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Synthesis of a Self-adjuvanting MUC1 Vaccine via Diselenide-Selenoester Ligation-Deselenization David M McDonald, Cameron C Hanna, Anneliese S Ashhurst, Leo Corcilius, Scott N Byrne, and Richard J Payne ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00675 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Synthesis of a Self-adjuvanting MUC1 Vaccine via Diselenide-Selenoester Ligation-Deselenization David M McDonald†‡, Cameron C Hanna†, Anneliese S Ashhurst†‡, Leo Corcilius†, Scott N Byrne‡ and Richard J Payne†* School of Chemistry, The University of Sydney, NSW 2006 (Australia). Infectious Diseases and Immunology, Faculty of Medicine and Health, The University of Sydney, NSW 2006 (Australia). * Correspondence: [email protected] † ‡

ABSTRACT Access to lipopeptide-based vaccines for immunological studies remains a significant challenge owing to the amphoteric nature of the molecules which makes them difficult to synthesize and purify to homogeneity. Here, we describe the application of a new peptide ligation technology – the diselenide-selenoester ligation (DSL) – to access self-adjuvanting glycolipopeptide vaccines. We show that rapid ligation of glycopeptides and lipopeptides is possible via DSL in mixed organic solvent-aqueous buffer and, when coupled with deselenization chemistry, affords rapid and efficient access to a vaccine candidate possessing a MUC1 glycopeptide epitope and the lipopeptide adjuvant Pam2Cys. This construct was shown to elicit MUC1-specific antibody and cytotoxic T lymphocyte responses in the absence of any other injected lipids or adjuvants. The inclusion of the helper T cell epitope PADRE boosted both the antibody response and resulted in elevated cytokine production. INTRODUCTION The transmembrane glycoprotein mucin 1 (MUC1) is over-expressed in a wide variety of epithelial cancers, including breast, pancreatic, lung and colorectal tumors. Due to alterations in the expression of glycosyltransferases during cancer progression, the extracellular variable number tandem repeat (VNTR) domain of tumor-associated MUC1 is typically decorated with truncated O-linked glycans in place of larger O-glycan chains that are found on MUC1 in healthy tissues (Figure 1).1 These truncated glycans are known as tumor-associated carbohydrate antigens (TACAs),

Figure 1. Illustration of the tumour-associated changes in MUC1 O-glycosylation.

and include the monosaccharide TN antigen (GalNAc-α-Ser/Thr) and disaccharide T antigen (Gal(13)GalNAc--Ser/Thr). Due to its exclusive over-expression and aberrant glycosylation in tumor cells, MUC1 was ranked 2nd out of 75 cancer antigens.2 More than 50 clinical trials investigating MUC1-based vaccines have been conducted over the past 20 years, however, as of yet none have advanced into the clinic. There are several factors that have likely contributed to the limited success of MUC1-based vaccines to date,3, 4 but one key feature that needs to be addressed in any vaccine candidate is the poor immunogenicity of MUC1. One approach to increase the quality of immune responses towards MUC1-based vaccines is the development of self-adjuvanting vaccines that contain both MUC1 glycopeptide moieties (i.e. the peptide sequence of the MUC1 VNTR with TACAs appended at specific Ser and Thr residues) and immunostimulatory adjuvant moieties in the same molecule.5, 6 This approach has been investigated by us and others in pre-clinical settings.7-16 Previously, we have reported the synthesis and immunological evaluation of self-adjuvanting vaccine candidates containing the adjuvants tripalmitoylcysteine 13 or macrophage-activating (Pam3Cys)10, 14 liked via a lipopeptide 2 (MALP-2) triethyleneglycolate spacer to 20 amino acid MUC1 glycopeptides, in which each of the five O-


