Synthesis and immunological evaluation ofN-acyl modified Globo H derivatives as anticancer vaccine candidates

Article abstract of DOI:10.1039/d1md00067e

Globo H is a tumor-associated carbohydrate antigen (TACA), which serves as a valuable target for antitumor vaccine or cancer immunotherapies. However, most TACAs are T-cell-independent, and they cannot induce powerful immune response due to their poor imm

Full text of DOI:10.1039/d1md00067e

RSC
Medicinal Chemistry
View Article Online
View Journal | View Issue
RESEARCH ARTICLE
Synthesis and immunological evaluation of N-acyl
modified Globo H derivatives as anticancer
vaccine candidates†
Cite this: RSC Med. Chem., 2021, 12,
1239
*
Canjia Zhai, ‡ Xiu-Jing Zheng,‡ Chengcheng Song and Xin-Shan Ye
Globo H is a tumor-associated carbohydrate antigen (TACA), which serves as a valuable target for
antitumor vaccine or cancer immunotherapies. However, most TACAs are T-cell-independent, and they
cannot induce powerful immune response due to their poor immunogenicity. To address this problem,
herein, several Globo H analogues with modification on the N-acyl group were prepared through a
preactivation-based glycosylation strategy from the non-reducing end to the reducing end. These modified
Globo H derivatives were then conjugated with carrier protein CRM197 to form glycoconjugates as
anticancer vaccine candidates, which were used in combination with adjuvant glycolipid C34 for
immunological studies. The immunological effects of these synthetic vaccine candidates were evaluated
on Balb/c mice. The results showed that the fluorine-modified N-acyl Globo H conjugates can induce
higher titers of IgG antibodies that can recognize the naturally occurring Globo H antigen on the surface
of cancer cells and can eliminate cancer cells in the presence of a complement, indicating the potential of
these synthetic glycoconjugates as anticancer vaccine candidates.
Received 2nd March 2021,
Accepted 17th May 2021
DOI: 10.1039/d1md00067e
rsc.li/medchem
α-L-Fuc-(1,2)-β-D-Gal
(1,3)-β-D-GalNAc-(1,3)-α-D-Gal-(1,4)-β-D-Glc-
Introduction
(1,4)-β-Cer, where the fucose at the non-reducing end and the
β-linkage at the CD junction play important roles in the
recognition with its antibody.12,14 Globo H antigen has been
conjugated with a protein carrier,15–17 monophosphoryl lipid A
(MPLA),18 or polysaccharide A1 (PS A1)19 for the development of
anticancer vaccines. Even though the Globo H-KLH vaccine has
been approved for clinical trials,20,21 there are still limitations in
their clinical application since this vaccine usually elicited higher
titers of IgM antibody than those of IgG antibodies, implying
that the T-cell-mediated immune response is weak, which is a
major challenge in carbohydrate-based vaccine design.22
Cancer cells can often express some unique carbohydrate
sequences on their surfaces because of the aberrant
glycosyltransferase activity within these cells. These unusual
glycans are known as tumor-associated carbohydrate antigens
(TACAs).1,2 TACAs are important targets for the development of
antitumor vaccines or cancer immunotherapies. Unfortunately,
most TACAs are T-cell-independent antigens, and they are also
expressed by normal tissues at a low level, and this leads to
their poor immunogenicity.3 Strategies to enhance the
immunogenicity of TACAs include conjugating TACAs with a
suitable immunogenic carrier such as proteins,4,5 modification
of TACAs through metabolic oligosaccharide engineering
(MOE) which focuses on the modification of sialic acid,6,7 and
introducing unnatural TACA analogues such as modification of
carbohydrate antigen structures.8–10
Globo H is a member of Globo series TACAs. It was first
separated and characterized from human breast cancer cell line
MCF-7 in 1983,11,12 and was later found to be overexpressed in
many kinds of cancer cells in the form of glycosphingolipid.13-
Fig. 1a shows the natural Globo H antigen with the structure of
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Xue Yuan Rd No. 38, Beijing 100191, China.
E-mail: xinshan@bjmu.edu.cn
† Electronic supplementary information (ESI) available: Compound
characterization and supplementary data. See DOI: 10.1039/d1md00067e
‡ These authors contributed equally to this work.
