Rhamnose modified bovine serum albumin as a carrier protein promotes the immune response against sTn antigen

Article abstract of DOI:10.1039/d0cc05263a

Rhamnose and sTn antigen were co-conjugated to bovine serum albumin (BSA), a weakly immunogenic carrier protein, for cancer vaccine development. The immune responses against sTn have been significantly augmented with the involvement of Rha-specific antibodies to enhance antigen uptake. This journal is

Full text of DOI:10.1039/d0cc05263a

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Rhamnose Modified Bovine Serum Albumin as Carrier Protein
Promotes the Immune Response against sTn Antigen
Han Lin,‡ Haofei Hong,‡ Jinfeng Wang, Chen Li, Zhifang Zhou * and Zhimeng Wu *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Rhamnose and sTn antigen were co-conjugated to bovine serum immune response against carrier protein, but not against the
albumin (BSA), a weakly immunogenic carrier protein, for cancer TACAs, is elicited. The large number of peptide epitopes
vaccine development. The immune responses against sTn have presented in carrier proteins may compete with carbohydrate
been significantly augmented with the involvement of Rha- hapten for a limited number of helper T cells and B cells, leading
specific antibodies to enhance antigen uptake.
to undesired activation or differentiation signals and factors that
suppress the immune response against carbohydrate antigen. For
example, in our previous study, we found that Globo H-KLH
conjugate elicited a much higher KLH-specific antibody titer
than that of Globo H-specific.9 In addition, only a few carrier
proteins are approved in clinical for vaccine industry and the
same carriers are used in different vaccines which may cause
vaccine interference and further lead to immune suppression
against carbohydrate antigens.10, 11 Therefore, new carriers for
vaccine construct are needed.
The aberrant glycans overexpressed on the cancer cell surface,
known as tumor-associated carbohydrate antigens (TACAs), are
attractive targets for the development of anti-cancer vaccines.1-3
However, carbohydrate antigens are usually T cell- independent
antigens which make them low immunogenicity and fail to elicit
antibody affinity maturation and immunological memorization.1,
2
To overcome this obstacle, the conventional strategy is to
conjugate TACAs to a carrier protein that provides T cell-
epitopes, allowing the TACAs to be taken up by antigen-
presenting cells (APCs) and presented to T helper cells by
MHCII complex together with the epitopes in the carrier.4 Thus,
the induced T cell help can prime an optimal B cell responses
and the immunogenicity of carbohydrate antigens can be
significantly enhanced.2 For example, the sTn (Neu5Ac-α2,6-
GalNAc-α-O-Ser/Thr) antigen has been conjugated with keyhole
limpet hemocyanin (KLH) as a vaccine Theratope which induced
predominantly humoral immune responses against breast cancer
To overcome this potential issue, novel strategies, employing
immunostimulatory molecules, such as Pam3CSK412-14
,
MPLA9, αGalCer15, 16, and others, to conjugate with TACAs as
fully synthetic vaccines have been developed and the immune
responses against TACAs are improved. Other non-protein
carriers,
including
dendrimers17,
polysaccharides18,
nanoparticles and virus-like particles19, also have been explored
to construct TACAs-based vaccines.
in
a
clinical trial.5 Intensive studies based on MUC1
Endogenous antibodies are naturally pre-existing antibodies in
human serum. Profiling studies have identified several abundant
antibodies ranging from 1% to 3% in human blood, such as anti-
galactose-α-(1,3)-galactose (α-Gal), anti-2,4-dinitrophenyl
group (DNP) and anti-rhamnose (Rha) antibodies etc.20
Recruiting these antibodies to cancer cells could trigger
antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC) to destruct target
cells.21 Notably, it has been demonstrated that vaccine constructs
containing one of the above mentioned haptens could form an in
situ immune complexes in vivo and selectively deliver antigen to
APCs by the binding between the endogenous antibody Fc
portion and the Fc receptors on APCs, leading to effective
internalization and enhanced cellular and humoral immune
response.20, 22, 23 For example, the studies initiated by Galili and
glycopeptide containing sTn and using tetanus toxoid or BSA as
carriers were developed as well and showed promising clinical
translational results.6-8 Globo H has been coupled with KLH or
diphtheria toxin mutant (CRM197) and exhibits efficacy in
clinical trials, as well.1
Although the great success of TACAs based glycoconjugate
vaccine has been achieved, it is often observed that a strong
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of
Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China †
email: zhifangzhou@jiangnan.edu.cn; zwu@jiangnan.edu.cn, Tel and Fax: 86-
510-85197582
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
‡ These authors contributed equally to the present work
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Journal Name
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DOI: 10.1039/D0CC05263A
Bleedings
OH
COOH
O
OH
HO
O
AcHN
HO
21
1
OH
-14
7
14
28
-21
-7
O
Day
HO
O
O
AcHN
O
N
NH
Group 5-7
4
Group 1: immunized with
H
n
Pre-immunized
with Rha-OVA
1
2
3
Group 2 and 5: immunized with
Group 3 and 6: immunized with
Group 4 and 7: immunized with
CP
H
NH
N
3
Figure 2. Immunization strategy.
