Syntheses and Immunological Evaluation of Self-Adjuvanting Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with a T-helper Cell Epitope as Antitumor Vaccine Candidates

Article abstract of DOI:10.1002/anie.201804437

Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen (TACA) rarely observed on healthy tissues. We synthesized two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn) vaccines possessing a T-helper epitope and a TLR2 agonist, since the clustered STn antigens are highly expressed on many cancer cells. Immunization of both vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine were less specific. These results illustrated that N-propionyl triSTn is a valuable unnatural TACA for anticancer vaccines.

Full text of DOI:10.1002/anie.201804437

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Accepted Article
Title: Syntheses and Immunological Evaluation of Self-Adjuvanting
Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with A T-
helper Cell Epitope as Antitumor Vaccine Candidates
Authors: Tsung-Che Chang, Yoshiyuki Manabe, Yukari Fujimoto,
Shino Ohshima, Yoshie Kametani, Kazuya Kabayama, Yuka
Nimura, Chun-Cheng Lin, and Koichi Fukase
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804437
Angew. Chem. 10.1002/ange.201804437
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Syntheses and Immunological Evaluation of Self-Adjuvanting
Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with A T-
helper Cell Epitope as Antitumor Vaccine Candidates
Tsung-Che Chang,a Yoshiyuki Manabe,a,b Yukari Fujimoto,c Shino Ohshima,d Yoshie Kametani,d
Kazuya Kabayama,a,b Yuka Nimura,a Chun-Cheng Lin,e Koichi Fukasea,b,*
Abstract: Sialyl-Tn (STn) is
a
tumor-associated carbohydrate
progression did not reach statistical significance.[8] Theratope
induced the high levels of anti-KLH and anti-STn IgGs but low
level of anti-mucin IgGs in patients (anti-KLH/anti-STn/anti-
ovine submaxillary mucin (OSM)=64:64:1), suggesting many
anti-STn IgGs could not recognize mucins and therefore cancer
cells.
antigen (TACA) rarely observed on healthy tissues. We synthesized
two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn)
vaccines possessing a T-helper epitope and a TLR2 agonist, since
the clustered STn antigens are highly expressed on many cancer
cells. Immunization of both vaccines in mice induced the anti-triSTn
IgG antibodies, which recognized triSTn-expressing cell lines PANC-
1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-
specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine
were less specific. These results illustrated that N-propionyl triSTn is
a valuable unnatural TACA for anticancer vaccines.
The use of the clustered STn should be necessary to provoke
the efficient anti-tumor response, since STn is expressed as
clusters on mucins. Here, we focused the trimeric STn clusters
observed in the mucins of most adenocarcinomas.[9] The
monoclonal antibodies B72.3 and MLS102, which primarily
recognize STn clusters, displayed a high specificity to tumor
cells against normal cells.[9a,b] The STn trimer (triSTn) is,
therefore, a promising target for recognizing epithelial cancers.
Self-adjuvanting strategy[10] offers an attractive approach to
circumvent the anti-carrier immune response by using a built-in
adjuvant without using carrier proteins. Toll-like receptor (TLR) 2
agonists, lipopeptides, have been employed in many self-
adjuvanting vaccines.[11] Boons et al. revealed that a tumor-
associated MUC1 glycopeptide vaccine composed of TLR1/2
ligand (Pam3CSK4) and a T-helper cell epitope induced high
titers of IgGs and Pam3CSK4 was more potent than TLR2/6
ligand (Pam2CSK4) as an adjuvant.[11a] In addition, TLR2
agonists proved to induce anti-tumor immune responses.[12] We
therefore used Pam3CSK4 as an adjuvant for the construction of
STn-based vaccines to overcome the poor immunogenicity of
STn.
