Synthesis and immunological evaluation of self-adjuvanting MUC1-macrophage activating lipopeptide 2 conjugate vaccine candidates

Article abstract of DOI:10.1039/c4cc03510k

We describe herein the synthesis and immunological evaluation of self-adjuvanting mucin 1 (MUC1)-macrophage activating lipopeptide 2 (MALP2) (glyco)peptide vaccine candidates. Vaccine constructs were shown to induce high titres of class-switched IgG antibodies in C57BL/6 mice after four immunisations despite the lack of a helper T cell epitope. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03510k

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Synthesis and immunological evaluation of
self-adjuvanting MUC1-macrophage activating
lipopeptide 2 conjugate vaccine candidates†
Cite this: Chem. Commun., 2014,
50, 10273
Received 9th May 2014,
Accepted 18th July 2014
David M. McDonald,ab Brendan L. Wilkinson,a Leo Corcilius,a
Morten Thaysen-Andersen,c Scott N. Byrneb and Richard J. Payne*a
DOI: 10.1039/c4cc03510k
www.rsc.org/chemcomm
We describe herein the synthesis and immunological evaluation vaccine development.7 Recently, a number of laboratories have
of self-adjuvanting mucin 1 (MUC1)–macrophage activating lipo- focussed on the development of multi-component vaccines to
peptide 2 (MALP2) (glyco)peptide vaccine candidates. Vaccine con- induce strong immune responses to MUC1 tumour-associated
structs were shown to induce high titres of class-switched IgG glycopeptides.8 In recent work, we and others have demonstrated
antibodies in C57BL/6 mice after four immunisations despite the that fully synthetic vaccines possessing the Toll-like receptor (TLR)
lack of a helper T cell epitope.
2/TLR1 agonist Pam3Cys can elicit a robust immune response
without the use of an external adjuvant such as complete Freund’s
Mucin 1 (MUC1) is a transmembrane glycoprotein that is adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) which is com-
normally expressed on the basal membrane of epithelial cells and monly employed in animal studies but not clinically approved.9
is known to be highly over-expressed on epithelial tumour cells.1 These studies have incorporated a range of helper T cell (Th)
The extracellular domain of MUC1 consists of a 20 amino acid epitopes to promote the CD4+ T cell responses required to induce
variable-number tandem repeat (VNTR) sequence that possesses antibody isotype class switching. In this study, we were interested
five potential sites of O-glycosylation.2 During tumour progression, in investigating self-adjuvanting MUC1 vaccine candidates posses-
alteration in the expression levels of glycosyltransferase enzymes sing macrophage activating lipopeptide 2 (MALP2) as an immuno-
leads to aberrant glycosylation patterns on MUC1 (and other adjuvant. MALP2 is a lipopeptide derived from Mycoplasma
proteins).3 The result is the presentation of truncated O-glycan fermentans and is a potent activator of TLR2/6 heterodimers
structures (appended to the side chain of serine (Ser) and threonine in dendritic cells, macrophages and B cells.10 The activation of
(Thr) residues) that are unique to cancer cells and are collectively TLR2/6 leads to the downstream production of pro-inflammatory
termed tumour-associated carbohydrate antigens (TACAs).4 cytokines TNFa, IL-1, and IL-6, and chemokines CCL2, CCL4, IL-8
Examples include the monosaccharide Tn (GalNAc-a-Ser/Thr) and CCL5.11 MALP2 has attracted significant interest as a novel
and disaccharide T (Gal(b1-3)GalNAc-a-Ser/Thr) TACAs. Since and efficacious immunoadjuvant in infectious disease vaccine
the TACA-bearing MUC1 VNTR domains are known to be highly development12 and displays direct anti-tumour activity through
over-expressed in over 90% of tumours and are not expressed in inflammation-associated pathways.13 However, to the best of our
healthy tissue, they have been widely investigated for use in cancer knowledge, MALP2 has not been investigated in the context of
immunotherapy as vaccine antigens.5 Importantly, this approach tumour vaccination. Importantly, it has been demonstrated that B
has been validated by the fact that MUC1-based vaccines have cell stimulation by MALP2 occurs without the need for accessory
entered clinical trials for the treatment of epithelial carcinomas of cells.10b We hypothesised that potent, direct stimulation of B cells
the breast, colon, pancreas, and lung, among others,6 and MUC1 with MALP2 would induce specific antibody responses without the
was ranked second out of 75 as a candidate antigen for cancer need for external Th epitopes. To this end, we set out to test this
hypothesis through the synthesis and immunological evaluation of
three self-adjuvanting MUC1–MALP2 conjugate vaccine candidates
1–3. These vaccines differed in the glycosylation state of the MUC1
VNTR and were covalently linked to the MALP2 adjuvant compo-
nent via a non-immunogenic triethyleneglycolate spacer (Fig. 1).
