Glycoconjugate Journal

, Volume 36, Issue 5, pp 399–408 | Cite as

Fluorine-modified sialyl-Tn-CRM197 vaccine elicits a robust immune response

  • Chengcheng Song
  • Xiu-Jing Zheng
  • Haili Guo
  • Yafei Cao
  • Fan Zhang
  • Qin Li
  • Xin-Shan YeEmail author
  • Yifa ZhouEmail author
Original Article


Even though a vaccine that targets tumor-associated carbohydrate antigens on epithelial carcinoma cells presents an attractive therapeutic approach, relatively poor immunogenicity limits its development. In this study, we investigated the immunological activity of a fluoro-substituted Sialyl-Tn (F-STn) analogue coupled to the non-toxic cross-reactive material of diphtheria toxin197 (CRM197). Our results indicate that F-STn-CRM197 promotes a greater immunogenicity than non-fluorinated STn-CRM197. In the presence or absence of adjuvant, F-STn-CRM197 remarkably enhances both cellular and humoral immunity against STn by increasing antigen-specific lymphocyte proliferation and inducing a mixed Th1/Th2 response leading to production of IFN-γ and IL-4 cytokines, as well as STn-specific antibodies. Furthermore, antisera produced from F-STn-CRM197 immunization significantly recognizes STn-positive tumor cells and increases cancer cell lysis induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) pathways. Our data suggest that this F-STn vaccine may be useful for cancer immunotherapy and possibly for prophylactic prevention of cancer.


TACAs STn Glycoconjugate vaccine Cancer immunotherapy 



We are grateful for the support by the grants (2017ZX09309025) from the Ministry of Science and Technology of China, the National Natural Science Foundation of China (21738001), the Scientific and Technologic Foundation of Jilin Province (20190103070JH), the Project funded by China Postdoctoral Science Foundation (2018 M640276), and the Fundamental Research Funds for the Central Universities (2412018QD012). We are grateful to Prof. Kevin H Mayo for critical reading and editing of this manuscript.

Compliance with ethical standards

Conflict of interest

Xiu-Jing Zheng and Xin-Shan Ye have an ownership interest in the patent (China patent No. ZL 2010 10,202,388.5, Japan patent No. 5815687, Int. Appl. No. PCT/CN2011/000610). No potential conflicts of interest were disclosed by the other authors.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the Animal Ethical Experimentation Committee of Peking University (Beijing, China).

Supplementary material

10719_2019_9884_MOESM1_ESM.pdf (539 kb)
ESM 1 (PDF 539 kb)


