A new model for the presentation of tumor-associated antigens and the quest for an anticancer vaccine: A solution to the synthesis challenge via ring-closing metathesis

Article abstract of DOI:10.1021/ja9052625

Fully synthetic, carbohydrate-based antitumor vaccine candidates have been synthesized in highly clustered modes. Multiple copies of tumor-associated carbohydrate antigens, Tn and STn, were assembled on a single cyclic peptide scaffold in a highly converg

Full text of DOI:10.1021/ja9052625

Published on Web 09/11/2009
A New Model for the Presentation of Tumor-Associated
Antigens and the Quest for an Anticancer Vaccine: A Solution
to the Synthesis Challenge via Ring-Closing Metathesis
Insik Jeon,† Dongjoo Lee,† Isaac J. Krauss,† and Samuel J. Danishefsky*,†,‡
Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York
AVenue, New York, New York 10065, and Department of Chemistry, Columbia UniVersity,
HaVemeyer Hall, 3000 Broadway, New York, New York 10027
Received June 26, 2009; E-mail: s-danishefsky@ski.mskcc.org
Abstract: Fully synthetic, carbohydrate-based antitumor vaccine candidates have been synthesized in highly
clustered modes. Multiple copies of tumor-associated carbohydrate antigens, Tn and STn, were assembled
on a single cyclic peptide scaffold in a highly convergent manner. Ring-closing metathesis-mediated
incorporation of an internal cross-linker was also demonstrated. In particular, this rigidified cross-linked
construct would enhance a cluster-recognizing antibody response by retaining an appropriate distance
between glycans attached to the peptide platform. Details of the design and synthesis of highly clustered
antigens are described herein.
Introduction
tumor-associated antigens are presented to the immune system
as glycoconjugates appended to immunogenic carrier molecules
Our laboratory is seeking the development of novel, fully
synthetic carbohydrate-based vaccines for the treatment of
cancer. This research program is based on the finding that
malignantly transformed cells often exhibit significant alteration
in the nature and quantity of carbohydrates presented on their
cell surfaces, either as glycosphingolipids or as glycoproteins.1
Presumably, when properly introduced to the immune system,
a tumor-associated carbohydrate-based antigen could set into
motion an exploitable immune response, leading to the genera-
tion of antibodies which would selectively bind to and hopefully
eliminate those tumor cells which overexpress the carbohydrates
in question. We are certainly not alone in this area. In particular,
impressive advances of carbohydrate-based anticancer vaccines
have been reported by Boons,2 Kunz,3 and Schmidt.4 The
viability of this carbohydrate vaccine concept has been con-
firmed experimentally in our laboratory. Thus, when these
(such as KLH carrier protein)5 and coadministered with an
immunological adjuvant (such as QS21),6 a carbohydrate-
specific antibody response may be induced. A number of
complex, fully synthetic carbohydrate-based constructs, syn-
thesized in our laboratories, have shown significant promise in
preclinical, and even clinical settings.7 In designing a carbohydrate-
based vaccine construct, it may be of considerable value to consider
the way in which the antigen is displayed in its natural environment,
i.e., on the surface of the transformed cell, to more directly mimic
this natural setting in the context of the vaccine.
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R. K.; Oettgen, H. F.; Livingston, P. O. Cancer Res. 1994, 54, 197–
203. (b) Helling, F.; Zhang, S.; Shang, A.; Adluri, S.; Calves, M.;
Koganty, R.; Longenecker, B. M.; Yao, T. J.; Oettgen, H. F.;
Livingston, P. O. Cancer Res. 1995, 55, 2783–2788.
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1991, 146, 431–437. (b) Marciani, D. J.; Press, J. B.; Reynolds, R. C.;
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May, R. D. Vaccine 2000, 18, 3141–3151. (c) Kim, S.-K.; Ragupathi,
G.; Musselli, C.; Choi, S. J.; Park, Y. S.; Livingston, P. O. Vaccine
1999, 18, 597–603. (d) Kim, S.-K.; Ragupathi, G.; Cappello, S.; Kagan,
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† Sloan-Kettering Institute for Cancer Research.
‡ Columbia University.
(1) (a) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 39,
836–863. (b) Livingston, P. O.; Zhang, S. Cancer Immunol.