Fig. 1 (a) Natural Globo H antigen; (b) modified Globo H derivatives in
this work.
This journal is © The Royal Society of Chemistry 2021
RSC Med. Chem., 2021, 12, 1239–1243 | 1239

View Article Online
Research Article
RSC Medicinal Chemistry
Because of the similar atom radius and lipophilicity of
fluorine compared with those of a hydrogen atom, and the
absence of fluorinated compounds in most organisms,
fluorinated modifications of TACAs have been applied as a
way to improve the immunogenicity in the development of
anticancer vaccines.23,24 More importantly, fluorination of
antigens has been shown to be a strategy to enhance the
enzymatic stability of synthetic vaccines.24 Previously, our
group conducted a series study of N-acyl modified STn
antigens and discovered that fluorinated modifications on
the STn can enhance the anti-STn IgG titers.25 Based on this
previous work, we want to design and synthesize N-acyl
modified Globo H derivatives (Fig. 1b) with the aim to
improve its immune efficacy. Herein, we describe the design,
synthesis, and immunological evaluation of these Globo H
analogue glycoconjugates.
Scheme
2
Synthesis of lactose building block 14. Reagents and
conditions: a) PhCH(OMe)2, CSA, DMF, 85%; b) BnBr, NaH, TBAI, DMF,
0 °C to r.t., 73%; c) NaBH3CN, HCl, THF, 0 °C to r.t, 82%.
4,6-hydroxyls on the galactose moiety, followed by the global
benzyl protection to get compound 13, which was further
converted to building block 14 as the last component in the
assembly of the Globo H hexasaccharide (Scheme 2).
The preparation of the globally protected Globo
H
Results and discussion
hexasaccharide is shown in Scheme 3. Starting from the non-
reducing end, first, the preactivation of fucose building block 1
at −72 °C with Ph2SO/Tf2O in dichloromethane (DCM), which
was followed by the addition of disaccharide acceptor 7,
Because of the biological importance of the Globo H antigen
in the development of anticancer vaccines, the preparation of
the Globo H carbohydrate antigen has attracted considerable
attention.
Different
and
strategies
chemoenzymatic
including
or
chemical
enzymatic
generated trisaccharide
8
in 82% isolated yield. The
synthesis,26–31
construction of tetrasaccharide 10 from trisaccharide 8 and
galactose building block 9 was conducted in the same way by
preactivation of 8 followed by the addition of 9. The last step
was the α-glycosylation of compound 10 and lactose building
block 14. The reaction was carried out, providing a coupled
product, but the ratio of the desired product/byproduct (15α/
15β) was only 1.2 when DCM was used as the solvent. To
improve the yield of the α linkage product, when the solvent
was switched to ether, the ratio of 15α/15β was increased to 3.7.
method17,32,33 have been developed for the construction of
Globo H hexasaccharides. The strategy we used in this work
mainly refers to the method reported by Huang's group31 with
some modifications, where the Globo H hexasaccharide was
retro-synthetically divided into four components: AB, C, DE,
and F. The synthesis was carried out from the non-reducing
end F to the reducing end AB through a preactivation-based
strategy.34 Monosaccharide building blocks 1, 2, 3 and 9 were
prepared according to the literature.31 The synthesis of DE
disaccharide building block 7 is shown in Scheme 1. Glycosyl
donor 2 was preactivated with Ph2SO/Tf2O followed by the
addition of glycosyl acceptor 3 to afford disaccharide 4, which
was subsequently converted to disaccharide building block 7
following a reported procedure.31
For the synthesis of lactose building block 14 (AB),
compound 11 (ref. 35) bearing a five-carbon amino linker was
reacted with benzaldehyde dimethyl acetal to protect the
Scheme 1 Synthesis of building block 7. Reagents and conditions: a)
Ph2SO, Tf2O, TTBP, DCM, preactivation of compound 2 at −72 °C for
15 min, then addition of compound 3, 85%; b) NaOMe, DCM/MeOH,
82%; c) 1,3-propanedithiol, Et3N, DCM/MeOH, 86%; d) TrocCl,
NaHCO3, THF, 64%.