O
O
O
O
HO
m
this study is to augment the immunogenicity of sTn while
reducing the potential adverse immune response caused by a
carrier protein.
HO
OH
1 CP=BSA sTn-BSA-Rha-0.5 (sTn loading=7.8%, Rha loading=0.5%)
2 CP=BSA sTn-BSA-Rha-1 (sTn loading=7.8%, Rha loading=1%)
3 CP=BSA sTn-BSA-Rha-2.8 (sTn loading=7.8%, Rha loading=2.8%)
4 CP=BSA sTn-BSA
5 CP=HSA sTn-HSA
(sTn loading=7.8%, Rha loading=0%)
(sTn loading=8.3%, Rha loading=0%)
The study was initiated by the construction of sTn-BSA-Rha
glycoconjugates (1-5) (Figure 1). The synthesis of sTn and Rha
derivatives, as well as their conjugation with BSA, is described
in Scheme 1. The sTn antigen 6 with an ethylamine at the
reducing end was synthesized according to a reported procedure
(Scheme S1 in SI).26 The sTn antigens were then conjugated with
protein BSA via the bifunctional glutaryl ester method which
was a well-established method used in our previous projects and
did not affect the immunological properties of conjugates.9, 27
Compound 6 reacted with a large excess (15 eq.) of
disuccinimidyl glutarate (DSG) in DMF to afford corresponding
monoesters 7, which was briefly purified by repeated
precipitation to remove excess DSG. Then, 7 was mixed with
BSA or HSA in PBS buffer to generate conjugate sTn-BSA 4 or
sTn-HSA 5 that were purified by centrifugal filter devices (MW
cut off: 10 kDa) to remove excess 7 and other small molecules.
The loading of sTn antigen in conjugates 4 and 5 was determined
by MALDI-TOF-MS (Table S1 and Figure S6 and S7), which
was 7.8% and 8.3%, respectively, suggesting that the
conjugating reactions were successful and the antigen loading
levels were suitable for biological studies.
Figure 1. Design and structure of sTn-BSA-Rha vaccine construct.
colleagues demonstrated that α-Gal epitope could significantly
augment the immunogenicity of tumor cells, HIV and influenza
vaccine.23 More recently, this strategy was utilized to improve
the immunogenicity of drug molecule successfully, such as
cocaine and fentany.24, 25 Sucheck and co-workers proved that
anchoring Rha hapten to a full synthetic anti-MUC1 cancer
vaccine, either in covalent or liposome form, could dramatically
enhance the immune responses in the presence of anti-Rha
antibodies.22
In the present work, we explore a novel strategy using Rha-and
sTn co-conjugated protein to construct TACAs-based vaccines
by recruiting the endogenous antibodies to improve the immune
efficacy. Instead of using conventional high immunogenicity
carrier protein, low immunogenicity and non-toxic protein, BSA,
is selected as a carrier protein. In this design, sTn, a promising
TACA antigen in anti-cancer vaccine development, which is
overexpressed in various carcinomas, such as breast, ovarian and
colon cancer, is conjugated to BSA at first; then followed by
conjugation with different loading of Rha to obtain sTn-BSA-
Rha glycoconjugates for immunological studies. The purpose of
The Rha antigen was synthesized starting from L-rhamnose 8
(Scheme 1). Firstly, rhamnose 8 was peracetylated to give 9,
which were following by a glycosylation reaction with azido-
triethylene glycol in the presence of BF3•Et2O to produce
compound 10. After removing the acetyl group in Zemplén
transesterification condition and subsequent hydrogenation to
reduce the azide group, 12 with an amine group at the reducing
end was obtained in good yield, which was transformed to
monoester 13 using DSG following the previous procedure.