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AdV. Exp. Med. Biol. 2001, 491, 369–402.
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Chem., Int. Ed. 1997, 36, 125–128. (b) Slovin, S. F.; Ragupathi, G.;
Adluri, S.; Ungers, G.; Terry, K.; Kim, S.; Spassova, M.; Bornmann,
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Jeon et al.
Figure 1. Representative anticancer vaccine constructs.
Along these lines, we have taken note of the mucin-bound
carbohydrate antigens, Tn and STn, which are overexpressed
on the surfaces of a variety of epithelial cancers, such as prostate,
breast, colon, and ovarian.8 On the tumor cell surface, Tn and
STn are presented in broadly conserved “clusters” of 2-4
carbohydrate units, O-linked to the mucin peptide through serine
or threonine residues.
Approaches using monomeric Tn or STn antigen, in which
one glycan unit is covalently appended to an immunogenic
carrier protein, have proven beneficial.9 However, it has been
shown that10 clustered vaccinesswherein multiple copies of the
carbohydrate are incorporated on a peptide backbone (Figure
1)sinduce higher titers against some carbohydrate epitopes.10a
Indeed, recent studies with the antitumor monoclonal antibody
(mAb) B72.3 revealed that it preferentially recognized clustered
STn in comparison with monovalent STn.9a,10b,c
interactions. However, the choice of template for multivalent
carbohydrate display may be crucial. Excessively flexible
scaffolds will permit attached glycans to remain far apart in
most conformations, thereby perhaps decreasing effective
clustering of the antigen. In considering a template for the
presentation of the clustered carbohydrates, we have been
attracted to the model, depicted in Figure 2, upon which the
clustered glycans would be displayed in a well-defined orienta-
tion. Our design, clearly inspired by Dumy11 and Robinson,12
is amenable to variations in the number and type of carbohy-
drates displayed, as well as the spacing of the carbohydrate
domains. Moreover, the promise of such templates is evidenced
by recent studies demonstrating their use for clustered antigen
(9) (a) Adluri, S.; Helling, F.; Ogata, S.; Zhang, S.; Itzkowitz, S. H.; Lloyd,
K. O.; Livingston, P. O. Cancer Immunol. Immunother. 1995, 41, 185–
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B. M. Cancer Immunol. Immunother. 1993, 36, 215–222. (c) MacLean,
G. D.; Reddish, M. A.; Bowen-Yacyshyn, M. B.; Poppema, S.;
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G. D.; Miles, D. W.; Rubens, R. D.; Reddish, M. A.; Longenecker,
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M. A.; Koganty, R. R.; Longenecker, B. M. J. Immunother. 1996, 19,
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To some extent, the lowered entropic penalty associated with
the increase in valency seems to enhance carbohydrate-protein
(8) (a) Springer, G. F. Science 1984, 224, 1198–1206. (b) Itzkowitz, S. H.;
Yuan, M.; Montgomery, C. K.; Kjeldsen, T.; Takahashi, H. K.; Bigbee,
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Gum, J.; Brockhausen, I. Glyconjugate J. 1996, 13, 693–707. (d)
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Figure 2. Cyclic peptide scaffold 1 and antibody response to multivalent-KLH conjugate. TH Cell ) T helper cell.
Figure 3. Protected glycosylamino acids and clustered antigens.
syntheses in our lab and elsewhere.13,14 In fact, we have recently
employed this scaffold in the context of a separate program,
directed toward the development of an HIV vaccine.15
The essential element of our cyclic peptide design is a pair
of ꢀ-turn-inducing D-Pro-L-Pro sequences16 at both ends of the
macrocycle. Positions A-F (red, with side chain projecting
“above” the plane of the scaffold) may contain handles for
glycan attachment, whereas position G is a cysteine residue
(blue, with side chain projecting from the “bottom” face of the
scaffold), suitable for linkage to a carrier protein or biological
marker. This scaffold is tunable in that differential placement
of aspartate residues in positions A-F permits variation in the
number and spacing of the glycan attachments (1, Figure 2).