Scheme 3 Synthesis of protected Globo H saccharide.
1240 | RSC Med. Chem., 2021, 12, 1239–1243
This journal is © The Royal Society of Chemistry 2021

View Article Online
RSC Medicinal Chemistry
Research Article
Scheme 4 Synthesis of Globo H derivatives. Reagents and conditions:
a) 1 M NaOH, THF, 50 °C, overnight; b) anhydride of corresponding
carboxylic acid, pyridine, DMAP; for compound G1, CH2FCOCl,
pyridine, DMAP; c) H2, Pd(OH)2/C, THF/HOAc/H2O, 64% for compound
G0, 47% for compound G1, 61% for compound G2, 66% for compound
G3, over three steps.
Scheme 5 Synthesis of Globo H-derived glycoconjugates.
resulting product was then purified through dialysis in PBS
buffer to afford the corresponding glycoconjugate. The
carbohydrate loading percentage was determined using the
MALDI-TOF MS spectra (Table S1, ESI†) and the protein
content was determined by the BCA assay.39
To synthesize the N-acyl modified derivatives, the
trichloroethoxycarbonyl (Troc) group was deprotected to get
the free amino functionality, which was further reacted with
the corresponding acetic anhydride or fluorinated acetyl
anhydride
(fluorinated
acetyl
chloride
for
the
The immunogenicity
of
these
Globo
H-derived
monofluorinated derivative). Finally, global deprotection was
conducted under catalytic hydrogenolysis conditions (H2/
Pd(OH)2) to generate the target molecules G0–G3 (Scheme 4),
which were purified by C18 reversed-phase column
chromatography (H2O → H2O/MeOH, 8/1, v/v) to give the
corresponding deprotected product. The final products were
characterized by NMR (1H, 13C, HMBC) spectroscopy and
glycoconjugates was evaluated on Balb/c mice. Groups of six
female mice were vaccinated intramuscularly four times at
biweekly intervals with Globo H analogue-CRM197 (2 μg of
Globo H analogue) and glycolipid adjuvant C34 (2 μg). The
capabilities of the induced antibodies to recognize the native
Globo H antigen and the modified Globo H antigens were
examined by measuring the antibody titers of the pooled sera of
each group or the serum antibody of each mouse using plates
coated with the unmodified G0-BSA or the modified
carbohydrate antigen-BSA glycoconjugates, respectively. The IgG
antibody titers against the native Globo H antigen after the 3rd
and 4th vaccinations are shown in Fig. 2. Compared with the
antibody level of the unmodified glycoconjugate (G0-CRM197),
the modified glycoconjugates showed enhanced antibody titers;
in particular for the glycoconjugates G2-CRM197 and G3-
CRM197, clear enhancement of antibody titers was observed.
Also, the modified Globo H antigen-CRM197 conjugates elicited
enhanced antibody titers against their corresponding modified
Globo H antigen compared with that of the unmodified
glycoconjugate (G0-CRM197) (Fig. S1, ESI†). Furthermore, the
subclasses of IgG antibodies were identified, in which the
reactivity of pooled sera IgG subclasses with G0-BSA was tested
by ELISA. The results indicated that the IgG1 antibody was the
predominant subtype (Table S2, ESI†), implying that T cell
response was induced. The generation of IgG1 antibodies is the
evidence of a type 2 T-helper (Th2) response.40,41
1
HRMS analyses (see the ESI†). The H NMR data of the native
compound G0 coincide with those reported in the
literature.17 These globally deprotected products were further
used to conjugate with protein to prepare glycoconjugates as
vaccine candidates.
Carrier protein diphtheria toxoid cross-reactive material
(CRM) 197 is a US FDA approved vaccine carrier protein.
Compared with the Globo H-KLH+QS21 vaccine used in
clinical phase III, the Globo H-CRM197+C34 vaccine has been
shown to induce higher titers of IgG antibodies, which has
been approved for clinical studies.16,36 Thus, we chose
CRM197 as a carrier protein and glycolipid C34 as an
adjuvant. With Globo H and its N-acyl modified derivatives in
hand, the conjugation with protein was performed to prepare
glycoconjugates. The glycoconjugates were prepared
according to a reported procedure.37 As shown in Scheme 5,
the Globo H analogues (G0–G3) were treated with an excess
amount of linker 16 (ref. 38) to generate the corresponding
ester intermediates. The reactions were monitored by TLC.