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glycosylation sites were derivatized with TACAs. When injected into mice, these vaccines generated strong humoral immune responses, but did not generate MUC1-specific CD8+ cytotoxic T lymphocyte (CTL) responses. We have since proposed that self-adjuvanting vaccine candidates containing longer MUC1 glycopeptide epitopes bearing fewer glycans might induce stronger CTL responses6 – an avenue that we explore in this work. One of the significant bottlenecks in the investigation of self-adjuvanting vaccines is the conjugation of (glyco)peptide antigens to lipopeptide or glycolipid adjuvants, which affords highly amphiphilic molecules, as well as the purification of these constructs to generate homogeneous molecules for immunological studies. Indeed, while lipidated adjuvants such as the TLR2 agonists di- and tri-palmitoylated glycerylcysteine (Pam2Cys and Pam3Cys, respectively) can be incorporated into short peptides via solid-phase peptide synthesis (SPPS),17 the synthesis of longer fragments leads to complex mixtures which are difficult to purify. Previously, we have performed fragment condensation of suitably protected (glyco)peptide fragments activated at the C-termini as pentafluorophenyl esters to generate target vaccines.18 While this approach generated the desired self-adjuvanting vaccine candidates in excellent yields, the method required the purification of protected lipopeptides by normal-phase HPLC in order to avoid undesired condensation reactions. An alternative approach for the synthesis of vaccine candidates is through the late stage assembly of fragments via native chemical ligation (NCL). First developed by Kent and colleagues,19 NCL has been used for preparation of numerous protein targets to date, but has only been used to assemble vaccines in a small number of cases. Boons and colleagues have reported the synthesis of vaccine constructs containing Pam3Cys from unprotected fragments via liposome-mediated NCL.8, 20 While this approach was chemoselective, it required formulation of each ligation fragment into liposomes, and the ligation products then had to be separated from the liposomal components for HPLC purification. Furthermore, this ligation approach was unsuccessful for the generation of similar compounds containing sialylated glycopeptides.21 Toth and colleagues have also used NCL of lipopeptides in aqueous solvent with the addition of sodium dodecyl sulfate (SDS)

as a detergent, which required separation post-ligation.22 We have also demonstrated that aqueous insoluble fragments can be ligated via NCL in a mixed solvent system consisting of aqueous ligation buffer and N-methyl-2-pyrrolidone (NMP).23 NCL in mixed solvent systems was successful, but reaction times were longer than equivalent ligations in aqueous buffer. We recently reported the development of a new ligation methodology between a peptide containing a C-terminal aryl selenoester fucntionality and a peptide bearing an N-terminal selenocystine moiety (the oxidized form of the 21st amino acid selenocysteine) dubbed the diselenide-selenoester ligation (DSL).24 The DSL technology has also been expanded to other amino acids through the use of synthetic selenol-derived amino acids. Importantly, these reactions reached completion in 1-5 min and proceeded smoothly in the absence of any additives, such as a reductant or aryl thiol that are normally required to accelerate NCL reactions. Following the ligation reaction, the Sec residue can also be cleanly converted to native alanine or serine through reductive deselenization or oxidative deselenization, respectively. Given the speed and efficiency of the DSL methodology, we envisaged that it may serve as an efficient means of synthesizing selfadjuvanting vaccine candidates. Towards this end, herein, we describe the adaptation of DSL to the rapid and efficient synthesis of an amphiphilic self-adjuvanting cancer vaccine candidate containing Pam2Cys and a MUC1 glycopeptide. We show that this synthetic vaccine was able to elicit MUC1 glycopeptide-specific antibody and CTL responses in vivo. RESULTS AND DISCUSSION Vaccine Design. We and others have previously prepared vaccines that incorporate the full-length 20 amino acid VNTR of MUC1. However, in the full length MUC1 glycoprotein the VNTR sequence is repetitive. Thus, any 20 amino acid VNTR sequence necessarily restricts the range of potential CD8+ T cell epitopes presented. As an example, the HLAA2-binding epitope STAPPAHGV25, 26 was absent from the glycopeptide epitopes we used in previous studies (GVTSAPDTRPAPGSTAPPAH). Furthermore, it is unknown how N- or C-terminal modification of these peptide epitopes affects their proteasomal processing. In addition to covering a wider range of potential CD8+ epitopes, longer