In an effort to identify an optimal presentation of the
carbohydrates on the cyclic peptide scaffold, we chose as
our targets the four constructs shown in Figure 3. Structures
(10) (a) Kudryashov, V.; Glunz, P. W.; Williams, L. J.; Hintermann, S.;
Danishefsky, S. J.; Lloyd, K. O. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 3264–3269. (b) Slovin, S. F.; Ragupathi, G.; Musselli, C.;
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21, 4292–4298. (c) Zhang, S.; Walberg, L. A.; Ogata, S.; Itzkowitz,
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Lloyd, K. O.; Livingston, P. O. Cancer Res. 1995, 55, 3364–3368.
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M. Tetrahedron Lett. 1995, 36, 1225–1258. (b) Dumy, P.; Renaudet,
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J.; Dumy, P. ChemBioChem 2006, 7, 1298–1314.
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HelV. Chim. Acta 2000, 83, 3097–3112. (b) Favre, M.; Moehle, K.;
Jiang, J.; Pfeiffer, B.; Robinson, J. A. J. Am. Chem. Soc. 1999, 121,
2679–2685, For a review, see. (c) Robinson, J. A. Synlett 2000, 429–
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V. Y.; Geng, X. J.; Warren, D.; Chastain, M.; Shiver, J. W.;
Danishefsky, S. J. J. Am. Chem. Soc. 2007, 129, 11042–11044. (b)
Joyce, J. G.; Krauss, I. J.; Song, H. C.; Opalka, D. W.; Grimm, K. M.;
Nahas, D. D.; Esser, M. T.; Hrin, R.; Feng, M.; Dudkin, V. Y.;
Chastain, M.; Shiver, J. W.; Danishefsky, S. J. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 15684–15689.
(13) Grigalevicius, S.; Chierici, S.; Renaudet, O.; Lo-Man, R.; Deriaud,
E.; Leclerc, C.; Dumy, P. Bioconjugate Chem. 2005, 16, 1149–1159.
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Scheme 1. Synthesis of Cyclic Peptide Scaffolds
4 and 5 incorporate six and four replicate copies of the Tn
antigen, respectively, and construct 6 presents four copies
of the STn disaccharide. We also sought to prepare a
multiantigenic construct, 7, incorporating both the Tn and
STn antigens.
Protected O-linked glycosylamino acids 2 and 3 (Figure
3) were prepared from the L-hydroxynorleucine benzyl ester,
according to our previously established protocol.17 These Tn
and STn “cassettes,” which we originally employed in earlier
approaches to the synthesis of clustered antigens, serve as
useful building blocks for glycal assembly. In this system,
the N-termini of both the Tn and STn cassettes serve as
handles for coupling to the peptide scaffold, and the
remaining carboxyl function may provide a handle for further
elaboration (i.e., as T-helper or additional B-epitope attach-
ments).
indication of the requisite hexavalent product, 14. Upon close
investigation, it was found that the HOAt, which is generally
used as an activation additive, along with HATU, plays a
significant role in promoting undesired aspartimide forma-
tion. Fortunately, activation of the aspartic acids with HATU
alone in the presence of DIEA in DMSO effectively min-
imized aspartimide formation, and consequently allowed high
yielding conversion to the desired hexavalent product, 14
(Scheme 2).
Having identified workable coupling conditions, we were
able to assemble the tetravalent constructs 15 and 16 in a
similar fashion. These less congested glycopeptides exhibit
different spatial arrangements, yet they still express the
epitopes in highly clustered fashions. Following global
deprotection, the highly clustered antigens 4, 5, and 6 were
in hand.
Cyclic peptides 9, 10, and 11 (containing 4 or 6 aspartate
residues) were prepared in parallel through automated solid-
phase synthesis from the prolinated trityl resin 8 (Scheme
1). Cleavage from the resin, macrocyclization,18 and depro-
tection of the tert-butyl esters of the aspartate and tyrosine
residues furnished 1, 12, and 13 in good overall yields.
With the components of the target structures in hand, we
directed our efforts toward the covalent attachment of the
carbohydrate antigens to the scaffolds. The Lansbury aspar-
tylation reaction19 is often employed in glycopeptide syn-
thesis. However, the standard Lansbury aspartylation protocol
employs a glycosidic amine as the coupling partner of
activated aspartic acid, rather than the primary amino acid
nitrogen, which would be required by our strategy. In
considering the application of this protocol to our own
system, there was concern that altered nucleophilicity of the
amine might favor the emergence of nonproductive pathways.
