Perfluoroalkylated amphiphilic MUC1 glycopeptide antigens as tools for cancer immunotherapy

Abstract

The synthesis of perfluoroalkylated glycopeptide antigens and their specific binding to anti-MUC1 mouse antibodies is reported.

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Most epithelial cancer cells over-express an abnormal form of the transmembrane glycoprotein MUC1 characterised by aberrant glycosylation patterns and the exposure of immunogenic peptide epitopes. Several studies have shown that these tumour-associated MUC1 glycoforms are of particular interest for diagnostic tools and represent attractive targets for the development of cancer vaccines.¹ For immunisation purposes, the carbohydrate antigen is traditionally coupled to a carrier protein which provides T cell help required for antibody production, and although this approach has proven to be successful,² it is also fraught with problems such as carrier-induced suppression.³

Alternative vaccine platforms delivering multiple copies of tumour antigens have been developed by functionalising peptide templates, dendrimers, nanoparticles and liposomes.⁴ With regard to the latter, self-assembled systems based on fluorinated amphiphiles have also been proposed as delivery systems.⁵ The unique hydrophobic and lipophobic characters of F-alkylated amphiphiles promote their self-aggregation into stable, organised molecular systems for different biomedical applications.⁶ Besides, hydrophobised glycoconjugates are valuable diagnostic tools for carbohydrate microarrays.⁷

Herein, the solid-phase syntheses of novel amphiphilic mucin-derived glyco(lipo)peptide antigens 9a–c for use in cancer immunotherapy are described. The antigens consist of complete tandem repeat domains of MUC1 glycosylated at Thr6 and/or Thr18 with a T_Nantigen residue. The latter is a tumour-related antigen particularly relevant for breast, colon and prostate cancer that has already found use in the preparation of various carbohydrate-based vaccine prototypes.⁸ The hydrophobic part of the conjugates is derived from tris(hydroxymethyl)aminomethane (TRIS) onto which three 1H,1H,2H,2H,3H,3H-perfluoroundecyl chains were crafted.⁹ The synthesis of the amphiphilic tail is depicted in Scheme 1 and involves a radical iodoperfluoroalkylation–hydrogenolysis reaction sequence. Thus, starting from Boc-protected allylated TRIS derivative 1,⁹ the radical F-alkyl iodide addition was initiated using Et₃B/O₂ in hexane,¹⁰ furnishing the triply iodoperfluoroalkylated adduct 2 in a reasonable yield of 60% after column chromatography. Subsequent hydrogenolysis of the carbon–iodine bond was performed with Pd/C at atmospheric pressure and afforded the polyfluorinated product 3 in 96% yield. Acidolytic cleavage of the Boc-protecting group followed by the addition to diglycolic anhydride and fluorous solid-phase extraction (F-SPE) finally provided the amphiphilic TRIS derivative 4 (80%, 2 steps) for attachment to the targeted glycopeptide building blocks.

Scheme 1 (a) Synthesis of the fluorinated membrane anchor 4. (b) Structure of the amphiphilic model peptide 5; for a description of the synthesis see Scheme 2.

The conjugation protocol of the fluorinated membrane anchor was established on a solid support using an amphiphilic 9-mer model peptide 5 (Scheme 1). The latter was obtained by employing the Fmoc strategy and a pre-loaded TentaGel S trityl resin, as reported previously.^11,12 Thus, coupling of the amino acids and the spacer 6¹³ (10 equiv., each) was achieved by activation with HBTU/HOBt¹⁴ and diisopropylethylamine (DIPEA) in DMF. Subsequent attachment of the perfluoroalkylated amphiphilic tail 4 proved to be difficult because of its reduced solubility and aggregation. Finally, conjugation of 4 (3 equiv.) was obtained by manual coupling in a Merrifield glass reactor over three days using HATU/HOAt¹⁵ in a solvent mixture of CH₂Cl₂, DMF and dioxane for activation. Detachment of the peptide from the resin and simultaneous cleavage of the acid-labile amino acid side chain protecting groups by treatment with a mixture of TFA, triisopropylsilane (TIS) and water provided 5 in a yield of 17% (based on the loaded resin) after semipreparative RP-HPLC. Next, amphiphilic mono- and diglycosylated antigen conjugates, with the T_Nantigen at position Thr6 and/or Thr18, were assembled (Scheme 2). In the light of the rather low yield of model peptide 5, the more reactive coupling reagents HATU and HOAt with N-methylmorpholine (NMM) in N-methylpyrrolidone (NMP) and extended reaction times of nine hours were employed for attachment of the sterically demanding T_N building block 7.¹² Besides, after Fmoc removal from the glycosylated peptides using piperidine in NMP, the following two amino acid residues were double coupled to minimise the amount of truncated peptides. Subsequent coupling of the remaining amino acids of the TR sequence and the triethylene glycol spacer 6 was then accomplished using the standard protocol (HBTU/HOBt, DIPEA in DMF), again. After manual attachment of the perfluoroalkyl anchor 4 using HATU/HOAt with DIPEA in CHCl₃/DMF/dioxane, the partially deblocked mono- and diglycosylated glyco(lipo)peptide analogs 8 were released from the resin (TFA/TIS/water) and purified by semi-preparative RP-HPLC, or more conveniently, by simple F-SPE (MeOH + 0.1% TFA). In the latter case, the targeted amphiphiles 8a, 8b, and 8c were obtained in overall yields of 17%, 27%, and 5%, respectively (based on the loaded resin).

