also imparts an additional functional handle
with which to explore future novel compositions of matter.
Glycosylation of the azido-sugar 6 with the trisaccharide
hemiacetal 8, derived from anomeric desilyation of the previ-
ously prepared trisaccharide 7,27 proceeded under the dehydra-
tive coupling protocol28 (Ph2SO, Tf2O) to afford the selectively
protected tetrasaccharide 9 (70%). Subsequent removal of the
tri-iso-propylsilyl acetal within 9 was accomplished with TBAF
to provide the corresponding hemiacetal (93%), which was then
converted to the anomeric R-trichloroacetimidate (10, 95%).
This constitutes a successful route to a fully elaborated tet-
Scheme 3a
a Reagents and conditions: (a) 21, Tf2O, Ph2SO, TBP, CH2Cl2, -45 °C;
add 20, -78f0 °C, 91% (1.1:1, R:ꢀ, SiO2 separation); (b) BaOH·8H2O,
MeOH, 23 °C, 81%.
rasaccharide fragment that incorporates the required galacto-
C4-azido group on which to anchor a variety of amide acyl
chains.
Initial efforts toward the preparation of acyl chain variants
of QS-21 focused on 19 (Scheme 2), 22, and 23 (Scheme 3) as
promising acylation agents to access novel hydrolytically stable
saponin adjuvants. The most complex amide acyl chain of these
(26) Shi, L.; Kim, Y. J.; Gin, D. Y. J. Am. Chem. Soc. 2001, 123, 6939–
6940.
(27) Deng, K.; Adams, M. M.; Gin, D. Y. J. Am. Chem. Soc. 2008, 130,
5860–5861.
(28) Garcia, B. A.; Poole, J. L.; Gin, D. Y. J. Am. Chem. Soc. 1997, 119,
7597–7598.
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A R T I C L E S
Adams et al.
is the glycosylated pseudodimeric amide 19 (Scheme 2),
incorporating the most conservative structural variations of the
acyl chain. This substrate serves as an isosteric mimic of the
natural acyl substituent, differing only in the central ester-to-
amide replacement, which should serve to further enhance
stability of this stereochemically elaborate substructure. In
addition, two simplified lipophilic amide acyl chains (Scheme
3), in the form of either a glycosylated (e.g., 22) or a
nonglycosylated (e.g., 23) linear aliphatic moiety, were also
pursued. Synthetic QS saponins bearing the latter two hydro-
phobic chains would not only be prepared by considerably
shorter synthetic sequences as compared to that of 19, but also
provide a clear indication of whether the elaborate stereochem-
ical array within the native acyl chain is required for adjuvant
activity.
The synthesis of the isosteric amide acyl chain 19 (Scheme
2) began with D-alloisoleucine (11), obtained from L-isoleucine
by the three-step epimerization-resolution protocol of Sakai.29
Following amino protection of 11 as its benzyl carbamate
(BnOCOCl, >99%), the carboxylic acid was subjected to
Arndt-Eistert homologation via its derivatization to the cor-
responding R-diazoketone and Wolff rearrangement to provide
the ꢀ-amino acid 12 (62%). Subsequent activation of the
carboxylic acid in 12 with carbonyldiimidazole allowed for
Claisen condensation with the Li-enolate of t-butyl acetate (13)
to provide ꢀ-keto ester 14 (60%), a suitable substrate for Noyori
catalytic asymmetric hydrogenation.30 This proceeded with
RuCl2 ·(S)-BINAP and H2, providing the ꢀ-hydroxy ester 15
(89%) with complete catalyst-controlled diastereoselectivity.
Hydroxyl group silylation (TBSOTf, 94%) was followed by
hydrogenolytic unmasking of carbamate 15 to afford amine 16
in 97% yield. Importantly, the deliberate selection of the t-butyl
ester protective group in this sequence precluded unproductive
lactamization of amine 16, allowing for its acylation (80%) with
the glycosylated acyl chain fragment 17, previously prepared
in the synthesis of QS-21.31-33 This transformation provided,
after acid-catalyzed t-butyl ester removal (76%), the selectively
protected fully intact acyl chain 19, the direct OfN amide
mimic of the QS-21 acyl chain.