Modifying the C5 N-substituent of the sialic acid in the sialic
acid-containing antigens has been shown to impact their
immunogenicity.[13] In those approaches, unfortunately, some of
the antibodies produced only recognized the modified antigens,
not the parent antigens. Whereas, N-propionyl STn
glycoconjugate vaccine induced the production of antibody
which can recognize natural STn epitope and was more
immunogenic.[13b-f] Here, we tested the use of unnatural N-
propionyl triSTn as a TACA antigen to construct a vaccine,
Tumor cells are distinguished from normal cells by the display of
aberrant cell surface levels and types of carbohydrate domains,
called tumor-associated carbohydrates antigens (TACAs).[1]
Because TACAs are prevalent on cancer cell surfaces, they
represent a promising class of active immunotherapy epitopes.[2]
However, these TACAs are T-cell independent antigens to
exhibit poor immunogenicity and difficult to induce
immunoglobulin (Ig) class switching. A typical strategy used to
address the disadvantages of TACAs involves conjugating them
to a strong immunogenic carrier protein, such as keyhole limpet
hemocyanin (KLH).[3] Conversely, carrier proteins might
suppress an antibody response against the TACA epitopes.[4]
The sialyl-Tn (STn, NeuAc(2,6)GalNAc-O-Ser/Thr) antigen is
a TACA richly expressed on mucins in a number of epithelium-
derived tumors (e.g., breast, pancreas, prostate, gastric and
ovarian) and is rarely observed on adult healthy tissues.[5,6] STn
correlates with invasion and aggression potential.[5] STn has
been used to develop anticancer vaccines such as STn-KLH
conjugate, Theratope, for treating metastatic breast cancer.[7]
However, Theratope was failed in a phase 3 clinical trial;
extensions in the overall survival and time to disease
[a]
Dr. T.-C. Chang, Dr. Y. Manabe, Dr. K. Kabayama, Ms. Y. Nimura,
Prof. Dr. K. Fukase
expected to generate
immunogenic response.
a
more effective triSTn-specific
Department of Chemistry, Graduate School of Science, Osaka
University.
1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
Koichi@chem.sci.osaka-u.ac.jp
We designed the anti-triSTn vaccines 1 and 2, which are
composed of the N-acetyl or N-propionyl triSTn antigens in the
middle, the T-helper cell peptide (TT830-843 from the tetanus
[b]
[c]
Dr. Y. Manabe, Dr. Kabayama, Prof. Dr. K. Fukase
Core for Medicine and Science Collaborative Research and
Education, Project Research Center for Fundamental Science,
Osaka University.
1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
Prof. Dr. Y. Fujimoto
[15]
toxoid, QYIKANSKFIGITE),[14] and Pam3CSK4 (Figure 1). The
universal human and mouse T-helper cell epitope was
indispensable for the production of IgG antibodies via the
adaptive immune response. In comparison to the construction of
other self-adjuvanting glycopeptide vaccines through the linear
solid phase synthesis,[11] the advantages of our design were
facilitation of assemblies of the triSTn antigens with the T-cell
epitope and the adjuvant via convergent synthesis. Copper click
azide-alkyne cycloaddition (CuAAC) and thioether ligation were
employed to build the vaccine 1 and 2, since the resulting
Department of Chemistry, Faculty of Science and Technology, Keio
University.
3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
Ms. S. Ohshima, Prof. Dr. Kametani
Faculty of Medicine, School of Medicine, Tokai University.
143 Shimokasuya, Isehara-shi, Kanagawa 259-1193, Japan
Prof. Dr. C.-C. Lin
[d]
[e]
Department of Chemistry, National Tsing Hua University.
101 Sec. 2, Kuang Fu Rd., Hsinchu 30013, Taiwan
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10.1002/anie.201804437
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Figure 1. The fully synthetic vaccines 1 and 2.
to obtain the N-TFA STn monomers in good yields with
reasonable stereoselectivity (/= 4/1). Here, glycosylation of 4
with C5-azido sialyl donor 3 by using ICl and In(OTf)3 as a
promoter afforded the C5-azido STn monomer 5 in good yield
with high -selectivity (/= 11/1) (Scheme 1).[17] The triSTn
antigens 6 and 7 were readily prepared from compound 5 in
good yields (6: 33% and 7: 22% in 13 steps) via modifications
of functional groups, introduction of linkers, and an iterative
peptide coupling process (Scheme S1). Conjugation between
6/7 and T cell peptide 8 by click reaction was then investigated
(Table S1). Compounds 6/7 smoothly reacted with 8 by using
appropriate amounts of Cu(SO4)2 and sodium ascorbate in 80%
DMSO(aq) (oxygen-free) to give 9 and 10 in moderate yields,
respectively. It should be noted that use of the non-degassed
solvents in the CuAAC reaction led to the significant production
of an unidentified by-product (Figure S9). Since the removal of
Fmoc group of 9 with 20% piperidine/DMF gave a considerable
amount of by-products, 9/10 were treated with 50 mM LiOH(aq)
to afford the mixture of the free thiol and the disulfide-linked
dimer, which was treated with tris(2-carboxyethyl)phosphine
(TCEP) to afford the free thiol 11/12 in good yields, respectively.
