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Cancer Immunology, Immunotherapy

, Volume 54, Issue 5, pp 424–430 | Cite as

Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn

  • Ella Kagan
  • Govind Ragupathi
  • San San Yi
  • Celso A. Reis
  • Jeff Gildersleeve
  • Daniel Kahne
  • Henrik Clausen
  • Samuel J. Danishefsky
  • Philip O. LivingstonEmail author
Original Article

Abstract

We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.

Keywords

Cancer vaccine Conjugate vaccine Glycosylated MUC1 MUC1 Tn antigen 

Abbreviations

BSA

Bovine serum albumin

dOSM

Desialylated ovine submaxillary mucin

ELISA

Enzyme-linked immunosorbent assay

FACS

Fluorescence-activated cell sorting

HPAEC-PAD

High-pH anion-exchange chromatography with pulsed amperometric detection

HSA

Human serum albumin

KLH

Keyhole limpet hemocyanin

MBS

m-Malemidobenzoyl-N-hydroxysuccinimide ester

PBS

Phosphate buffer saline

QS21

Quillaja saponaria Molina–based immunological adjuvant

Notes

Acknowledgements

This work is supported by NIH grants CA33049 and CA52477, and the Breast Cancer Research Fund. C.A.R. is supported by FCT/POCTI 363767/99.