VNTR peptides are more likely to retain the residues flanking such epitopes, which may be important for processing. The repetitive nature of the MUC1 VNTR also enables the choice of N- and C-terminal residues that are favorable for the assembly of vaccine constructs via ligation technologies. We envisaged that the 27mer MUC1 VNTR peptide AHGVTSAPDTRPAPGSTAPPAHGVTSA would encompass a wider range of CD8+ T cell epitopes, while possessing an N-terminal Ala which could be readily accessed via DSL-deselenization at selenocystine. Number and position of glycans. The only MUC1 VNTR glycopeptide that has been unequivocally demonstrated to bind murine MHC is SAPDT(TN)RPAP.27 Additionally, to the best of our knowledge, the only MUC1 VNTR vaccine that has demonstrated MUC1 glycopeptide-specific CTL induction in vivo contained SAPDT(TN)RPAP, conjugated to Pam3Cys and a poliovirus helper T cell epitope.12 Combined with the finding that O-glycosylation of the SAP region can inhibit MUC1 VNTR processing by the immunoproteasome28, these observations led us to propose that per-glycosylation of MUC1 VNTR vaccine candidates may hinder induction or activation of MUC1-specific CTLs.6

As such, we sought to design a MUC1VNTR sequence that, when incorporated into a self-adjuvanting vaccine, could maintain high levels of humoral reactivity, while also inducing MUC1-specific CTLs. Of the 7 potential sites of O-glycosylation in the 27 residue MUC1 VNTR peptide proposed above, glycosylation of both Thr in PDTR and Ser in GSTA epitopes have been implicated in the generation of tumor-specific antibodies via stabilization of highly immunogenic conformations of the peptide backbone.29, 30 We hypothesized that 27mer MUC1 VNTR glycopeptide 1, glycosylated with TN at these two sites, would retain the H-2Kb-binding epitope SAPDT(TN)RPAP without sacrificing antibody reactivity (Scheme 1A). Synthesis of Vaccine Components. In order to determine whether a self-adjuvanting vaccine candidate containing the 27 mer MUC1 VNTR glycopeptide 1 could induce strong CTL activity, we designed self-adjuvanting vaccine candidate 2, bearing the adjuvant Pam2CysSKKKK17 covalently attached to MUC1 VNTR glycopeptide 1 via a triethyleneglycolate linker (Scheme 1B). Retrosynthetically, we envisaged that 2 could be rapidly accessed via DSL-deselenization between lipopeptide selenoester 3, possessing an N-terminal Pam2Cys moiety, and glycopeptide diselenide 4, bearing an N-terminal selenocystine.

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Scheme 1: A) Structure of MUC1 glycopeptide 1. B) Retrosynthesis of self-adjuvanting glycopeptide vaccine candidate 2.


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Synthesis of lipopeptide selenoester 3 was achieved via Fmoc-strategy SPPS on 2-chlorotrityl chloride resin (Scheme 2). The N-terminal amine was protected as the tert-butyl carbamate by on-resin treatment with di-tert-butyl dicarbonate and pyridine in DMF, and the side chain-protected peptide was subsequently removed from resin by mild acidic cleavage with hexafluoroisopropanol (HFIP). The crude, protected lipopeptide was converted to the C-terminal phenyl selenoester by treatment with diphenyl diselenide (DPDS, 30 eq.) and tributyl phosphine (30 eq.) in DMF for 2 h at room temperature.24 The protecting groups were then removed by global cleavage with an acidic cocktail consisting of trifluoroacetic acid (TFA), triisopropylsilane, and water. Semi-preparative HPLC on C18 stationary phase at 50 °C furnished 3 in 9% yield over 16 steps (equating to 86% yield per step).

Global acidic cleavage and deprotection provided crude glycopeptide 5, bearing an N-terminal selenazolidine and two per-acetylated TN glycans (Scheme 3). Treatment of crude 5 with aqueous hydrazine effected the concomitant removal of the O-acetyl groups and conversion of the N-terminal selenazolidine to selenocysteine, which rapidly oxidized to form the desired diselenide dimer glycopeptide 4 (Scheme 3). Brief treatment with tris(2-carboxyethyl)phosphine (TCEP) to reduce diselenides (~10 s), and immediate purification by semi-preparative HPLC led to successful isolation of 4 as a selenol, which immediately re-oxidized to provide pure diselenide 4 in 32% yield over 53 steps (98% yield per step). Glycopeptide 1 (required for immunological studies) was also synthesized via a similar strategy (see Supporting Information for details).