Coupling of thiol 11/12 with 13 was then carried out according
to Kunz and Li (Table S2).[11d] The use of excess amount of
nucleophile 11/12 compared to electrophile 13 in DMSO
(oxygen-free) in the presence of Et3N was critical to obtain the
coupling products. The vaccines 1 and 2 were thus obtained in
approximately 30% yields. Although significant amount of the
disulfide-linked dimers was formed, they were readily converted
to 11/12 by TCEP treatment.
Scheme 1. Synthesis of the vaccines 1 and 2. (a) ICl, In(OTf)3, EtCN, MS4A,
-78 oC, 3 h, 94% (based on recovery of 4), / = 11/1; (b) see the Supporting
Information; (c) 8, CuSO4, sodium ascorbate, 80% DMSO(aq), rt, 5 h, and then
DOTA, rt, 40 min; 67% for 9, 68% for 10; (d) i) 50 mM LiOH, H2O, rt, 30 min;
ii) 90 mM HOAc/H2O; iii) 20 mM TCEP, H2O, rt, 30 min, 84% for 11, 72% for
12; (e) 13, Et3N, DMSO, 40 oC, 15 h, 30% for 1, 31% for 2.
To examine the necessity of covalent attachment among the
three components (trisTn or N-propionyl-triSTn, T-helper cell
epitope, and adjuvant) in vaccines 1 and 2, compounds 14-16
were synthesized for control experiments (Figure 2A). TriSTn,
N-propionyl-triSTn, STn, and linker were conjugated to bovine
serum albumin (BSA) through a squarate linker[18] to afford
BSA-triSTn, -N-propionyl-triSTn, -STn, and -linker for ELISA
assays (Figure 2B). (The synthesis of 14-16 and the
preparation of the BSA glycoconjugates are described in the
Supporting Information).
triazole[11e] and thioether[16] moieties have the low
immunogenicity.
A simple and reliable method to construct the N-acetyl and N-
propionyl STn monomer is of primary importance in the
preparation of the triSTn antigens. Previously, Guo et al.[13c] and
Ye et al.[13f] employed the N-trifluoroacetyl (N-TFA) sialyl donor
Vaccines 1, 2, mixture (8+14+15), and mixture (8+14+16) in
phosphate-buffered saline (PBS) were intraperitoneally
administrated to wild-type BALB/c mice on day 1, 14, 28, and
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42. The first immunizations were performed using complete
Freund’s adjuvant (CFA), the others with incomplete Freund’s
adjuvant (IFA). Blood was collected from each mouse before
immunization on day 0 (blank controls) and on days 8, 21, 35,
and 49. The blood samples were used to prepare plasma by
the standard method. The immunogenicities of vaccine 1,
mixture (8+14+15), 2, and mixture (8+14+16) were evaluated
using the corresponding plasma samples A-D, respectively
(Figure 2C). Mouse immunized with vaccine 1 in PBS without
an external adjuvant provided plasma E.
than plasma A. The results were consistent with the studies by
Kunz and Li et al, i.e., the inclusion of CFA led to inhibition of
the antibody production with self-adjuvanting vaccines.[19, 11d-e]
These results indicated that self-adjuvanted vaccines have high
potential for the practical application.
Noteworthy, the binding epitope of anti-triSTn IgGs induced by
vaccine 1 was carbohydrate part of 14 because anti-linker IgG
titers were very low in A and E. Although the anti-triSTn IgGs
elicited by vaccine 1 recognize extensively triSTn epitope but
concomitantly monoSTn. As mentioned above, STn antigen
was observed on a few normal cells, though it was more highly
expressed on cancer cells.[6] To avoid the problem of
autoimmunity in a new STn-based vaccine, the desired immune
response should be specific against the triSTn epitope.
Plasma C produced by vaccine 2 possessing unnatural-type N-
propionyl triSTn also exhibited a significant immune response
against N-propionyl and N-acetyl triSTn epitopes with the
higher specificity to triSTn and a negligible immune response to
the linker epitope (Figure 3B). The IgGs raised by vaccine 2,
therefore, have the cross-reactivity to natural-type N-acetyl
triSTn epitope. Though the titer of plasma C was lower than
plasma A, the IgG titer ratio of triSTn to monoSTn (7.3:1, Figure
3B) in plasma C was 2.2-fold higher than the same ratio in
plasma A (3.3:1, Figure 3A). The self-adjuvanting strategy was
also effective in N-propionyl triSTn antigen (plasma C versus D,
Figure 3B). The IgM antibody titers of plasma A-D on day 49
also gave similar results (see the Supporting Information).