The late-stage convergent assembly of the amide acyl chain
variants of QS-21 (Scheme 4) capitalized on the recent
disclosure of our semisynthetic approach to access homogeneous
samples of QS-saponin adjuvants.27 Although the acyl chain
and linear tetrasaccharide quadrants of the QS saponins are
synthesized de novo, the remaining half of the natural product,
comprising the trisaccharide-triterpene conjugate, could be
isolated by controlled chemical degradation of QS-extracts and
selective protection to yield the protected prosapogenin 24
(Scheme 4) in only three steps.27 Thus, Schmidt glycosylation35
of the C28 carboxylic acid in 24 with the tetrasaccharide
glycosyl trichloroacetimidate donor 10 provided the glycosyl
ester 25 (82%) with complete anomeric selectivity. The lone
azide group in 25 was responsive to reduction with benzene-
selenol to reveal the corresponding amine 26 (91%), onto which
various acyl chains could be appended. In the cases of
N-acylation with the glycosylated acyl chain carboxylic acid
derivatives 19 and 22, couplings were accomplished by initial
carboxylate activation with ethyl chloroformate. For introduction
of the aliphatic lauryl chain, direct acylation with lauryl chloride
(23) could be accomplished. Subsequent sequential hydro-
genolysis and acid hydrolysis effected global deprotection of
the resulting advanced intermediates to provide the amide acyl
chain SQS-saponin variants 27 (SQS-0101), 28 (SQS-0102),
and 29 (SQS-0103) in 79%, 69%, and 68% yields, respectively,
from the amine precursor 26.
Evaluation of Immune Response Augmentation. Currently,
there exists no rapid in vitro biological screen for assessing the
potential efficacy of saponin vaccine adjuvants, given that the
mechanism by which saponins augment immune response is
unknown. As a result, evaluation of these novel saponins as
immunostimulants proceeded directly to preclinical studies
involving mouse vaccination with the melanoma antigen GD3
ganglioside conjugated to the KLH carrier protein (GD3-KLH,
Chart 2). This is a clinically relevant vaccine model that has
proven useful for comparing the immunopotentiating ability of
various adjuvants.36,37 Monitoring antibody responses to both
the carbohydrate antigen and the protein carrier provides a useful
assessment of adjuvant performance to antigens of different
immunogenicity, ranging from a poorly immunogenic glycolipid
(GD3) to a highly immunogenic protein (KLH). Moreover,
previous experience with this vaccination protocol has consis-
tently correlated antibody titers against KLH to that of T-cell
response to this antigen.37
Fortunately, synthetic access to the two remaining acyl chain
fragments 22 and 23 (Scheme 3) was significantly less effort-
intensive given the absence of stereochemical complexity within
these substructures. For example, the glycosylated acyl chain
22 could be readily obtained in a three-step sequence34 involving
dehydrative glycosylation of allyl 12-hydroxydodecanoate (20)
with 2,3,5-tri-O-TBS-arabinofuranose (21)31,32 to provide the
intermediate glycoside (91%) as a separable mixture of anomers
(1.1:1, R:ꢀ). The R-anomer was then advanced via Ba(OH)2-
mediated saponification of the allyl ester to provide the acyl
chain 22 (81%). Finally, the nonglycosylated aliphatic acyl chain
variant was the easiest to procure given that lauryl chloride (23)
is commercially available.
Groups of five mice (C57BL/6J, female, 6-8 weeks of age)
were vaccinated with GD3-KLH at a 10 µg dose (Figure 1).
The antigen was coadministered with the adjuvant of interest
in a vaccination protocol involving three subcutaneous injections
at 1-week intervals (days 0, 7, and 14) plus a booster at day 65.