Scheme 2: Synthesis of Pam2Cys-containing lipopeptide selenoester fragment 3.

Synthesis of MUC1 VNTR glycopeptide diselenide 4 was carried out via Fmoc-strategy SPPS on Rink amide resin (see Scheme 3 and Supporting Information). -GalNAc-derived serine and threonine were incorporated as synthetic peracetylated building blocks as described previously.31 A protected selenocysteine building block, N-Boc protected (4R)-1,3-selenazolidine-4carboxylic acid (Boc-Sez-OH),32 was coupled onto the nascent glycopeptide in the presence of HATU, hydroxyazabenzotriazole (HOAt) and iPr2NEt.

Scheme 3: Synthesis of diselenide dimer MUC1 VNTR glycopeptide fragment 4.

Synthesis of Vaccine via DSL-deselenization. With lipopeptide selenoester 3 and diselenide dimer MUC1 VNTR glycopeptide 4 in hand, the assembly of the target Pam2Cys-MUC1VNTR vaccine candidate 2 via DSL-deselenization could commence (Scheme 4). Due to the insolubility of 3



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Scheme 4: A) Synthesis of vaccine candidate 2 by DSL-deselenization of lipopeptide selenoester 3 and MUC1 glycopeptide diselenide 4. B) Characterization of 2 by HPLC, ESI and MALDI-TOF mass spectrometry. See the supporting information for full characterization data.

in aqueous ligation buffer (6 M guanidine hydrochloride, 1 M HEPES, pH ~7), we investigated the use of NMP as an organic co-solvent (20 vol.%) or Tween®-20 (1 vol.%) as a non-ionic surfactant in the above ligation buffer to solubilize the lipopeptide selenoester component to enable ligation. Selenoester 3 proved to be soluble under both conditions and, gratifyingly, when 3 and 4 were simply dissolved in these buffers, ligation product was observed as the major species in both cases. However, we noted that Tween-20 co-eluted with both selenoester fragment 3 and ligation product 6 on reverse-phase HPLC and, as such, ligation reactions employing the mixed solvent system of NMP:ligation buffer (1:4 v/v) were deemed optimal for the generation of the vaccines. In order to optimize the yield of the reaction, two equivalents of lipopeptide selenoester 3 were added with respect to monomeric 4 (i.e. one equivalent with respect to the diselenide dimer). This was necessary due to a minor side reaction in which some of fragment 3 was consumed through intramolecular

macrolactamization between one of the four lysine side-chains in the tetralysine motif and the reactive selenoester moiety. Pleasingly, under these conditions the ligation was rapid, and diselenide dimer fragment was fully consumed within 2 min to afford target ligation product 6 as the major project. It is important to note that the DSL reaction liberates phenylselenolate that typically rapidly oxidizes to DPDS when the ligation is performed in a purely aqueous buffer system. However, in the mixed solvent system utilized here, DPDS remained soluble, leading to diselenide exchange with symmetrical diselenide product 6 to afford some asymmetric diselenide product containing an equivalent of phenylselenolate.24, 33, 34 This was inconsequential for the overall efficiency of the reactions, however, as the crude reaction mixture could be reduced with TCEP and extracted with hexane to remove the phenylselenol, which would otherwise inhibit the ensuing deselenization reaction.


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Having optimized the DSL reaction we next subjected the diselenide dimer product 6 to deselenization. Specifically, the crude ligation mixture was degassed under a stream of argon for 5 min, then TCEP and dithiothreitol (DTT) were added in one volume of mixed solvent ligation buffer, and the deselenization reaction was allowed to proceed at pH 4.0 overnight at 37 °C which led to clean conversion to the target vaccine. Following HPLC purification and lyophilization, self-adjuvanting vaccine candidate 2 was obtained in 62% yield over two steps. Importantly, both the speed and efficiency of this DSL-deselenization approach represents a substantial improvement to prior approaches employed for vaccine synthesis in our labs.