Finally, sTn-BSA conjugate 4 was incubated with different
amounts of monoester 13 in PBS buffer (pH = 7.4), respectively,
to generate sTn-BSA-Rha glycoconjugates 1, 2 and 3. The
loading of Rha hapten in conjugate 1-3 was determined by
MALDI-TOF-MS (Table S1 and Figure S8-S10 in SI), which
were 0.5%, 1% and 2.8%, respectively. The low to high loading
of Rha in conjugates sTn-BSA-Rha 1-3 with a fixed amount of
sTn could help to reveal the capability of anti-Rha antibody-
mediated vaccination against sTn.
OH
COOH
OH
HO
OH
COOH
HO
OH
HO
O
HO
a
AcHN
b
4
and
5
O
O
AcHN
OH
O
OH
O
O
HO
AcHN
O
O
O
HO
AcHN
O
N
O N
O
H
NH2
6
7
O
N3
3
N3
3
OH
OAc
O
O
d
e
c
O
O
O
O
HO
AcO
AcO
HO
HO
AcO
AcO
HO
OH
OAc
OAc
OH
11
8
9
10
f
O
N
O
NH2
3
H
N
3
O
O
h
g
O
1-3
O
HO
O
O
O
HO
HO
OH
HO
OH
13
12
Scheme 1. (a) disuccinimidyl glutarate, DMF:PBS (4:1), rt; (b)
BSA/HSA, PBS, rt; (c) Ac2O, pyridine, rt; (d) azido-triethylene
glycol, BF3·Et2O, CH2Cl2, rt, 68%; (e) MeONa, MeOH; (f) 10%
Pd/C, H2, MeOH, rt; (g) disuccinimidyl glutarate, DMF:PBS (4:1),
rt; (h) 4, PBS, rt.
To evaluate the immunological activities of our vaccine
conjugates, we set seven groups of mice (groups 1-7) for these
studies (Figure 2). Earlier studies have proved that laboratory
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also interesting to observe that the anti-sTn titer was closely
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DOI: 10.1039/D0CC05263A
relevant to the loading level of Rha in glycoconjugate (group 2-
4 and group 5-7). Conjugation of Rha hapten on the vaccine does
not suppress the immunogenicity of sTn, whereas higher loading
of Rha in vaccine generally lead to better uptake of the sTn
antigen in the presence of anti-Rha antibodies and enable more
anti-sTn specific antibody production.
We also analyzed the anti-sTn and anti-Rha specific immune
response progress in groups 2-4 and 5-7. As presented in Figure
3B and Figure S2-A, mice in group 2-4 generated low titers of
anti-sTn specific antibody on day 21 and relative higher anti-Rha
antibody. However, both the anti-sTn and anti-Rha specific
antibody was increased dramatically on day 28. This interesting
finding indicated that sTn-BSA-Rha vaccine, especially for high
loading Rha conjugate 3, might have a self-adjuvanting effect at
the late stage of immunization, which was attributed to anti-Rha
antibody-mediated antigen uptake mechanism. While for group
5-7, high titers of anti-Rha antibody were maintained in the
vaccination period (Figure S2-B). Consequently, a faster anti-
sTn immune response was provoked with the help of endogenous
anti-Rha antibodies (Figure 3B). For example, the elicited anti-
sTn specific antibody on day 21 in group 5-7 was comparable to
group 2-4 on day 28.
Figure 3 Immunological evaluation of the synthetic vaccine: (A)
Antibody titers of different groups at Day 28. Conjugate 5 as coating
antigens to detect sTn-specific antibodies. (B) The average titers of
sTn-specific total antibodies in pooled antisera collected on day 0,
14, 21 and 28 from mice of group 1-7. (C) Evaluations of BSA-
specific and sTn-specific antibodies in pooled antisera of group 1,
4, and 7. (D) Antibody isotypes and subtypes of the conjugate
vaccine. The mean of antibody titers of three parallel experiments is
shown for each sample, and the error bar shows the standard error
of three replicate experiments. (* represents p < 0.05, ** represents
P< 0.01, *** represents P<0.005.)