These ester intermediates were subsequently incubated with
proteins (CRM197 or BSA) in PBS buffer (pH 7.6). The
To investigate the capabilities of mouse sera induced by
G0-CRM197, G1-CRM197, G2-CRM197 and G3-CRM197 to
This journal is © The Royal Society of Chemistry 2021
RSC Med. Chem., 2021, 12, 1239–1243 | 1241

View Article Online
Research Article
RSC Medicinal Chemistry
Fig. 2 Serum antibody titers against G0-BSA of individual mouse
vaccinated with G0-CRM197, G1-CRM197, G2-CRM197 and G3-
CRM197 after the (A) 3rd and (B) 4th vaccinations. Each data point
represents the average titers of two detections of an individual mouse,
and the black line in each series represents the median serum titer of
six mice in each group. The data of IgG titers were plotted in the form
of log 10.
Fig. 4 Antibodies elicited by glycoconjugates mediate complement-
dependent cytotoxicity (CDC) to eliminate Globo H-positive MCF-7
cancer cells. Cell lysis of pooled sera (1 : 10 dilution) after the 4th
recognize the native Globo H antigen on cancer cells, the
binding affinity of the immune serum antibodies for Globo
H-positive MCF-7 human breast cancer cells was analyzed by
flow cytometry. As shown in Fig. 3, the antisera elicited by
the unmodified G0-CRM197 or the modified conjugates (G1-
CRM197, G2-CRM197, G3-CRM197) can react well with the
Globo H-positive MCF-7 cancer cells and the antisera induced
by G2-CRM197 and G3-CRM197 bind better to the cells than
those by G0-CRM197, which is consistent with the trend of
the serum titer, indicating that the induced antibodies are
specific to the Globo H antigen on the cell surface.
vaccination. Data are the mean
SEM of three independent
experiments. Cytotoxicity is shown with percent cell lysis determined
by the lactate dehydrogenase (LDH) assay. Three parallel holes were
arranged for each measurement. One-way ANOVA analysis was
performed. Asterisks represent p < 0.05.
Overall, our results showed that fluorination of the Globo
H antigen can enhance its immunogenicity. This may be
attributed to the exogenetic properties of the fluorinated
compounds and the increased enzymatic stability in vivo.23
Complement-dependent cytotoxicity (CDC) was then
investigated to determine whether the modified Globo H-
CRM197-provoked immune response is useful for cancer Conclusions
immunotherapy. As shown in Fig. 4, the antisera obtained
In conclusion, different N-acyl modified Globo H derivatives
from vaccination with G2-CRM97 and G3-CRM197 can induce
much stronger cancer cell cytotoxicity compared with those
of the untreated mice (pre-immune mice), G0-CRM97 and
G1-CRM197 immunized mice.
and their carrier protein conjugates were designed and
synthesized. The immunological properties of these
glycoconjugates were evaluated using Balb/c mice. According
to the results, among the three N-fluoroacetyl modified
analogues, the difluorinated glycoconjugate G2-CRM197 and
trifluorinated glycoconjugate G3-CRM197 induced obviously
enhanced titers of IgG antibodies compared with the
unmodified Globo H conjugate G0-CRM197, thus enhancing
the immunogenicity. This study demonstrates that the N-acyl
modification of Globo H holds potential for the development
of anticancer vaccines.
Ethical statement
All experiments were carried out according to the International
Association for the Study of Pain ethical guidelines
(Zimmermann, 1983) and approved by the Institutional Animal
Care and Use Committee of Peking University.
Author contributions
C. Z. conducted all the synthesis and compound
characterization. X. Z. conducted all biological work. C. S.
Fig. 3 Serological IgG analysis results of MCF-7 human breast cancer
cells after the 4th vaccination by flow cytometry.