MUC1-labelled and control splenocytes were adoptively transferred into vaccinated mice 16 h prior to euthanasia. Cytotoxic activity was measured by the specific lysis of MUC1-labelled cells. Mice treated with self-adjuvanting MUC1 vaccine candidate 2 exhibited significantly higher MUC1-specific cellular cytotoxicity than mice treated with PBS (Figure 2A). The overall magnitude of the lysis was low (~20%) but, to the best of our knowledge, this was the first demonstration that a single-agent self-adjuvanting vaccine candidate can induce MUC1 glycopeptide-specific CTL activity in the absence of an external adjuvant such as complete Freund’s adjuvant or formulation with surfactants. Serum MUC1-specific antibodies were enumerated by standard indirect ELISA, using plates coated with MUC1 glycopeptide 1 as the capture antigen. Sera from vaccinated mice had significantly higher MUC1-specific IgM titers than PBS-treated controls, but not IgG (see Supporting Information). This lack of isotype class switching was attributed to the lack of a helper T cell response (see Supporting Information). However, the antibodies in the sera of vaccinated mice bound to the MUC1-expressing human breast cancer cell line MCF-7 as demonstrated by flow cytometry (see Supporting

Immunological evaluation. With self-adjuvanting vaccine candidate 2 in hand, we sought to investigate its immunological properties. Female C57BL/6 mice were vaccinated via sub-cutaneous injection with 2 (5 nmol) dissolved in PBS or PBS alone as a negative control. Vaccinations were repeated 14 d after the initial injections, and the spleens and inguinal lymph nodes were collected 7 d after the second vaccination. Blood was collected weekly for enumeration of MUC1-specific serum antibodies. To evaluate the activation of MUC1-specific CTLs, a mixed population of

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Figure 2: A) in vivo CTL activity towards glycopeptide 1 induced by vaccination with self-adjuvanting vaccine candidate 2. Bars represent mean (± SEM) of n = 15 mice per group from three independent experiments. Results were analyzed using an unpaired twotailed t test (p = 0.011). B) Serum concentration of MUC1-specific IgM and IgG at day 21 post-immunization. C) PADRE-induced cytokine production. Cells isolated from the inguinal lymph nodes of immunized mice were activated in vitro with PADRE peptide overnight, then the concentration of cytokine in the supernatant was measured by multiplex bead assay. Bars represent the mean (± SEM) of n = 6 C57BL/6 mice. Results were analyzed by Kruskall-Wallis test with Dunn’s multiple comparison test.



Information). Previously, we and others have improved the MUC1-specific IgG titers by the covalent inclusion of a helper T cell epitope from an unrelated protein into self-adjuvanting vaccine candidates.10, 12, 35 To test whether an external helper epitope could improve the MUC1-specific antibody response towards vaccine candidate 2, mice received sub-cutaneous injection of 2 admixed with pan-DR epitope (PADRE), a universal helper T cell epitope.13, 36 Mice injected with admixed 2 and PADRE produced significantly higher titres of MUC1-specific IgM than mice vaccinated with 2 alone, but still no IgG (Figure 2B). This increase in IgM was associated with an increase in cytokine production by PADRE-specific helper T cells, including IFN-γ, IL-2, IL-17a, IL-6, and IL-22 (Figure 2C and Supporting Information). This cytokine response was not generated when PADRE was used in the absence of 2, suggesting that the helper T cell epitope worked in conjunction with the self-adjuvanted MUC1 glycopeptide epitope. While it is not completely clear why 2 elicited a CTL response, and only a weak antibody response, we speculate that this may be owing to the lack of a conjugated T-cell helper epitope to promote a Thelper 2-type response, or perhaps the absence of an external depot adjuvant such as a liposomal formulation or Complete Freund’s Adjuvant (CFA) that have been employed in other vaccine strategies.5 Future work will therefore test whether covalent incorporation of a helper T-cell epitope into the vaccine candidate, with or without an associated liposomal adjuvant, is able to further enhance these responses and aid in class switching. Conclusions Synthesis and purification of long (glyco)lipopeptides is extremely challenging and has, in some cases, been the bottleneck in the design, synthesis and immunological evaluation of synthetic self-adjuvanting vaccine candidates. Here, we describe the application of the DSL technology in mixed organic solvent-aqueous buffer, in concert with deselenization chemistry, to effect the rapid and efficient synthesis of self-adjuvanting vaccine candidate 2, possessing a 27 amino acid MUC1 glycopeptide epitope and the adjuvant Pam2Cys. When injected into mice, this vaccine candidate induced MUC1-specific IgM and CTL responses in the absence of any other injected lipids or adjuvants.