The elicited anti-BSA and anti-sTn specific antibodies in groups
1, 4 and 7 were analysed by ELISA. It was found that the
produced anti-sTn-specific antibodies were significantly higher
than that of BSA-specific in group 4 and 7 (Figure 3C). This
result suggested that immune response against carrier protein
was dramatically reduced because of the weak immunogenicity
characters of BSA, which was different from other commonly
used TACAs based vaccine design, where strong immunogenic
carrier protein, such as KLH, was applied and anti-carrier protein
immune response was dominated. Moreover, it is worth noting
that with the help of endogenous Rha antibodies, the immune
response of anti-sTn was enhanced without suppressing, even
weak immunogenic carrier protein was used. This result inspired
us that with hapten modification strategy, carrier proteins with
low immune-stimulating abilities could be promisingly applied
in carbohydrate-based vaccines in the future.
mice do not contain significant titer of naturally occurring anti-
Rha antibodies.28 Therefore, we pre-immunized mice in groups
5-7 with Rha-OVA to establish high levels of anti-Rha
antibodies which were considered as endogenous antibodies.
Figure S1 in the supporting information shows that after
immunization with Rha-OVA, high titers of Rha-specific
antibody (about 200000) were detected in the mice sera of groups
5-7. After that, all the groups of mice were immunized with sTn-
BSA-Rha conjugate 1-4 according to Figure 2 on day 1 and
boosts on day 7, 14 and 21. Mouse blood samples were collected
on day 7, 14, 21 and 28 after the first immunization. The
antibodies were determined by enzyme-linked immunosorbent
assays (ELISA). The antibody titer is set as the dilution factor at
the optical density (OD) 415 nm reaches 0.2 from the regression
analysis of the curves of the OD value against serum dilution
number.
The antibody isotypes and subtypes are detected in groups 1, 4
and 7. IgG1 and IgG2b are the major subtypes in groups 4 and 7
which indicated that T cell immunity is successfully provoked
by the Rha-modified BSA carrier protein (Figure 3D). In cancer
immunotherapy, IgG1 usually mediated potent effector functions,
such as ADCC and CDC, to destruct cancer cells. The preferred
IgG1 was abundantly generated in this strategy, indicating that
the vaccine may have a promising therapeutic effect.
The ELISA results of seven groups on day 28 antisera were
summarized in Figure 3A. As expected, group 1 immunized
with sTn-BSA conjugate 4 without Rha produced the lowest
sTn-specific antibody (about 534). For groups 2-4 immunized
with sTn-BSA-Rha 1-3, significant high titers of anti-sTn
specific antibodies were elicited, which were 10809, 19491 and
22685, respectively. The elicited antibodies were improved
approximately 20- to 42-fold compared with group 1. For groups
5-7 that was pre-immunized with Rha-OVA and then immunized
with sTn-BSA-Rha 1-3, notably higher anti-sTn specific
antibodies were generated that were 23901, 29501 and 34306.
Compared with group 1, the production of anti-sTn antibody was
enhanced by 45, 55 and 64 fold, respectively. These results
suggested that the endogenous anti-Rha antibodies pre-existing
in mice serum could target delivery of glycoconjugate 1-3 to
APC cells and result in more efficient transportation and
internalization as well as the overall immune response. It was
To explore this possibility, the properties of antisera induced by
these conjugates were further studies by a tumor cell binding
assay detected by flow cytometry. The human breast cancer cell
MCF-7 which has sTn expression was incubated with the pooled
antisera collected from mice immunized with groups 1, 4 and 7,
respectively. After the labelling by the fluorescent second
antibody, the cell samples were measured by flow cytometry.
Figure 4A revealed that the antisera induced by groups 4 and 7
could successfully recognize the sTn structure expressed on
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In conclusion, we developed a new strategy for targeting
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immunological studies demonstrated that in the presence of
endogenous anti-Rha antibodies, sTn-BSA-Rha conjugates can
form immune complexes by recruiting pre-generated anti-Rha
antibodies to deliver the vaccine to APCs. The structure-activity
study showed that the high loading of Rha hapten on the vaccine
could mediate a better delivery to APCs, thereby triggering a
stronger and faster immune response. This strategy provides a
simple but efficient approach to augment the immunogenicity of
carbohydrate antigen using weak and non-toxic carrier protein
that otherwise is difficult to achieve. Considering the widely
used carbohydrate antigen in vaccine research and industry, this
strategy is potentially applicable to construct new
glycoconjugates for vaccine development.
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This work was supported by the National Natural Science
Foundation of China (No. 21472070, 21602084) and Social
Development Key Project of Jiangsu Province [BE2019632], the
project was partly funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions, the 111
Project (No. 111-2-06), China Postdoctoral Science Foundation
Grant (BX20200153, 2018M632227), the Open Foundation of
Key Laboratory of Carbohydrate Chemistry & Biotechnology
Ministry of Education (No. KLCCB-KF202006) and National
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Notes and references
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