We envisaged that the vaccine constructs containing MALP2
and a single copy of the unglycosylated MUC1 VNTR or the Tn- and
T-perglycosylated variants 1–3 could be readily obtained through
iterative condensation reactions using three fragments, each readily
a School of Chemistry, The University of Sydney, NSW 2006, Australia.
E-mail: richard.payne@sydney.edu.au
b Discipline of Infectious Diseases and Immunology, The University of Sydney,
NSW 2006, Australia
c Department of Chemistry and Biomolecular Sciences, Macquarie University,
NSW 2109, Australia
† Electronic supplementary information (ESI) available: Full synthetic procedures,
characterisation of novel compounds and immunological methods and data. See
DOI: 10.1039/c4cc03510k
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 10273--10276 | 10273

View Article Online
Communication
ChemComm
Fig. 1 Structure of MALP2-containing di-component vaccine candidates 1–3.
Scheme 1 (A) Synthesis of vaccine candidates 1–3 from fragments 4–7 and 8. IS = Ile-Ser(cMe,MePro) pseudoproline residue. See ESI† for detailed
conditions. (B–D) MALDI-TOF mass spectra of vaccine candidates 1–3.
synthesised by standard Fmoc-strategy solid-phase peptide syn- Following cleavage from the resin with global side chain deprotec-
thesis (Fmoc-SPPS, Scheme 1). These included the fully deprotected tion, de-O-acetylation of the carbohydrate moieties and purification
MUC1 VNTR (glyco)peptides 4–6, the side chain and N-terminally by HPLC provided 5 and 6 in good yields over the iterative steps.
protected peptide fragment of MALP2 containing a triethylene- The MALP2 peptide fragment was also synthesised by Fmoc-SPPS
glycolate spacer that was activated at the C terminus as a on 2-Cl-Trt-Cl resin and incorporated a Ile-Ser(cMe,MePro) pseudo-
pentafluorophenyl (Pfp) ester (7), and finally Fmoc-protected proline dipeptide moiety in order to prevent on-resin aggregation
Pam2CysGly-OPfp (8) (Scheme 1) (see ESI† for synthetic details). and improve the yield of the peptide synthesis. Liberation of
(Glyco)peptide fragments 4–6 were synthesised on 2-chlorotrityl the peptide from the solid support was effected via treatment
chloride (2-Cl-Trt-Cl) resin. For the synthesis of 5 and 6, per- with hexafluoroisopropanol (HFIP) in dichloromethane to afford
acetylated glycosylamino acid building blocks, corresponding to N-Fmoc- and side chain-protected peptide 9. Treatment of crude 9
suitably masked variants of the Tn and T antigen-derived amino with pentafluorophenyl trifluoroacetate (Pfp-TFA) and pyridine pro-
acids, were incorporated at the Ser and Thr sites within the MUC1 vided protected peptidyl Pfp-ester 7 in 12% isolated yield following
VNTR sequence using conditions reported previously (see ESI†).9b purification by normal-phase HPLC. Similarly, Fmoc-Pam2CysGly-OH
10274 | Chem. Commun., 2014, 50, 10273--10276
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
was obtained by coupling synthetic Fmoc-Pam2Cys-OH to Gly
immobilised on 2-Cl-Trt-Cl resin followed by cleavage with HFIP.
The crude lipopeptide was subsequently converted to the C-terminal
Pfp-ester using the conditions described above. Following normal-
phase HPLC purification, 8 was afforded in 63% yield. The
fragments were next assembled through pentafluorophenyl
ester-mediated fragment condensation chemistry14 to afford the
target vaccines 1–3. Specifically, (glyco)peptides 4–6 were first
reacted with Pfp ester 7 in the presence of 1-hydroxybenzotriazole
(HOBt) and N,N-diisopropylethylamine (DIEA) followed by
deprotection of the N-Fmoc carbamate in situ to provide partially
protected conjugates 10–12 in moderate to good yields following
purification by semi-preparative C4 HPLC. Finally, 10–12 were
reacted with Pfp-ester 8 in the presence of HOBt and DIEA.