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We also show that the inclusion of a helper T cell epitope promoted an increase in the IgM response and led to a significant increase in cytokine production that are suggestive of a mixed helper T cell response. While the immunological properties of this vaccine remain to be optimized, importantly, the DSL-deselenization synthetic strategy now provides a robust method to access libraries of self-adjuvanting vaccine candidates. Although the vaccine candidate described here failed to elicit strong humoral responses, this new technique will enable the inclusion of several immune stimulating components and/or adjuvants with a view to discovering synthetic vaccines capable of inducing both strong cell-mediated and strong, class-switched humoral responses. Studies toward this end are currently underway in our laboratories. Outside of vaccine research, the DSL-deselenization technology should also serve as a useful tool for the chemical synthesis of lipoproteins, many of which are beyond the scope of current chemical methodology. METHODS Mixed-solvent DSL. Glycopeptide diselenide 2 (2.9 mg, 0.48 μmol) and lipopeptide selenoester 3 (3.0 mg, 1.93 μmol, 2 eq. w.r.t. the monomer of 2) were lyophilized together into a microcentrifuge tube and then NMP:ligation buffer (1:4 v/v, 193 μL, 5 mM w.r.t. the monomer of 2) was added. The ligation buffer consisted of aqueous HEPES buffer (1.0 M, pH 7.4) containing guanidine hydrochloride (6 M). After addition of the ligation mixture, the pH was 5.2. After 2 min, the ligation reaction was complete as determined by HPLC-MS analysis. Deselenization. TCEP (12 mg, 48 μmol, 50 eq.) was added to the ligation mixture. This solution was extracted with hexane (3 × 1 mL). A slight emulsion formed at the interface, which was retained with the aq. layer for the next step. Further TCEP (12 mg, 48 μmol, 50 eq.) and dithiothreitol (DTT, 0.7 mg, 4.8 μmol, 5 eq.) were added in 1 volume of mixed solvent buffer (final pH = 4.0), and the reaction mixture was heated to 37 °C. After 3 h, the emulsion had resolved, and deselenization was complete as determined by HPLC-MS analysis. Purification by semi-preparative HPLC (50-100% B over 40 min, 7 mL min-1, 40 °C), followed by lyophilisation, afforded pure 3 as a white solid (2.6 mg, 62% yield).


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See the supporting information for a detailed description of all materials, experimental methods, and compound characterization. Supporting Information Available: This material is available free of charge via the Internet. Acknowledgement We would like to thank the Australian Postgraduate Award and John A. Lamberton Scholarship schemes for funding (DMM and CHH). Funding Sources We thank Cure Cancer Australia (Grant no. 1049757) for funding this research. References 1. Kufe, D. W. (2009) Mucins in cancer: function, prognosis and therapy, Nat. Rev. Cancer 9, 874-885. 2. Cheever, M. A., Allison, J. P., Ferris, A. S., Finn, O. J., Hastings, B. M., Hecht, T. T., Mellman, I., Prindiville, S. A., Viner, J. L., Weiner, L. M., and Matrisian, L. M. (2009) The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research, Clin. Cancer Res. 15, 5323-5337. 3. Kimura, T., and Finn, O. J. (2013) MUC1 immunotherapy is here to stay, Expert Opin. Biol. Ther. 13, 35-49. 4. Rivalland, G., Loveland, B., and Mitchell, P. (2015) Update on Mucin-1 immunotherapy in cancer: a clinical perspective, Expert Opin. Biol. Ther.15, 1773-1787. 5. Gaidzik, N., Westerlind, U., and Kunz, H. (2013) The development of synthetic antitumour vaccines from mucin glycopeptide antigens, Chem. Soc. Rev. 42, 4421-4442. 6. McDonald, D. M., Byrne, S. N., and Payne, R. J. (2015) Synthetic self-adjuvanting glycopeptide cancer vaccines, Front. Chem. 3, 60. 7. Cremer, G. A., Bureaud, N., Piller, V., Kunz, H., Piller, F., and Delmas, A. F. (2006) Synthesis and biological evaluation of a multiantigenic Tn/TFcontaining glycopeptide mimic of the tumor-related MUC1 glycoprotein, ChemMedChem 1, 965-968. 8. Ingale, S., Wolfert, M. A., Gaekwad, J., Buskas, T., and Boons, G.-J. (2007) Robust immune responses elicited by a fully synthetic threecomponent vaccine, Nature chemical biology 3, 663. 9. Kaiser, A., Gaidzik, N., Becker, T., Menge, C., Groh, K., Cai, H., Li, Y. M., Gerlitzki, B., Schmitt, E., and Kunz, H. (2010) Fully synthetic vaccines consisting of tumor-associated MUC1 glycopeptides and a lipopeptide ligand of the Toll-