As the negative control, mice were vaccinated with the GD3-
KLH antigen only. As a positive control, vaccinations were
performed with naturally derived QS-21 (NQS-21), obtained
by fractionating
a mixture of saponins from Quillaja
saponaria.38 The adjuvant dose employed in these comparative
studies was 10 µg, a quantity of QS-21 known to induce
measurable antibody responses with acceptable toxicity effects
(29) Noda, H.; Sakai, K.; Murakami, H. Tetrahedron: Asymmetry 2002,
13, 2649–2652.
(30) Noyori, R.; Ohkuma, T.; Kitamura, M.; Takaya, H.; Sayo, N.;
Kumobayashi, H.; Akutagawa, S. J. Am. Chem. Soc. 1987, 109, 5856–
5858.
(31) Wang, P.; Kim, Y. J.; Navarro-Villalobos, M.; Rohde, B. D.; Gin,
D. Y. J. Am. Chem. Soc. 2005, 127, 3256–3257.
(35) Schmidt, R. R.; Kinzy, W. AdV. Carbohydr. Chem. Biochem. 1994,
50, 21–123.
(32) Kim, Y. J.; Wang, P.; Navarro-Villalobos, M.; Rohde, B. D.;
Derryberry, J.; Gin, D. Y. J. Am. Chem. Soc. 2006, 128, 11906–11915.
(33) Deng, K.; Adams, M. M.; Damani, P.; Livingston, P. O.; Ragupathi,
G.; Gin, D. Y. Angew. Chem., Int. Ed. 2008, 47, 6395–6398.
(34) Lee, R. E.; Mikusova, K.; Brennan, P. J.; Besra, G. S. J. Am. Chem.
Soc. 1995, 117, 11829–11832.
(36) Kim, S. K.; Ragupathi, G.; Musselli, C.; Choi, S. J.; Park, Y. S.;
Livingston, P. O. Vaccine 2000, 18, 597–603.
(37) Kim, S. K.; Ragupathi, G.; Cappello, S.; Kagan, E.; Livingston, P. O.
Vaccine 2001, 19, 530–537.
(38) Kensil, C. R. Methods Mol. Med. 2000, 42, 259–271 (Vaccine
Adjuvants).
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Potent Quillaja Saponin Vaccine Adjuvants
A R T I C L E S
Scheme 4a
a Reagents and conditions: (a) BF3 ·OEt2, 4 Å ms, CH2Cl2, -78f23 °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).
Chart 2
expressing GD3 antigen (Figure 1E). Sera drawn 7 days after
the booster were evaluated by FACS using the SK-Mel-28 (GD3
positive) cell line. The individual FACS results are presented
at 10 µg SQS-adjuvant dose for direct comparison. Prevacci-
nation sera from mice showed less than 10% positive cells, while
sera obtained after vaccination with all three synthetic adjuvants
showed significant positive reactivity with SK-Mel-28. The
median percent positive cell reactivities were 15% for the no-
adjuvant control, 87% for NQS-21 (1 and 2), 78% for SQS-
0101 (27), 83% for SQS-0102 (28), and 64% for SQS-0103
(29). These cell-surface reactivity results further reinforce the
comparable adjuvant activity of synthetic SQS-0101, -0102, and
-0103 relative to that of NQS-21. In addition, cell surface
in mice. Antibody titers against GD3 (IgM and IgG) and KLH
(IgG) were determined by ELISA. Remarkably, all of the
synthetic SQS-adjuvants (27-29) are at least as active as NQS-
21 in immunopotentiating ability, as illustrated by the IgM and
reactivity against SK-Mel-28 was further characterized by
IgG response to GD3 (Figure 1A and B) measured 1 week
complement-dependent cytotoxicity (CDC) assays (Figure 1F)
following the booster (day 72). In each case, antibody titers
using rabbit complement and sera from the mice vaccinated with
were significantly higher than the group with GD3-KLH alone.
GD3-KLH (10 µg) plus SQS-101 (27), SQS-102 (28), or SQS-
103 (29), each at 20 µg doses. Less than 6% median reactivity
was detected in prevaccination sera and in mice vaccinated with
GD3-KLH alone, both serving as negative controls. However,
sera obtained from mice immunized with GD3-KLH plus SQS-
101, SQS-102, and SQS-103 all showed significantly enhanced
cytotoxicity against SK-Mel-28 over the negative controls.