References

  1. 1.
    Livingston PO, Wong GYC, Adluri S, Tao Y, Padavan M, Parente R, Hanlon C, Helling F, Ritter G, Oettgen HF, Old LJ (1994) Improved survival in AJCC stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside. J Clin Oncol 12:1036–1044PubMedGoogle Scholar
  2. 2.
    Chapman PB, Morrissey DM, Panageas KS, Hamilton WB, Zhan C, Destro AN, Williams L, Israel RJ, Livingston PO (2000) Induction of antibodies against GM2 ganglioside by immunizing melanoma patients using GM2-KLH+QS21 vaccine: a dose-response study. Clin Cancer Res 6:874–879PubMedGoogle Scholar
  3. 3.
    Helling F, Shang Y, Calves M, Oettgen HF, Livingston PO (1994) Increased immunogenicity of GD3 conjugate vaccines: comparison of various carrier proteins and selection of GD3-KLH for further testing. Cancer Res 54:197–203PubMedGoogle Scholar
  4. 4.
    Ragupathi G, Meyers M, Adluri S, Howard L, Musselli C, Livingston PO (2000) Induction of antibodies against GD3 ganglioside in melanoma patients by vaccination with GD3-lactone-KLH conjugate plus immunological adjuvant QS-21. Int J Cancer 85:659–666CrossRefPubMedGoogle Scholar
  5. 5.
    Dickler MN, Ragupathi G, Liu NX, Musselli C, Martino DJ, Miller VA, Kris MG, Brezicka FT, Livingston PO, Grant SC (1999) Immunogenicity of the fucosyl-GM1-keyhole limpet hemocyanin (KLH) conjugate vaccine in patients with small cell lung cancer. Cancer Res 5:2773–2779Google Scholar
  6. 6.
    Ragupathi G, Slovin S, Adluri S, Sames D, Kim IJ, Kim HM, Spassova M, Bornmann WG, Lloyd K, Scher HI, Livingston PO, Danishefsky SJ (1999) A fully synthetic globo H carbohydrate vaccine induces a focused humoral response in prostate cancer patients: a proof of principle. Angewandte Chemie 38:563–566CrossRefGoogle Scholar
  7. 7.
    Gilewski T, Ragupathi G, Bhuta S, Williams LJ, Musselli C, Zhang XF, Bencsath KP, Panageas KS, Chin J, Norton L, Houghton AN, Livingston PO et al (2001) Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: a phase I trial. Proc Natl Acad Sci U S A 98:3270–3275Google Scholar
  8. 8.
    Sabbatini P, Kudryashov V, Ragupathi G, Danishefsky S, Livingston PO, Bornmann W, Spassova M, Spriggs D, Aghajanian C, Soignet S, Peyton M, O’Flaherty C et al (2000) Immunization of ovarian cancer patients with a synthetic LewisY–protein conjugate vaccine: clinical and serological results. Int J Cancer 87:79–85CrossRefPubMedGoogle Scholar
  9. 9.
    Adluri S, Gilewski T, Zhang S, Ramnath V, Ragupathi G, Livingston PO (1999) Specificity analysis of sera from breast cancer patients vaccinated with Muc1-KLH plus QS-21. Br J Cancer 79:1806–1812CrossRefPubMedGoogle Scholar
  10. 10.
    Gilewski T, Adluri S, Ragupathi G, Zhang S, Yao TJ, Panageas K, Moynahan M, Houghton A, Norton L, Livingston PO (2000) Vaccination of high risk breast cancer patients with Mucin-1 keyhole limpet hemocyanin conjugate plus QS-21. Clin Cancer Res 6:1693–1701PubMedGoogle Scholar
  11. 11.
    Zhang S, Walberg LA, Ogata S, Itzkowitz SH, Koganty RR, Reddish M, Gandhi SS, Longenecker BM, Lloyd KO, Livingston PO (1995) Immune sera and monoclonal antibodies define two configurations for the sialyl Tn tumor antigen. Cancer Res 55:3364–3368PubMedGoogle Scholar
  12. 12.
    Dicker M, Gilewski T, Ragupathi G, Adluri R, Koganty RR, Longenecker M, Houghton AN, Norton L, Livingston PO (1997) Vaccination of breast cancer patients (pts) with no evidence of disease (NED) with sialyl Tn cluster (sTa(c))-keyhole limpet hemocyanin (KLH) conjugate plus adjuvant QS-21: preliminary results. Proc ASCO 16:1572Google Scholar
  13. 13.
    Nakada H, Inoue M, Numata Y, Tanaka N, Funakoshi J, Fukui S, Mellors A, Yamashina I (1993) Epitopic structure of Tn glycophorin A for an anti-Tn antibody (MLS 128). Proc Natl Acad Sci U S A 90:2495–2499Google Scholar
  14. 14.
    Ragupathi G, Howard L, Cappello S, Koganty RR, Qiu D, Longenecker BM, Reddish MA, Lloyd KO, Livingston PO (1999) Vaccines prepared with sialyl-Tn and sialyl-Tn trimers using the 4-(4-maleimidomethyl) cyclohexane-1-carboxyl hydrazide linker group result in optimal antibody titers against ovine submaxillary mucin and sialyl-Tn-positive tumor cells. Cancer Immunol Immunother 48:1–8Google Scholar
  15. 15.
    Liang R, Yan L, Loebach J, Ge M, Uozumi Y, Sekanina K, Horan N, Gildersleeve J, Thompson C, Smith A, Biswas K, Still WC et al (1996) Parallel synthesis and screening of a solid phase carbohydrate library. Science 274:1520–1522CrossRefPubMedGoogle Scholar
  16. 16.
    Liang R, Loebach J, Horan N, Ge M, Thompson C, Yan L, Kahne D (1997) Polyvalent binding to carbohydrates immobilized on an insoluble resin. Proc Natl Acad Sci U S A 94:10554–10559Google Scholar
  17. 17.
    Ragupathi G, Koganty RR, Qui DS, Lloyd KO, Livingston PO (1998) A novel and efficient method for synthetic carbohydrate conjugate vaccine preparation: synthesis of sialyl Tn KLH conjugate using a 4-(4-N-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linker arm. Glycoconjugate J 15:217–221CrossRefGoogle Scholar
  18. 18.
    White T, Bennett EP, Takio K, Sorensen T, Bonding N, Clausen H (1995) Purification and cDNA cloning of a human UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem 270:24156–24165CrossRefPubMedGoogle Scholar
  19. 19.
    Bennett EP, Hassan H, Mandel U, Mirgorodskaya EP, Burchell J, Taylor-Papadimitriou J, Hollingsworth MA, Merkx G, Vankessel AG, Eiberg H, Steffensen R, Clausen H (1998) Cloning of a human UDP-N-acetyl-alpha-D-alactosaminepolypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat. J Biol Chem 273:30472–30481CrossRefPubMedGoogle Scholar
  20. 20.
    Zhang S, Graeber LA, Helling F, Ragupathi G, Adluri S, Lloyd KO, Livingston PO (1996) Augmenting the immunogenicity of synthetic MUC1 peptide vaccines in mice. Cancer Res 56:3315–3319PubMedGoogle Scholar
  21. 21.
    Rohrer JS, Miller HI (2003) Detecting O-linked oligosaccharides on glycoproteins. Anal Biochem 316:131–134CrossRefPubMedGoogle Scholar
  22. 22.
    Ogata S, Chen A, Itzkowitz SH (1994) Use of model cell lines to study the biosynthesis and biological role of cancer-associated sialosyl-Tn antigen. Cancer Res 54:4036–4044PubMedGoogle Scholar
  23. 23.
    Huntsberger DV, Leaverton PE (ed) (1970) In: Statistical inference in the biomedical sciences. Allyn Bacon, Boston, pp 138–140, 337–338Google Scholar
  24. 24.
    Kurosaka A, Kitagawa H, Fukui S, Numata Y, Nakada H, Fuakoshi I, Kawasaki T, Ogawa T, Iijima H, Yamashina I (1988) A monoclonal antibody that recognizes a cluster of a disaccharide, NeuAcα2→6GalNAc, in mucin-type glycoproteins. J Biol Chem 263:8724–8726PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Ella Kagan
    • 1
  • Govind Ragupathi
    • 1
  • San San Yi
    • 1
  • Celso A. Reis
    • 2
  • Jeff Gildersleeve
    • 4
  • Daniel Kahne
    • 5
  • Henrik Clausen
    • 3
  • Samuel J. Danishefsky
    • 1
  • Philip O. Livingston
    • 1
    Email author
  1. 1.Memorial Sloan-Kettering Cancer CenterNew YorkUSA
  2. 2.Institute of Molecular Pathology and ImmunologyUniversity of PortoPortoPortugal
  3. 3.School of DentistryUniversity of CopenhagenCopenhagenDenmark
  4. 4.Princeton UniversityPrincetonUSA
  5. 5.Harvard Medical SchoolBostonUSA

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