like receptor 2, Angew. Chem. Int. Ed. 49, 36883692. 10. Wilkinson, B. L., Day, S., Malins, L. R., Apostolopoulos, V., and Payne, R. J. (2011) Selfadjuvanting multicomponent cancer vaccine candidates combining per-glycosylated MUC1 glycopeptides and the Toll-like receptor 2 agonist Pam3CysSer, Angew. Chem. Int. Ed. 50, 1635-1639. 11. Cai, H., Huang, Z. H., Shi, L., Zhao, Y. F., Kunz, H., and Li, Y. M. (2011) Towards a fully synthetic MUC1-based anticancer vaccine: efficient conjugation of glycopeptides with mono-, di-, and tetravalent lipopeptides using click chemistry, Chem. Eur. J. 17, 6396-6406. 12. Lakshminarayanan, V., Thompson, P., Wolfert, M. A., Buskas, T., Bradley, J. M., Pathangey, L. B., Madsen, C. S., Cohen, P. A., Gendler, S. J., and Boons, G. J. (2012) Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine, Proc. Natl. Acad. Sci. U. S. A. 109, 261-266. 13. Wilkinson, B. L., Day, S., Chapman, R., Perrier, S., Apostolopoulos, V., and Payne, R. J. (2012) Synthesis and immunological evaluation of self-assembling and self-adjuvanting tricomponent glycopeptide cancer-vaccine candidates, Chem. Eur. J. 18, 16540-16548. 14. McDonald, D. M., Wilkinson, B. L., Corcilius, L., Thaysen-Andersen, M., Byrne, S. N., and Payne, R. J. (2014) Synthesis and immunological evaluation of self-adjuvanting MUC1-macrophage activating lipopeptide 2 conjugate vaccine candidates, Chem. Commun. 50, 10273-10276. 15. Liu, Y., Zhang, W., He, Q., Yu, F., Song, T., Liu, T., Zhang, Z., Zhou, J., Wang, P. G., and Zhao, W. (2016) Fully synthetic self-adjuvanting MUC1fibroblast stimulating lipopeptide 1 conjugates as potential cancer vaccines, Chem. Commun. 52, 10886-10889. 16. Glaffig, M., Stergiou, N., Hartmann, S., Schmitt, E., and Kunz, H. (2018) A synthetic MUC1 anticancer vaccine containing mannose ligands for targeting macrophages and dendritic cells, ChemMedChem 13, 25-29. 17. Metzger, J. W., Wiesmuller, K. H., and Jung, G. (1991) Synthesis of N alpha-Fmoc protected derivatives of S-(2,3-dihydroxypropyl)cysteine and their application in peptide synthesis, Int. J. Pept. Prot. Res. 38, 545-554. 18. Wilkinson, B. L., Malins, L. R., Chun, C. K. Y., and Payne, R. J. (2010) Synthesis of MUC1lipopeptide chimeras, Chem. Commun. 46, 62496251. 19. Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. (1994) Synthesis of proteins by native chemical ligation, Science 266, 776-779. 20. Ingale, S., Buskas, T., and Boons, G. J. (2006) Synthesis of glyco(lipo)peptides by