  1. 1.
    Pinho, S.S., Reis, C.A.: Glycosylation in cancer: mechanisms and clinical implications. Nat. Rev. Cancer. 15(9), 540–555 (2015)PubMedGoogle Scholar
  2. 2.
    Dube, D.H., Bertozzi, C.R.: Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4(6), 477–488 (2005)PubMedGoogle Scholar
  3. 3.
    Guo, Z., Wang, Q.: Recent development in carbohydrate-based cancer vaccines. Curr. Opin. Chem. Biol. 13(5–6), 608–617 (2009)PubMedGoogle Scholar
  4. 4.
    Bonam, S.R., Partidos, C.D., Halmuthur, S.K.M., Muller, S.: An overview of novel adjuvants designed for improving vaccine efficacy. Trends Pharmacol. Sci. 38(9), 771–793 (2017)PubMedGoogle Scholar
  5. 5.
    Astronomo, R.D., Burton, D.R.: Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat. Rev. Drug Discov. 9(4), 308–324 (2010)PubMedGoogle Scholar
  6. 6.
    Shi, M., Kleski, K.A., Trabbic, K.R., Bourgault, J.P., Andreana, P.R.: Sialyl-Tn polysaccharide A1 as an entirely carbohydrate immunogen: synthesis and immunological evaluation. J. Am. Chem. Soc. 138(43), 14264–14272 (2016)PubMedGoogle Scholar
  7. 7.
    Ingale, S., Wolfert, M.A., Gaekwad, J., Buskas, T., Boons, G.J.: Robust immune responses elicited by a fully synthetic three-component vaccine. Nat. Chem. Biol. 3(10), 663–667 (2007)PubMedGoogle Scholar
  8. 8.
    Richichi, B., Thomas, B., Fiore, M., Bosco, R., Qureshi, H., Nativi, C., Renaudet, O., BenMohamed, L.: A cancer therapeutic vaccine based on clustered Tn-antigen mimetics induces strong antibody-mediated protective immunity. Angew. Chem., Int. Ed. Engl. 53(44), 11917–11920 (2014)Google Scholar
  9. 9.
    Slovin, S.F., Ragupathi, G., Musselli, C., Fernandez, C., Diani, M., Verbel, D., Danishefsky, S., Livingston, P., Scher, H.I.: Thomsen-Friedenreich (TF) antigen as a target for prostate cancer vaccine: clinical trial results with TF cluster (c)-KLH plus QS21 conjugate vaccine in patients with biochemically relapsed prostate cancer. Cancer Immunol. Immunother. 54(7), 694–702 (2005)PubMedGoogle Scholar
  10. 10.
    Jennings, H.J., Roy, R., Gamian, A.: Induction of meningococcal group B polysaccharide-specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J. Immunol. 137(5), 1708–1713 (1986)PubMedGoogle Scholar
  11. 11.
    Liu, C.C., Ye, X.S.: Carbohydrate-based cancer vaccines: target cancer with sugar bullets. Glycoconj. J. 29(5–6), 259–271 (2012)PubMedGoogle Scholar
  12. 12.
    Bruge, J., Bouveret-Le Cam, N., Danve, B., Rougon, G., Schulz, D.: Clinical evaluation of a group B meningococcal N-propionylated polysaccharide conjugate vaccine in adult, male volunteers. Vaccine. 22(9–10), 1087–1096 (2004)PubMedGoogle Scholar
  13. 13.
    Doores, K.J., Fulton, Z., Hong, V., Patel, M.K., Scanlan, C.N., Wormald, M.R., Finn, M.G., Burton, D.R., Wilson, I.A., Davis, B.G.: A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity. Proc. Natl. Acad. Sci. U. S. A. 107(40), 17107–17112 (2010)PubMedGoogle Scholar
  14. 14.
    Ragupathi, G., Meyers, M., Adluri, S., Howard, L., Musselli, C., Livingston, P.O.: Induction of antibodies against GD3 ganglioside in melanoma patients by vaccination with GD3-lactone-KLH conjugate plus immunological adjuvant QS-21. Int. J. Cancer. 85(5), 659–666 (2000)PubMedGoogle Scholar
  15. 15.
    Ragupathi, G., Livingston, P.O., Hood, C., Gathuru, J., Krown, S.E., Chapman, P.B., Wolchok, J.D., Williams, L.J., Oldfield, R.C., Hwu, W.J.: Consistent antibody response against ganglioside GD2 induced in patients with melanoma by a GD2 lactone-keyhole limpet hemocyanin conjugate vaccine plus immunological adjuvant QS-21. Clin. Cancer Res. 9(14), 5214–5220 (2003)PubMedGoogle Scholar
  16. 16.
    Krug, L.M., Ragupathi, G., Hood, C., George, C., Hong, F., Shen, R., Abrey, L., Jennings, H.J., Kris, M.G., Livingston, P.O.: Immunization with N-propionyl polysialic acid-KLH conjugate in patients with small cell lung cancer is safe and induces IgM antibodies reactive with SCLC cells and bactericidal against group B meningococci. Cancer Immunol. Immunother. 61(1), 9–18 (2012)PubMedGoogle Scholar
  17. 17.
    Zheng, X.J., Yang, F., Zheng, M., Huo, C.X., Zhang, Y., Ye, X.S.: Improvement of the immune efficacy of carbohydrate vaccines by chemical modification on the GM3 antigen. Org. Biomol. Chem. 13(22), 6399–6406 (2015)PubMedGoogle Scholar