In addition, the IgG antibody response against KLH was also
strikingly elevated (Figure 1C) with all of the novel SQS-saponin
adjuvants. Antibody subtyping of the anti-GD3 IgG isotype
(Figure 1D) revealed a significant bias toward the mouse IgG2b
subtype with all of our synthetic saponin adjuvants (SQS-0101,
-0102, and -0103), a result similar to that of naturally derived
NQS-21. Production of other mouse anti-GD3 IgG subtypes,
including IgG1, IgG2a, and IgG3, was low or negligible as
indicated by class-specific ELISA employing the standard 0.1
absorbance level threshold for positive reactivity.
As a standard initial overall assessment of toxicity, the weight
loss of the mice was monitored at 0, 1, 2, 3, and 7 days after
the first vaccination (Figure 2). For the negative control
involving vaccinations with GD3-KLH only (no adjuvant), no
appreciable weight loss was observed. For the positive control,
the presence of NQS-21 (10 µg) elicited notable and expected
median weight loss of 9% after the initial injection. The group
The activity of these anti-GD3 antibodies was further
investigated for their ability to effect tumor cell binding and
lysis. These properties were first assessed by flow cytometry,
monitoring the cell surface reactivity of a tumor cell-line
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A R T I C L E S
Adams et al.
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 five 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 3 weeks (days 0, 7, and 14), followed by a booster at day
65. Postboost serological data at day 72 are presented. (A) Anti-GD3 titers (IgM) after vaccination; median values in curly brackets. (B) Anti-GD3 titers
(IgG) after vaccination; median values in curly brackets. (C) Anti-KLH titers (IgG) after vaccination; median values in curly brackets. (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 curly brackets. (F) Complement-dependent cytotoxicity
(CDC) activity of anti-GD3 against SK-Mel-28 tumor cell line expressing GD3 antigen.
which include scarcity, difficulty in purification to homogeneity,
dose-limiting toxicity, and chemical instability. The challenges
associated with QS-21 have prompted the generation of alternate
structures inspired by QS saponins for the discovery of adjuvants
with greater stability and more favorable therapeutic profiles.
With the current semisynthetic approach to complex QS
saponins,27 the preparation of novel adjuvants with unpre-
cedented control over molecular structure is now possible. Using
this strategy, the first generation of SQS-amide analogues
(27-29) has been successfully prepared. Each of these structures
exhibits at least comparable adjuvant activity to that of natural
NQS-21, as evidenced by initial ELISA, FACS, and CDC data
(Figure 1). For the strongly immunogenic KLH protein carrier,
expected high antibody titers were evident. Like that of NQS-
21, the anti-KLH titers for all of the SQS-analogues were at
least 20-fold higher than the negative control group comprising
GD3-KLH antigen in the absence of a saponin adjuvant. For
the weakly immunogenic GD3 antigen, initial IgM production
followed by class switching to IgG was observed with all three
synthetic saponins at levels comparable to that of NQS-21. Upon
subtyping these antibodies, the IgG2b subclass was shown to
predominate. The mouse IgG2b and IgG2a subclasses are known
to induce potent immunotherapeutic effector functions,39 includ-
ing complement-dependent cytotoxicity (CDC) and antibody-
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).
vaccinated with SQS-0102 had an 11% median weight loss,
while the maximum weight loss of the remaining groups
incorporating SQS-0101 and SQS-0103 was less than 5%.
Overall, weight loss with 10 µg SQS-0102 was slightly, but
not significantly, more than with NQS-21 (p > 0.15), while
weight loss with SQS-0101 (p ) 0.05) and SQS-0103 (p <
0.025) was significantly less than that with NQS-21.
Discussion
QS-21 remains the immunopotentiator of choice in many
cancer and infectious disease vaccine trials despite its liabilities,
(39) Nimmerjahn, F.; Ravetch, J. V. Science 2005, 310, 1510–1512.