Figure 3. IgG antibody titers of (A) plasma A, B, and E, and (B) plasma C
and D after four immunizations (day 49) with various preparations and before
immunization (day 0). ELISA plates were coated with BSA-triSTn for the anti-
triSTn IgG titers, BSA-N-propionyl-triSTn for the anti-N-propionyl-triSTn IgG
titers, BSA-STn for the anti-STn IgG titers, BSA-linker for the anti-linker IgG
titers, or BSA for the nonspecific-binding IgG titers. Error bar represented
standard error of the mean.
Figure 2. Various preparations for immunization and BSA-glycoconjugates
for ELISA assays. (A) compounds 14-16. (B) BSA-triSTn, -N-propionyl-triSTn,
-STn, and -linker conjugates. (C) The vaccines were evaluated in wild-type
BALB/c mice to generate corresponding immune responses (plasma samples
A-E). *The first immunization was administered with complete Freund’s
adjuvant (CFA), the others with incomplete Freund’s adjuvant (IFA). ** All
progress of the immunization was administered only with PBS buffer without
CFA or IFA.
The differences in the immune responses induced by vaccines
1 and 2 were further explored by measuring the IgG and IgM
titers in plasma A and C on days 0, 8, 21, 35, and 49 (Figure
4A-D). The IgG and IgM titers against the triSTn epitope in
The IgG antibody titers on day 0 and day 49 are shown in
Figure 3. No detectable anti-triSTn IgG titers were presented
before immunization. As depicted in plasma A (Figure 3A), the
high anti-triSTn IgG titer was induced by vaccine 1. On the
contrary, the mixture (8+14+15) afforded a negligible immune
response to the triSTn epitope (plasma B). These results clearly
demonstrated that the covalent conjugation in vaccine 1 was
critical for the production of IgGs. Moreover, plasma E obtained
by administration with vaccine 1 in the absence of an external
adjuvant (CFA) showed the slightly higher anti-triSTn IgG titer
plasma
A and C gradually increased with the boost
immunizations. In plasma A, IgG titers were boosted after third
and fourth immunizations on days 28 and 42, whereas IgM
titers were boosted after second immunization on day 14.
Interestingly, on days 35 and 49 in plasma A, IgGs revealed
higher selectivity to triSTn against STn than IgMs (Figure 4A
and 4C), suggesting affinity maturation in IgGs was promoted
after third and fourth immunizations of vaccine 1.
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In plasma C, both IgG and IgM titers induced by vaccine 2 were
boosted after first and second immunizations (Figure 4B and
4D). On the day 21, plasma C showed 2-fold higher triSTn-
specific IgG titers (3151 for triSTn) compared to plasma A
(titers: 1467 for triSTn). These results suggested that N-
propionyl triSTn is more immunogenic than N-acetyl triSTn.
However, the same phenomenon was not observed after third
and fourth immunization. Although the mechanistic details were
not clear, the slight structural difference between N-propionyl
and N-acetyl triSTns resulted in the significant difference in
immune responses.
(mean fluorescence intensity (MFI); PANC-1-A: 830,339,
PANC-1-C: 1,008,248, HepG2-A: 925,487, HepG2-C: 889,697).
PANC-1 expresses clustered STn and STn,[21] whereas HepG2
shows specific expression of clustered STn.[22] Our results
indicated that the IgGs induced by vaccines 1 and 2 recognized
clustered STn antigens on cancer cells, though the IgGs might
also recognize monoSTn on cancer cells. In addition, the IgGs
induced by 1 and 2 showed similar affinity to PANC-1 and
HepG2, despite of the lower titers of anti-triSTn IgGs elicited by
2 (Figure 3 versus Figure 5). As described, the IgG ratio of anti-
KLH/anti-monoSTn/anti-OSM obtained from the Theratope
phase 3 clinical trial was 64:64:1. These results suggested that
anti-monoSTn IgGs barely recognize the native STn and the
clustered STn, since OSM contains both clustered and
unclustered forms of the STn and is considered to be
representative of native STn present on tumor-associated
mucin.[8a] The present study indicates the triSTn antigens can
induce antibodies that can recognized the clustered STn on
cancer cells and therefore provides the opportunity to solve the
issue of Theratope.