For example, the competing, relatively facile cyclization of
the peptide itself might lead to the formation of aspartimide.20
Indeed, this side reaction was found to be a problem in the
coupling of peptide 12 with glycosylamino acid 2. Under
standard conditions (HOAt, HATU, DIEA/DMSO), formation
of the undesired aspartimide was predominant, with little
We next turned to the synthesis of the unimolecular
multiantigenic construct, 7,21 wherein both the Tn and STn
carbohydrate antigens are displayed on the peptidic backbone.
This type of multivalent construct is intended to reflect the
degree of carbohydrate heterogeneity associated with most
cancers.22 There is significant variation in the types of
carbohydrates which are overexpressed on the tumor cell
surface, even within a particular cancer type. By combining
clusters of both the Tn and STn antigens within a single cyclic
peptide scaffold, we would hope to induce a more effective
immune response, in which the antibodies raised would target
a greater proportion of transformed tumor cells. It is not
unlikely that careful strategic considerations in the peptide
scaffold design could well influence the effectiveness of a
multiantigenic construct.
(20) (a) Bodanszky, M.; Natarajan, S. J. Org. Chem. 1975, 40, 2495–2499.
(b) Bodanszky, M.; Kwei, J. Z. Int. J. Pept. Protein Res. 1978, 12,
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1988, 1, 6–18. (d) Mergler, M.; Dick, F.; Sax, B.; Weiler, P.; Vorherr,
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Wilson, R. M.; Danishefsky, S. J. J. Am. Chem. Soc. 2006, 128, 2715–
2725. (b) Allen, J. R.; Harris, C. R.; Danishefsky, S. J. J. Am. Chem.
Soc. 2001, 123, 1890–1897. (c) Ragupathi, G.; Coltart, D. M.;
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(18) The macrocyclization was essentially instantaneous at room tempera-
ture, indicating that cross-strand hydrogen bonding may preorganize
the acyclic peptide for cyclization. Moreover, downfield 1H NMR
chemical shifts of selected NH resonances, slow D2O exchange times,
and the temperature profile of these chemical shifts provided prelimi-
nary evidence of the desired ꢀ-sheet character of the peptide scaffold.
(19) Cohen-Anisfeld, S. T.; Lansbury, P. T. J. Am. Chem. Soc. 1993, 115,
10531–10537.
(22) (a) Zhang, S. L.; Cordon-Cardo, C.; Zhang, H. S.; Reuter, V. E.; Adluri,
S.; Hamilton, W. B.; Lloyd, K. O.; Livingston, P. O. Int. J. Cancer
1997, 73, 42–49. (b) Zhang, S. L.; Zhang, H. S.; Cordon-Cardo, C.;
Reuter, V. E.; Singhal, A. K.; Lloyd, K. O.; Livingston, P. O. Int. J.
Cancer 1997, 73, 50–56.
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Scheme 2. Syntheses of Clustered Glycopeptides
Initial efforts to reach cyclic peptide 17 were unsuccessful,
due to complications arising from aspartimide formation at
position 8, leading to the undesired product, 17a (Scheme
3). Unlike the bulky Asp-ꢀ-tert-butyl esters, the less hindered
Asp-ꢀ-allyl ester is susceptible to undesired aspartimide
formation, arising from intramolecular nucleophilic attack
by the amide nitrogen at the aspartyl C-terminus.23 However,
aspartimide formation of the Asp-ꢀ-allyl ester at position 5
was impeded by the presence of proline in position 6. Thus,
we initiated a design decision to transpose the -ꢀ-allyl and
-ꢀ-tert-butyl protecting groups of positions 8 and 12.
Gratifyingly, no evidence of aspartimide formation was found
in the synthesis of cyclic peptide 18.
Following selective tert-butyl deprotection of 18 under the
conditions described in Scheme 1, intermediate 13 was in
hand. We then undertook the sequential attachment of the
Tn and STn carbohydrate antigens. Eventually, coupling
of Tn 2 with peptide 13 proceeded efficiently to produce the
Tn glycopeptide construct, as shown in Scheme 4. The
liberation of additional reaction sites by palladium-mediated
allyl deprotection24 resulted in a high yield of divalent
product 20. Addition of STn 3 to activated 20 and subsequent
global deacetylation completed the synthesis of tetravalent
antigen 7.