1242 | RSC Med. Chem., 2021, 12, 1239–1243
This journal is © The Royal Society of Chemistry 2021

View Article Online
RSC Medicinal Chemistry
Research Article
helped with some biological work. X. Y. designed and
supervised the project.
20 T. Gilewski, G. Ragupathi, S. Bhuta, L. J. Williams, C. Musselli,
X. F. Zhang, K. P. Bencsath, K. S. Panageas, J. Chin, C. A.
Hudis, L. Norton, A. N. Houghton, P. O. Livingston and S. J.
Danishefsky, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 3270–3275.
21 C.-S. Huang, A. L. Yu, L.-M. Tseng, L. W. C. Chow, M.-F.
Hou, S. A. Hurvitz, R. B. Schwab, J. L. Murray, H.-K. Chang,
H.-T. Chang, S.-C. Chen, S.-B. Kim, J.-T. Hung, S.-H. Ueng,
S.-H. Lee, C.-C. Chen and H. S. Rugo, J. Immunother. Cancer,
2020, 8, e000342.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 21738001 and
81821004) and the National Key Research and Development
Program of China (Grant No. 2018YFA0507602).
22 F. Khatun, I. Toth and R. J. Stephenson, Adv. Drug Delivery
Rev., 2020, 165–166, 117–126.
23 A. Hoffmann-Röder, A. Kaiser, S. Wagner, N. Gaidzik, D.
Kowalczyk, U. Westerlind, B. Gerlitzki, E. Schmitt and H.
Kunz, Angew. Chem., Int. Ed., 2010, 49, 8498–8503.
24 J. Jiménez-Barbero, B. Linclau, A. Ardá, N. C. Reichardt, M.
Sollogoub, L. Unione and S. P. Vincent, Chem. Soc. Rev.,
2020, 49, 3863–3888.
Notes and references
1 S. I. Hakomori and Y. Zhang, Chem. Biol., 1997, 4, 97–104.
2 A. B. T-cell, S. J. Danishefsky and J. R. Allen, Angew. Chem.,
Int. Ed., 2000, 39, 836–863.
3 R. Mettu, C.-Y. Chen and C.-Y. Wu, J. Biomed. Sci., 2020, 27,
9.
4 J. A. Speir, U. M. Abdel-Motal, M. Jondal and I. A. Wilson,
Immunity, 1999, 10, 51–61.
5 C. Brocke and H. Kunz, Bioorg. Med. Chem., 2002, 10,
3085–3112.
6 Y. Pan, P. Chefalo, N. Nagy, C. Harding and Z. Guo, J. Med.
Chem., 2005, 48, 875–883.
7 J. Wu and Z. Guo, Bioconjugate Chem., 2006, 17, 1537–1544.
8 Z. Yin and X. Huang, J. Carbohydr. Chem., 2012, 31, 143–186.
9 H. J. Jennings, R. Roy and A. Gamian, J. Immunol., 1986, 137,
1708–1713.
10 C. C. Liu and X. S. Ye, Glycoconjugate J., 2012, 29, 259–271.
11 R. Kannagis, S. B. Leverys, F. Ishigamis, S. Hakomorisj, H.
Lynne, B. B. Knowlesll and D. Solterb, J. Biol. Chem.,
1983, 8934–8942.
12 E. G. Bremer, S. B. Levery, S. Sonnino, R. Ghidoni, S.
Canevari, R. Kannagi and S. Hakomori, J. Biol. Chem.,
1984, 259, 14773–14777.
13 P. O. Livingston, Semin. Cancer Biol., 1995, 6, 357–366.
14 I. J. Kim, T. K. Park, S. Hu, K. Abrampah, S. Zhang, P. O.
Livingston and S. J. Danishefsky, J. Org. Chem., 1995, 60,
7716–7717.
15 G. Ragupathi, T. K. Park, S. Zhang, I. J. Kim, L. Graber, S.
Adluri, K. O. Lloyd, S. J. Danishefsky and P. O. Livingston,
Angew. Chem., Int. Ed. Engl., 1997, 36, 125–128.