  18. 18.
    Song, C., Sun, S., Huo, C.X., Li, Q., Zheng, X.J., Tai, G., Zhou, Y., Ye, X.S.: Synthesis and immunological evaluation of N-acyl modified Tn analogues as anticancer vaccine candidates. Bioorg. Med. Chem. 24(4), 915–920 (2016)PubMedGoogle Scholar
  19. 19.
    Sun, S., Zheng, X.J., Huo, C.X., Song, C., Li, Q., Ye, X.S.: Synthesis and evaluation of Glycoconjugates comprising N-Acyl-Modified Thomsen-Friedenreich antigens as anticancer vaccines. ChemMedChem. 11(10), 1090–1096 (2016)PubMedGoogle Scholar
  20. 20.
    Lee, H.Y., Chen, C.Y., Tsai, T.I., Li, S.T., Lin, K.H., Cheng, Y.Y., Ren, C.T., Cheng, T.J., Wu, C.Y., Wong, C.H.: Immunogenicity study of Globo H analogues with modification at the reducing or nonreducing end of the tumor antigen. J. Am. Chem. Soc. 136(48), 16844–16853 (2014)PubMedGoogle Scholar
  21. 21.
    Sahabuddin, S., Chang, T.C., Lin, C.C., Jan, F.D., Hsiao, H.Y., Huang, K.T., Chen, J.H., Horng, J.C., Ho, J.A.A., Lin, C.C.: Synthesis of N-modified sTn analogs and evaluation of their immunogenicities by microarray-based immunoassay. Tetrahedron. 66(38), 7510–7519 (2010)Google Scholar
  22. 22.
    Cao, Y., Stosiek, P., Springer, G.F., Karsten, U.: Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study. Histochem. Cell Biol. 106(2), 197–207 (1996)PubMedGoogle Scholar
  23. 23.
    Holmberg, L.A., Sandmaier, B.M.: Vaccination with Theratope (STn-KLH) as treatment for breast cancer. Expert Rev. Vaccines. 3(6), 655–663 (2004)PubMedGoogle Scholar
  24. 24.
    Miles, D., Roche, H., Martin, M., Perren, T.J., Cameron, D.A., Glaspy, J., Dodwell, D., Parker, J., Mayordomo, J., Tres, A., Murray, J.L., Ibrahim, N.K.: Phase III multicenter clinical trial of the sialyl-TN (STn)-keyhole limpet hemocyanin (KLH) vaccine for metastatic breast cancer. Oncologist. 16(8), 1092–1100 (2011)PubMedGoogle Scholar
  25. 25.
    Ibrahim, N.K., Murray, J.L., Zhou, D., Mittendorf, E.A., Sample, D., Tautchin, M., Miles, D.: Survival advantage in patients with metastatic breast cancer receiving endocrine therapy plus Sialyl Tn-KLH vaccine: post hoc analysis of a large randomized trial. J. Cancer. 4(7), 577–584 (2013)PubMedGoogle Scholar
  26. 26.
    Chang, T.C., Manabe, Y., Fujimoto, Y., Ohshima, S., Kametani, Y., Kabayama, K., Nimura, Y., Lin, C.C., Fukase, K.: 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. Angew. Chem., Int. Ed. Engl. 57(27), 8219–8224 (2018)Google Scholar
  27. 27.
    Ress, D.K., Baytas, S.N., Wang, Q., Munoz, E.M., Tokuzoki, K., Tomiyama, H., Linhardt, R.J.: Synthesis of double C-glycoside analogue of sTn. J. Org. Chem. 70(20), 8197–8200 (2005)PubMedGoogle Scholar
  28. 28.
    Kuberan, B., Sikkander, S.A., Tomiyama, H., Linhardt, R.J.: Synthesis of a C-glycoside analogue of sTn: an HIV- and tumor-associated antigen. Angew. Chem. Int. Ed. 42(18), 2073–2075 (2003)Google Scholar
  29. 29.
    Huo, C.X., Zheng, X.J., Xiao, A., Liu, C.C., Sun, S., Lv, Z., Ye, X.S.: Synthetic and immunological studies of N-acyl modified S-linked STn derivatives as anticancer vaccine candidates. Org. Biomol. Chem. 13(12), 3677–3690 (2015)PubMedGoogle Scholar
  30. 30.
    Yang, F., Zheng, X.J., Huo, C.X., Wang, Y., Zhang, Y., Ye, X.S.: Enhancement of the immunogenicity of synthetic carbohydrate vaccines by chemical modifications of STn antigen. ACS Chem. Biol. 6(3), 252–259 (2011)PubMedGoogle Scholar
  31. 31.
    Song, C., Zheng, X.J., Liu, C.C., Zhou, Y., Ye, X.S.: A cancer vaccine based on fluorine-modified sialyl-Tn induces robust immune responses in a murine model. Oncotarget. 8(29), 47330–47343 (2017)PubMedGoogle Scholar
  32. 32.
    Harris, J.R., Markl, J.: Keyhole limpet hemocyanin: molecular structure of a potent marine immunoactivator. A review. Eur. Urol. 37(Suppl 3), 24–33 (2000)PubMedGoogle Scholar
  33. 33.
    Lin, K.H., Liang, J.J., Huang, W.I., Lin-Chu, S.Y., Su, C.Y., Lee, Y.L., Jan, J.T., Lin, Y.L., Cheng, Y.S., Wong, C.H.: In vivo protection provided by a synthetic new alpha-galactosyl ceramide analog against bacterial and viral infections in murine models. Antimicrob. Agents Chemother. 54(10), 4129–4136 (2010)PubMedGoogle Scholar
  34. 34.
    Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150(1), 76–85 (1985)PubMedGoogle Scholar
  35. 35.
    Svennerholm, L.: Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta. 24(3), 604–611 (1957)PubMedGoogle Scholar
  36. 36.
    Guttormsen, H.K., Paoletti, L.C., Mansfield, K.G., Jachymek, W., Jennings, H.J., Kasper, D.L.: Rational chemical design of the carbohydrate in a glycoconjugate vaccine enhances IgM-to-IgG switching. Proc. Natl. Acad. Sci. U. S. A. 105(15), 5903–5908 (2008)PubMedGoogle Scholar
  37. 37.
    Qiao, J., Ghani, K., Caruso, M.: Diphtheria toxin mutant CRM197 is an inhibitor of protein synthesis that induces cellular toxicity. Toxicon. 51(3), 473–477 (2008)PubMedGoogle Scholar
  38. 38.
    Usonis, V., Bakasenas, V., Lockhart, S., Baker, S., Gruber, W., Laudat, F.: A clinical trial examining the effect of increased total CRM(197) carrier protein dose on the antibody response to Haemophilus influenzae type b CRM(197) conjugate vaccine. Vaccine. 26(35), 4602–4607 (2008)PubMedGoogle Scholar
  39. 39.
    Jones, C.: Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An. Acad. Bras. Cienc. 77(2), 293–324 (2005)PubMedGoogle Scholar
  40. 40.
    Xiong, Q., Wei, Y., Xie, H., Feng, Z., Gan, Y., Wang, C., Liu, M., Bai, F., Xie, F., Shao, G.: Effect of different adjuvant formulations on the immunogenicity and protective effect of a live mycoplasma hyopneumoniae vaccine after intramuscular inoculation. Vaccine. 32(27), 3445–3451 (2014)PubMedGoogle Scholar
  41. 41.
    Qiu, L., Gong, X., Wang, Q., Li, J., Hu, H., Wu, Q., Zhang, J., Guo, Z.: Combining synthetic carbohydrate vaccines with cancer cell glycoengineering for effective cancer immunotherapy. Cancer Immunol. Immunother. 61(11), 2045–2054 (2012)PubMedGoogle Scholar
  42. 42.
    Julien, S., Picco, G., Sewell, R., Vercoutter-Edouart, A.S., Tarp, M., Miles, D., Clausen, H., Taylor-Papadimitriou, J., Burchell, J.M.: Sialyl-Tn vaccine induces antibody-mediated tumour protection in a relevant murine model. Br. J. Cancer. 100(11), 1746–1754 (2009)PubMedGoogle Scholar
  43. 43.
    Slovin, S.F., Keding, S.J., Ragupathi, G.: Carbohydrate vaccines as immunotherapy for cancer. Immunol. Cell Biol. 83(4), 418–428 (2005)PubMedGoogle Scholar
  44. 44.
    Coffman, R.L., Sher, A., Seder, R.A.: Vaccine adjuvants: putting innate immunity to work. Immunity. 33(4), 492–503 (2010)PubMedGoogle Scholar
  45. 45.
    Wu, T.N., Lin, K.H., Chang, Y.J., Huang, J.R., Cheng, J.Y., Yu, A.L., Wong, C.H.: Avidity of CD1d-ligand-receptor ternary complex contributes to T-helper 1 (Th1) polarization and anticancer efficacy. Proc. Natl. Acad. Sci. U. S. A. 108(42), 17275–17280 (2011)PubMedGoogle Scholar
  46. 46.
    Galli, G., Pittoni, P., Tonti, E., Malzone, C., Uematsu, Y., Tortoli, M., Maione, D., Volpini, G., Finco, O., Nuti, S., Tavarini, S., Dellabona, P., Rappuoli, R., Casorati, G., Abrignani, S.: Invariant NKT cells sustain specific B cell responses and memory. Proc. Natl. Acad. Sci. U. S. A. 104(10), 3984–3989 (2007)PubMedGoogle Scholar
  47. 47.
    Lehuen, A., Fazilleau, N.: Innate iNKT cell help to B cells: fast but does not last. Nat. Immunol. 13(1), 11–13 (2011)PubMedGoogle Scholar
  48. 48.
    Billiau, A., Matthys, P.: Modes of action of Freund's adjuvants in experimental models of autoimmune diseases. J. Leukoc. Biol. 70(6), 849–860 (2001)PubMedGoogle Scholar
  49. 49.
    Slansky, J.E., Rattis, F.M., Boyd, L.F., Fahmy, T., Jaffee, E.M., Schneck, J.P., Margulies, D.H., Pardoll, D.M.: Enhanced antigenspecifc antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex. Immunity. 13(4), 529–538 (2000)PubMedGoogle Scholar
  50. 50.
    Pongdee, R., Liu, H.W.: Elucidation of enzyme mechanisms using fluorinated substrate analogues. Bioorg. Chem. 32(5), 393–437 (2004)PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Chengcheng Song
    • 1
    • 2
  • Xiu-Jing Zheng
    • 2
  • Haili Guo
    • 1
  • Yafei Cao
    • 2
  • Fan Zhang
    • 1
  • Qin Li
    • 2
  • Xin-Shan Ye
    • 2
    Email author
  • Yifa Zhou
    • 1
    Email author
  1. 1.School of Life SciencesNortheast Normal UniversityChangchunChina
  2. 2.State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical SciencesPeking UniversityBeijingChina

Personalised recommendations