Scheme 2 Solid-phase synthesis of amphiphilic glycopeptide antigens 9a–c; NMP = N-methylpyrrolidone, HBTU = O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HOBt = N-hydroxybenzotriazole, DIPEA = diisopropylethylamine, HATU = O-(7-aza-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HOAt = N-hydroxy-7-azabenzotriazole, NMM = N-methylmorpholine, TIS = triisopropylsilane.

The decrease in the yield of glycopeptides 8a and 8c, each carrying a GalNAc residue at position 3 of the growing peptide chain, is most presumably a result of insufficient couplings due to steric hindrance exerted by the glycan.^8e The final O-deacetylation of the glycan chains was accomplished by transesterification using catalytic amounts of NaOMe in methanol at pH 9.5–10.0 to furnish glycopeptide antigens 9a, 9b, and 9c after F-SPE in 58%, 87%, and 83% yield, respectively. The amphiphiles 9 were characterised by RP-HPLC, HR-ESI and MALDI-TOF mass spectrometry, as well as ¹⁹F NMR spectroscopy (see ESI†). All compounds show in their 400 MHz ¹H NMR spectra characteristic singlets for the C(CH₂O) and C(O)CH₂O protons of the perfluoroalkyl anchor which, in the case of glycopeptide 9b,¹⁶ resonate at 3.76 ppm, 4.01 ppm, and 4.05 ppm, respectively.

In a preliminary study, the use of the perfluoroalkylated glycoconjugates as potential diagnostic tools, e.g., for mapping the specificity of anti-MUC1 antibodies was investigated. For this purpose, recognition of the antigens by Balb/c-mouse serum antibodies was determined by means of an ELISA performed on polystyrene microtitre plates coated with the amphiphiles 9. The serum antibodies were raised after immunisation with a structurally related glyco-⁶TF-MUC1 TTox vaccine¹⁷ carrying the larger tumour-associated TF antigen. Previous studies^2b,8f,18 revealed that antibodies elicited by such fully synthetic glyco-MUC1 TTox conjugates, although being specific, also show some desirable flexibility with regard to the nature and position of the glycan chains. The serum, increasingly diluted, was added and the antibody concentrations were detected photometrically by using a secondary anti-mouse antibody conjugated to horseradish peroxidase.¹⁹Fig. 1 shows that the antibodies from the serum bind specifically to the immobilised amphiphiles 9 which are also recognised by the monoclonal antibody SM3.²⁰ The SM3 antibody results from immunisation with (partially) deglycosylated mucin from human milk and preferentially binds to the unglycosylated PDTRPAP region of the MUC1 tandem repeat but also accepts short glycans in various positions. Moreover, strong binding was observed for immobilised amphiphile 9b which carries a single glycan chain in the same position as the vaccine used for immunisation, while the related amphiphiles 9a and 9c were less well recognised. The perfluoroalkylated glycoconjugates are therefore considered promising tools for generating microarray platforms that display the required epitopes with high loadings and structural integrity. Besides, the initial results encourage further exploration of the amphiphilic glycopeptide antigens toward the development of antigen-delivery systems and fluorous microarray formats for tumour immunotherapy.

Fig. 1 (a) Binding of immobilised glycoconjugate 9b to the monoclonal antibody SM3²⁰ and to mouse serum antibodies obtained from vaccination with a glyco-⁶TF-MUC1 TTox vaccine.¹⁷ (b) Antibody recognition of structurally related immobilised glycoconjugates 9a–c. Optical density at λ = 410 nm.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the Fonds der Chemischen Industrie.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures and characterisation data for all new compounds. See DOI: 10.1039/c0cc02250k

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