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


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.


Cancer vaccine Conjugate vaccine Glycosylated MUC1 MUC1 Tn antigen 



Bovine serum albumin


Desialylated ovine submaxillary mucin


Enzyme-linked immunosorbent assay


Fluorescence-activated cell sorting


High-pH anion-exchange chromatography with pulsed amperometric detection


Human serum albumin


Keyhole limpet hemocyanin


m-Malemidobenzoyl-N-hydroxysuccinimide ester


Phosphate buffer saline


Quillaja saponaria Molina–based immunological adjuvant



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.


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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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