Deprotection of the N-Fmoc group, followed by cleavage of the
side chain protecting groups on the MALP2 peptide fragment
through treatment with an acidic cocktail provided the desired
conjugate vaccine targets 1–3 in excellent yields (82–87%) following
purification by semi-preparative C4 HPLC.
In order to investigate the immunological response to the
self-adjuvanting MALP2 vaccine candidates, C57BL/6 mice were
immunised weekly via sub-cutaneous injections of each vaccine
candidate (or PBS as a control). MUC1-specific serum antibo-
dies were enumerated by ELISA throughout the four week
immunisation course. Immunised mice exhibited high titres
of MUC1-specific IgM antibodies after the first injection
which were sustained throughout the immunisation schedule
(Fig. 2a). In addition, high levels of class-switched IgG1, IgG2b
and IgG3 antibodies were elicited, titres of which increased
after each immunisation (Fig. 2b). In all cases, mice immunised
with the unglycosylated vaccine candidate 1 exhibited the
highest specific antibody titres (Fig. 2c). Antibodies isolated
from all mice were capable of recognising and binding to the
MUC1 VNTR (glyco)peptide epitopes against which they were
raised. These interactions were selective, with the exception of
antibodies raised against the Tn-containing vaccine, which also
reacted with unglycosylated and T antigen-containing MUC1
VNTR (glyco)peptides, as determined by ELISA, (see ESI†).
This cross-reactivity of antibodies raised against Tn-containing
MUC1 vaccines to unglycosylated and T-containing MUC1 (glyco)-
peptide antigens mirrors observations by Clausen and co-workers.2
Humoral immunity to MUC1 is considered a key factor controlling
the growth and metastasis of human cancer. The ability of MALP2
vaccines 1–3 to promote robust IgG1, IgG2b and IgG3 MUC1-
specific antibodies is likely to be particularly important given their
role in mediating antibody-dependent cell-mediated cytotoxicity
and complement activation.15 CD8+ and CD4+ T cell responses
were examined by in vivo CTL assay and in vitro cytokine staining,
but specific cytotoxic activity against glycoforms of the H-2Kb-
binding epitope SAPDT*RPAP16 was not observed (see ESI†). In
addition, there was no increase in IL-4, IFN-g or CD25-expressing
CD4+ or CD8+ T cells compared to PBS-treated controls (see ESI†).
This lack of observed Th response, together with the sustained high
levels of IgM observed,17 leads us to propose that these vaccines
induced T cell-independent class switching. Future experiments
involving CD4-depleted mice will explore this hypothesis further.
Fig. 2 Total MUC1-specific titres of (a) IgM and (b) IgG over time. (c) Day
27 IgG isotype and IgA titres from the sera of mice immunised with PBS or
vaccine candidates 1–3. Mice were immunised on days 1, 7, 14, and 21 of the
experiment. Plotted points represent median (Æ interquartile range) endpoint
titres of n = 6 C57BL/6 mice. See ESI† for experimental conditions.
In summary, we have successfully synthesised a number of
(glyco)lipopeptide self-adjuvanting MUC1–MALP2 conjugate
vaccine candidates. These self-adjuvanting vaccine candidates
induced robust humoral immune responses in animal models
with class switched antibodies of several isotypes, indicative of
poly-functional humoral immune responses. Importantly, this
response occurred in the absence of an external adjuvant or
helper T cell epitope. Future work in our laboratory will involve
the use of MALP2 in conjunction with a Th epitope to investigate
the role of T cell help in immune responses to MALP2 vaccines.
Notes and references
1 M. A. Tarp and H. Clausen, Biochim. Biophys. Acta, 2008, 1780, 546–563.
2 M. A. Tarp, A. L. Sorensen, U. Mandel, H. Paulsen, J. Burchell, J. Taylor-
Papadimitriou and H. Clausen, Glycobiology, 2007, 17, 197–209.
3 (a) E. Meezan, H. C. Wu, P. H. Black and P. W. Robbins, Biochemistry,
1969, 8, 2518–2524; (b) J. W. Dennis, M. Granovsky and C. E. Warren,
Biochim. Biophys. Acta, 1999, 1473, 21–34.
4 (a) S. Hakomori, Adv. Cancer Res., 1989, 52, 257–331; (b) D. S. Sanders
and M. A. Kerr, Mol. Pathol., 1999, 52, 174–178.