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Potent Quillaja Saponin Vaccine Adjuvants
A R T I C L E S
dependent cellular cytotoxicity (ADCC). Importantly, the
cytotoxic activity of these anti-GD3 antibodies was unambigu-
ously verified to effect CDC on the SK-Mel-28 (GD3 positive)
tumor cell line.
has also been suggested to react with putative T-cell surface
receptors via Schiff base formation, providing T-cells with a
costimulatory signal for T-cell activation.40 Within the limited
available data concerning the mechanism of action of QS-21,
there is strong evidence to suggest that a depot effect is not
operative5 and that the saponin is not a ligand for Toll-like
receptors 2 and 4 (TLR-2, TLR-4),41 proteins that activate innate
immune responses by recognizing pathogen-associated molec-
ular patterns. While these findings are highly significant, there
remain an infinite number of mechanistic possibilities to
consider. Despite the striking efficacy of QS-21 and related
saponins as immunological adjuvants, there is little guidance
(beyond broad immunological intuition) with which to marshal
exploratory efforts on their mechanisms of action. This paucity
of data has plagued the saponin adjuvant field, whereby seminal
mechanistic information in this arena is essentially nonexistent,
despite the decades-long use of saponin adjuvants in clinical
trials. Thus, this demonstration of high adjuvant potency with
simpler, hydrolytically stable SQS-adjuvants (27-29) not only
signals the potential for discovery of novel immunotherapeutics,
but also should enable the controlled installation of chemical,
optical, and radio-isotopic reporters as valuable molecular probes
on the adjuvant molecule to address long-standing mechanistic
questions concerning saponin immunopotentiation.
On the basis of these initial immunological data, our first
few hydrolytically stable structural variants (27-29) clearly
show that the elaborate stereochemical array within the native
QS-21 acyl chain is not essential to maintain adjuvant activity
comparable to the natural saponin. This finding, in combination
with the integration of semisynthetic prosapogenin 2427 into the
synthesis, shortens the synthetic route to potent saponin
adjuvants like 28 and 29 by more than 30 steps when compared
to the original chemical synthesis of QS-21.31–33 Finally,
differences in toxicity profiles between the amide variants and
NQS-21, based on tracking the weight loss in mice during
vaccination, are also notable. Whereas toxicity of SQS-0102
(28) is at least as pronounced as that of NQS-21 (1 and 2),
SQS-0101 (27) and SQS-0103 (29) exhibit lower overall toxicity
effects. Indeed, the specific reasons behind this structure-
dependent toxicity remain to be defined. Future investigations
on this front would entail adjuvant dose-escalation studies,
followed by detailed biochemical toxicity evaluations of those
mice exhibiting >10% weight loss upon vaccination. Neverthe-
less, the weight loss differential observed even at the 10 µg
adjuvant dose (Figure 2) is a strong indication that toxicity of
the saponin could be independently modulated through specific
structural perturbations while maintaining potent adjuvant
activity. These initial results speak to the likelihood of the future
discovery of even simpler molecular variants that possess potent
immunostimulatory activities and attenuated toxicity.
Acknowledgment. This work was supported by grants from
the NIH (R01 GM058833, PO1 CA052477, P50 AT002779), the
DOD Prostate Cancer Research Program #W81XWH-05-1-0085,
and Mr. William H. and Mrs. Alice Goodwin and the Com-
monwealth Foundation for Cancer Research and The Experimental
Therapeutics Center of Memorial Sloan-Kettering Cancer Center.
It is worth noting that the mechanism by which QS-21 (or
our SQS-analogues) potentiates immune response is unknown.
Hypotheses have been put forth that the saponin, through lectin-
mediated cell membrane interactions, may facilitate uptake of
the antigen into antigen-presenting cells (APCs), leading to
specific cytokine profiles that enhance T- and/or B-cell
responses.5,40 The lone aldehyde group within the QS triterpene
Supporting Information Available: Experimental details for
synthetic procedures and analytical data for isolable synthetic
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA9082842
(40) Marciani, D. J. Drug DiscoVery Today 2003, 8, 934–943.
(41) Pink, J. R.; Kieny, M.-P. Vaccine 2004, 22, 2097–102.
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