liposome-mediated native chemical ligation, Org. Lett. 8, 5785-5788. 21. Thompson, P., Lakshminarayanan, V., Supekar, N. T., Bradley, J. M., Cohen, P. A., Wolfert, M. A., Gendler, S. J., and Boons, G. J. (2015) Linear synthesis and immunological properties of a fully synthetic vaccine candidate containing a sialylated MUC1 glycopeptide, Chem. Commun. 51, 10214-10217. 22. Moyle, P. M., Olive, C., Good, M. F., and Toth, I. (2006) Method for the synthesis of highly pure vaccines using the lipid core peptide system, J. Pept. Sci. 12, 800-807. 23. Santhakumar, G., and Payne, R. J. (2014) Total synthesis of Polydiscamides B, C, and D via a convergent native chemical ligation-oxidation strategy, Org. Lett. 16, 4500-4503. 24. Mitchell, N. J., Malins, L. R., Liu, X. Y., Thompson, R. E., Chan, B., Radom, L., and Payne, R. J. (2015) Rapid Additive-Free SelenocystineSelenoester Peptide Ligation, J. Am. Chem. Soc. 137, 14011-14014. 25. Apostolopoulos, V., Haurum, J. S., and McKenzie, I. F. (1997) MUC1 peptide epitopes associated with five different H-2 class I molecules, Eur. J. Immunol. 27, 2579-2587. 26. Brossart, P., Heinrich, K. S., Stuhler, G., Behnke, L., Reichardt, V. L., Stevanovic, S., Muhm, A., Rammensee, H. G., Kanz, L., and Brugger, W. (1999) Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies, Blood 93, 4309-4317. 27. Apostolopoulos, V., Yuriev, E., Ramsland, P. A., Halton, J., Osinski, C., Li, W., Plebanski, M., Paulsen, H., and McKenzie, I. F. (2003) A glycopeptide in complex with MHC class I uses the GalNAc residue as an anchor, Proc. Natl. Acad. Sci. U. S. A. 100, 15029-15034. 28. Ninkovic, T., and Hanisch, F. G. (2007) Oglycosylated human MUC1 repeats are processed in vitro by immunoproteasomes, J. Immunol. 179, 2380-2388. 29. Gaidzik, N., Kaiser, A., Kowalczyk, D., Westerlind, U., Gerlitzki, B., Sinn, H. P., Schmitt, E., and Kunz, H. (2011) Synthetic Antitumor Vaccines Containing MUC1 Glycopeptides with Two Immunodominant Domains-Induction of a Strong Immune Response against Breast Tumor Tissues, Angew. Chem. Int. Edit. 50, 9977-9981. 30. Movahedin, M., Brooks, T. M., Supekar, N. T., Gokanapudi, N., Boons, G. J., and Brooks, C. L. (2017) Glycosylation of MUC1 influences the binding of a therapeutic antibody by altering the conformational equilibrium of the antigen, Glycobiology 27, 677-687. 31. Wilkinson, B. L., Malins, L. R., Chun, C. K., and Payne, R. J. (2010) Synthesis of MUC1lipopeptide chimeras, Chem. Commun. 46, 62496251.

32. Reddy, P. S., Dery, S., and Metanis, N. (2016) Chemical synthesis of proteins with nonstrategically placed cysteines using selenazolidine and selective deselenization, Angew. Chem. Int. Ed. 55, 992-995. 33. Rasmussen, B., Sorensen, A., Gotfredsen, H., and Pittelkow, M. (2014) Dynamic combinatorial chemistry with diselenides and disulfides in water, Chem. Commun. 50, 3716-3718. 34. Ji, S., Cao, W., Yu, Y., and Xu, H. (2014) Dynamic diselenide bonds: exchange reaction induced by visible light without catalysis, Angew. Chem. Int. Ed. 53, 6781-6785. 35. Westerlind, U., Hobel, A., Gaidzik, N., Schmitt, E., and Kunz, H. (2008) Synthetic vaccines consisting of tumor-associated MUC1 glycopeptide antigens and a T-cell epitope for the induction of a highly specific humoral immune response, Angew. Chem. Int. Ed. 47, 7551-7556. 36. Alexander, J., del Guercio, M. F., Maewal, A., Qiao, L., Fikes, J., Chesnut, R. W., Paulson, J., Bundle, D. R., DeFrees, S., and Sette, A. (2000) Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses, J. Immunol. 164, 1625-1633.


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