5 (a) T. Buskas, P. Thompson and G. J. Boons, Chem. Commun., 2009,
5335–5349; (b) P. Beatty, S. Ranganathan and O. J. Finn, Oncoimmunology,
2012, 1, 263–270; (c) N. Gaidzik, U. Westerlind and H. Kunz, Chem. Soc.
Rev., 2013, 42, 4421–4442.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 10273--10276 | 10275

View Article Online
Communication
ChemComm
6 C. K. Tang, M. Katsara and V. Apostolopoulos, Expert Rev. Vaccines, 10 (a) P. F. Muhlradt, M. Kiess, H. Meyer, R. Sussmuth and G. Jung,
2008, 7, 963–975.
J. Exp. Med., 1997, 185, 1951–1958; (b) S. Borsutzky, K. Kretschmer,
P. D. Becker, P. F. Muhlradt, C. J. Kirschning, S. Weiss and C. A. Guzman,
J. Immunol., 2005, 174, 6308–6313.
7 M. A. Cheever, J. P. Allison, A. S. Ferris, O. J. Finn, B. M. Hastings,
T. T. Hecht, I. Mellman, S. A. Prindiville, J. L. Viner, L. M. Weiner
and L. M. Matrisian, Clin. Cancer Res., 2009, 15, 5323–5337.
11 (a) P. F. Muhlradt and U. Schade, Infect. Immun., 1991, 59, 3969–3974;
(b) U. Deiters and P. F. Muhlradt, Infect. Immun., 1999, 67, 3390–3398.
¨
8 (a) A. Hoffmann-Roder, A. Kaiser, S. Wagner, N. Gaidzik, D. Kowalczyk,
U. Westerlind, B. Gerlitzki, E. Schmitt and H. Kunz, Angew. Chem., 12 F. Rharbaoui, B. Drabner, S. Borsutzky, U. Winckler, M. Morr,
Int. Ed., 2010, 49, 8498–8503; (b) N. Gaidzik, A. Kaiser, D. Kowalczyk,
U. Westerlind, B. Gerlitzki, H. P. Sinn, E. Schmitt and H. Kunz, Angew.
B. Ensoli, P. F. Muhlradt and C. A. Guzman, Eur. J. Immunol., 2002,
32, 2857–2865.
Chem., Int. Ed., 2011, 50, 9977–9981; (c) H. Cai, Z. H. Huang, L. Shi, 13 C. Schneider, T. Schmidt, C. Ziske, K. Tiemann, K. M. Lee,
Z. Y. Sun, Y. F. Zhao, H. Kunz and Y. M. Li, Angew. Chem., Int. Ed., 2012,
51, 1719–1723.
V. Uhlinsky, P. Behrens, T. Sauerbruch, I. G. Schmidt-Wolf, P. F.
Muhlradt, J. Schmidt and A. Marten, Gut, 2004, 53, 355–361.
9 (a) S. Ingale, M. A. Wolfert, J. Gaekwad, T. Buskas and G. J. Boons, 14 B. L. Wilkinson, L. R. Malins, C. K. Chun and R. J. Payne, Chem.
Nat. Chem. Biol., 2007, 3, 663–667; (b) B. L. Wilkinson, S. Day, L. R. Commun., 2010, 46, 6249–6251.
Malins, V. Apostolopoulos and R. J. Payne, Angew. Chem., Int. Ed., 15 (a) C. I. Bindon, G. Hale, M. Bruggemann and H. Waldmann, J. Exp.
2011, 50, 1635–1639; (c) V. Lakshminarayanan, P. Thompson, M. A.
Wolfert, T. Buskas, J. M. Bradley, L. B. Pathangey, C. S. Madsen,
Med., 1988, 168, 127–142; (b) S. Von Mensdorff-Pouilly, M. Moreno
and R. H. Verheijen, Cancers, 2011, 3, 3073–3103.
P. A. Cohen, S. J. Gendler and G. J. Boons, Proc. Natl. Acad. Sci. 16 V. Apostolopoulos, J. S. Haurum and I. F. McKenzie, Eur. J. Immunol.,
U. S. A., 2012, 109, 261–266; (d) B. L. Wilkinson, S. Day, R. Chapman, 1997, 27, 2579–2587.
S. Perrier, V. Apostolopoulos and R. J. Payne, Chem. – Eur. J., 2012, 17 J. J. Mond, A. Lees and C. M. Snapper, Annu. Rev. Immunol., 2005, 13,
18, 16540–16548.
655–692.
10276 | Chem. Commun., 2014, 50, 10273--10276
This journal is ©The Royal Society of Chemistry 2014