An intriguing question arose concerning the clustered
antigen design with regard to the potential flexibility of
linkers that attach the glycans to the cyclic peptide scaffold.
Cross-linking elements between carbohydrates would rigidify
the construct and prevent undesired spreading of glycan units.
This should better mimic a tightly clustered antigen and
enhance the cluster-recognizing antibody response. Ring-
closing metathesis (RCM) has proven to be a powerful tool
for the formation of peptide macrocycles in numerous
settings.25 In particular, fascinating work of Kiessling26 on
the synthesis of multivalent glycoconjugates has further
encouraged us to explore the ruthenium-catalyzed metathesis
for the incorporation of cross-linker in our system, which
would lead to highly clustered cross-linked constructs. Our
(25) (a) Blackwell, H. E.; Sadowsky, Jack., D.; Howard, R. J.; Sampson,
J. N.; Chao, J. A.; Steinmetz, W. E.; O’Leary, D. J.; Grubbs, R. H.
J. Org. Chem. 2001, 66, 5291–5302. (b) Miller, S. J.; Blackwell,
H. E.; Grubbs, R. H. J. Am. Chem. Soc. 1996, 118, 9606–9614. (c)
Blackwell, H. E.; Grubbs, R. H. Angew. Chem., Int. Ed. 1998, 37,
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Scheme 3. Cyclic Peptide Scaffold Design for Multi-Antigen Attachments
Scheme 4. Synthesis of Unimolecular Multiantigenic Glycopeptide 7
initial attempted RCM of di-Tn precursor 23 employing
Grubbs catalyst (24) proceeded to generate spacer-linked
construct 27 in modest yield (Scheme 5). Variation of the
reaction conditions, including higher catalyst loading, was
unable to induce complete conversion of 23. Additionally,
mass spectra of isolated RCM products indicated the exist-
ence of inseparable contaminants in which a methylene group
was deleted,27a presumably due to the ruthenium complex-
mediated isomerization27 of the terminal olefin of 23 (Figure
4, top) prior to ring closure. Although a significant increase
in yield was observed when the Hoveyda-Grubbs II catalyst
(25)28 was employed, terminal olefin isomerization could not
be suppressed. Gratifyingly, no isomerization of 23 occurred
with the use of Hoveyda-Grubbs I catalyst (26),29 which
afforded the desired RCM product 27 in good yield (Scheme
5 and Figure 4 (bottom)). The product olefins, a mixture of
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Figure 4. Low-resolution mass spectra of RCM products.
Scheme 5. Synthesis of Cross-Linked Glycopeptide 28
E- and Z-isomers, were then hydrogenated to furnish 28. The
protected aspartyl residues of 28 may serve as additional
handles for further modification of the construct; i.e. attach-
ment of multiple different antigens, to appropriately reflect
the heterogeneity of target cancers.
Conclusions
In summary, we have described the design and synthesis of
multivalent anticancer vaccine constructs. The individual car-
bohydrate-based antigens were prepared in well-defined and
highly clustered modes by convergent synthesis. In addition,
the successful introduction of a cross-linker provides a wider
scope of future modifications in epitope design either through
spacer length variations or additional glycan attachments on the
scaffolds. Upon conjugation to KLH carrier protein, the
anticipated higher molecular ratio of Tn or STn versus KLH
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could offer an additional benefit in terms of immunogenicity.
We expect that these highly clustered anticancer constructs will
help in gaining further insight into the way in which carbohy-
drate clusters are recognized by the immune system. Conjugation
to KLH carrier protein and immunological investigations will
be reported in due course.
Acknowledgment. This research was supported by a grant from
the National Institutes of Health (CA28824 to S.J.D.). D.L. is
grateful for support from the Terri Brodeur Breast Cancer Founda-
tion. Special thanks go to Rebecca Wilson for editorial consultation
and to Dana Ryan for assistance with the preparation of the
manuscript.
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Supporting Information Available: Experimental procedures
and characterization data. This material is available free of
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