Figure 4. The IgG and IgM antibody titers measured in plasma samples A
and C on days 0, 8, 21, 35, and 49 (A-D) and the anti-triSTn IgG antibody
subtypes in plasma A and C on days 0 and 49 (E-F). ELISA plates were
coated with BSA-triSTn to measure the anti-triSTn antibody titers and BSA-
STn to measure the anti-STn antibody titers. Error bar represented standard
error of the mean.
Figure 5. Flow cytometry analysis of the binding between plasma A and C
(1:50 diluted) collected on day 49 and the human pancreatic cancer cell line
PANC-1 (A and C) or human hepatic cancer cell line HepG2 (B and D). Alexa
Fluro®488-goat anti-mouse IgG (1:50 diluted) was used as a secondary
antibody for staining. Control: mice plasma before immunization (black color);
Plasma A or C: mixed plasma from a group (n = 5) of mice (red color).
IgG subclasses exhibit substantial differences in immune
functions and cytotoxicity against the target tumors. The IgG2b
and IgG3 isotypes of the anti-triSTn antibodies were observed
predominantly in both plasma A and C, whereas a small
amount of the IgG2a was also found (Figure 4E-F). The mouse
IgG3 is primarily anti-carbohydrate antibodies.[20] The
production of IgG2b and IgG2a suggested vaccines 1 and 2
induced the adaptive T cell-mediated immunity to implement an
IgG subclass switching.
Flow cytometric analysis revealed that the anti-triSTn IgGs in
plasma A and C effectively recognized human pancreatic
cancer cell line PANC-1 and human hepatic cancer cell line
HepG2 (Figure 5). Strong fluorescence intensities were
observed in PANC-1 and HepG2 treated with plasma A or C
The use of glycopeptide vaccines provides another possibility to
solve the issue of Theratope. Previously, Kunz et al. reported
that MUC1 glycopeptide vaccines having monoSTn induced
IgG1-type antibodies that recognized human breast cancer cell
lines T47D and MCF-7 as well as the breast tumor tissues,
which expressed tumor associated MUC1.[23] They also
generated IgG1-type monoclonal antibody GGSK-1/30 based
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Cai, Z.-Y. Sun, M.-S. Chen, Y.-F. Zhao, H. Kunz, Y.-M. Li, Angew.
Chem. Int. Ed. 2014, 53, 1699-1703; f) P. Thompson, V.
Lakshminarayanan, N. T. Supekar, J. M. Bradley, P. A. Cohen, M. A.
Wolfert, S. J. Gendler, G.-J. Boons, Chem. Commun. 2015, 51,
10214-10217.
on MUC1 glycopeptide vaccination. GGSK-1/30 bound to
MUC1-expressing tumor cells T47D, MCF-7, and PANC-1.
GGSK-1/30 also bound to the pancreatic cancer tissues,
whereas the normal cells showed almost no binding.[24] High
specificity of GGSK-1/30 to tumor associated MUC1-
expressing cancer cells is provably owing to the specific
recognition to the glycopeptide epitope.
In summary, N-propionyl triSTn vaccine 2 induced anti-triSTn
IgG antibodies that efficiently recognize cancer cells expressing
clustered STn. Additionally, vaccine 2 elicited a triSTn-specific
immune response that can minimize autoimmunity. Self-
adjuvanting N-propionyl triSTn fulfills the requirements for a
next-generation STn-based vaccine without the use of a carrier
protein as a means for avoiding the weaknesses of Theratope.
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This work was financially supported in part by JSPS KAKENHI
Grant Numbers 13F03329, 15H05836 in Middle Molecular
Strategy, 16H01885, 16H05924, 17K19201, 17H03571, JSPS
NEXT Program Grant Number LR025.
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This article is protected by copyright. All rights reserved.

10.1002/anie.201804437
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
The effective anti-STn antibodies
were generated by two fully synthetic
N-acetyl and N-propionyl triSTn
vaccines including the built-in T-
helper epitope and adjuvant. The N-
propionyl triSTn vaccine elicited a
triSTn-specific immune response.
Tsung-Che Chang, Yoshiyuki Manabe,
Yukari Fujimoto, Shino Ohshima,
Yoshie Kametani, Kazuya Kabayama,
Yuka Nimura, Chun-Cheng Lin, Koichi
Fukase*
Page 1. – Page 6.
Synthesis
Evaluation
and
of
Immunological
Self-Adjuvanting
Clustered N-Acetyl and N-Propionyl
Sialyl-Tn Combined with A T-helper
Cell Epitope as Antitumor Vaccine
Candidates
This article is protected by copyright. All rights reserved.