16 Y. L. Huang, J. T. Hung, S. K. C. Cheung, H. Y. Lee, K. C.
Chu, S. T. Li, Y. C. Lin, C. T. Ren, T. J. R. Cheng, T. L. Hsu,
A. L. Yu, C. Y. Wu and C. H. Wong, Proc. Natl. Acad. Sci. U. S.
A., 2013, 110, 2517–2522.
25 F. Yang, X. J. Zheng, C. X. Huo, Y. Wang, Y. Zhang and X. S.
Ye, ACS Chem. Biol., 2011, 6, 252–259.
26 M. T. Bilodeau, T. K. Park, S. Hu, J. T. Randolph, S. J.
Danishefsky, P. O. Livingston, S. Zhang and S. J.
Danishefsky, J. Am. Chem. Soc., 1995, 117, 7840–7841.
27 T. K. Park, I. J. Kim, S. Hu, M. T. Bilodeau, J. T. Randolph, O.
Kwon and S. J. Danishefsky, J. Am. Chem. Soc., 1996, 118,
11488–11500.
28 J. M. Lassaletta and R. R. Schmidt, Liebigs Ann., 1996, 1996,
1417–1423.
29 F. Burkhart, Z. Zhang, S. Wacowich-Sgarbi and C. H. Wong,
Angew. Chem., Int. Ed., 2001, 40, 1274–1277.
30 F. Bosse, L. A. Marcaurelle and P. H. Seeberger, J. Org.
Chem., 2002, 67, 6659–6670.
31 Z. Wang, L. Zhou, K. El-Boubbou, X. S. Ye and X. Huang,
J. Org. Chem., 2007, 72, 6409–6420.
32 D. M. Su, H. Eguchi, W. Yi, L. Li, P. G. Wang and C. Xia, Org.
Lett., 2008, 10, 1009–1012.
33 Z. Wang, M. Gilbert, H. Eguchi, H. Yu, J. Cheng, S. Muthana,
L. Zhou, P. G. Wang, X. Chen and X. Huang, Adv. Synth.
Catal., 2008, 350, 1717–1728.
34 X. Qin and X. S. Ye, Chin. J. Chem., 2021, 39, 531–542.
35 G. Biswas, O. Y. Jeon, W. S. Lee, D. C. Kim, K. T. Kim, S. Lee,
S. Chang and S. K. Chung, Chem. – Eur. J., 2008, 14,
9161–9168.
36 S. J. Danishefsky, Y. K. Shue, M. N. Chang and C. H. Wong,
Acc. Chem. Res., 2015, 48, 643–652.
37 C. X. Huo, X. J. Zheng, A. Xiao, C. C. Liu, S. Sun, Z. Lv and
X. S. Ye, Org. Biomol. Chem., 2015, 13, 3677–3690.
38 K. Guo, C. C. Chu, E. Chkhaidze and R. Katsarava, J. Polym.
Sci., Part A: Polym. Chem., 2005, 43, 1463–1477.
17 H. Y. Lee, C. Y. Chen, T. I. Tsai, S. T. Li, K. H. Lin, Y. Y.
Cheng, C. T. Ren, T. J. R. Cheng, C. Y. Wu and C. H. Wong,
J. Am. Chem. Soc., 2014, 136, 16844–16853.
18 Z. Zhou, G. Liao, S. S. Mandal, S. Suryawanshi and Z. Guo,
Chem. Sci., 2015, 6, 7112–7121.
39 P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H.
Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke,
B. J. Olson and D. C. Klenk, Anal. Biochem., 1985, 150, 76–85.
40 F. D. Finkelman, J. Holmes and W. E. Paul, Annu. Rev.
Immunol., 1990, 8, 303–333.
19 S. Ghosh, K. R. Trabbic, M. Shi, S. Nishat, P. Eradi, K. A.
Kleski and P. R. Andreana, Chem. Sci., 2020, 11,
13052–13059.
41 M. Skwarczynski, M. Zaman, C. N. Urbani, I. C. Lin, Z. Jia,
M. R. Batzloff, M. F. Good, M. J. Monteiro and I. Toth,
Angew. Chem., Int. Ed., 2010, 49, 5742–5745.
This journal is © The Royal Society of Chemistry 2021
RSC Med. Chem., 2021, 12, 1239–1243 | 1243