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Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer

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Abstract

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

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References

  1. Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and disease. Cell 126:855–867. doi:10.1016/j.cell.2006.08.019

    Article  PubMed  CAS  Google Scholar 

  2. Baldus SE, Engelmann K, Hanisch FG (2004) MUC1 and the MUCs: a family of human mucins with impact in cancer biology. Crit Rev Clin Lab Sci 41:189–231. doi:10.1080/10408360490452040

    Article  PubMed  CAS  Google Scholar 

  3. Kufe D (2009) Mucins in cancer: function, prognosis and therapy. Nat Rev Cancer 9:874–885. doi:10.1038/nrc2761

    Article  PubMed  CAS  Google Scholar 

  4. Tsuboi S, Hatakeyama S, Ohyama C, Fukuda M (2012) Two opposing roles of O-glycans in tumor metastasis. Trends Mol Med 18:224–232. doi:10.1016/j.molmed.2012.02.001

    Article  PubMed  CAS  Google Scholar 

  5. Rabinovich GA, van Kooyk Y, Cobb BA (2012) Glycobiology of immune responses. Ann N Y Acad Sci 1253:1–15. doi:10.1111/j.1749-6632.2012.06492.x

    Article  PubMed  CAS  Google Scholar 

  6. Ten Hagen KG, Fritz TA, Tabak LA (2003) All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13:1R–16R. doi:10.1093/glycob/cwg007

    Article  PubMed  Google Scholar 

  7. Brockhausen I (2006) Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO Rep 7:599–604. doi:10.1038/sj.embor.7400705

    Article  PubMed  CAS  Google Scholar 

  8. Ju T, Cummings RD (2002) A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase. Proc Natl Acad Sci USA 99:16613–16618. doi:10.1073/pnas.262438199

    Article  PubMed  CAS  Google Scholar 

  9. Mi R, Song L, Wang Y, Ding X, Zeng J, Lehoux S, Aryal RP, Wang J, Crew VK, van Die I, Chapman AB, Cummings RD, Ju T (2012) Epigenetic silencing of the chaperone Cosmc in human leukocytes expressing tn antigen. J Biol Chem 287:41523–41533. doi:0.1074/jbc.M112.371989

    Article  PubMed  CAS  Google Scholar 

  10. Schietinger A, Philip M, Yoshida B, Azadi P, Liu H, Meredith S, Schreiber H (2006) A mutant chaperone converts a wild-type protein into a tumor-specific antigen. Science 314:304–308. doi:10.1126/science.1129200

    Article  PubMed  CAS  Google Scholar 

  11. Ju T, Lanneau GS, Gautam T, Wang Y, Xia B, Stowell SR, Willard MT, Wang W, Xia JY, Zuna RE, Laszik Z, Benbrook DM, Hanigan MH, Cummings RD (2008) Human tumor antigens Tn and sialyl Tn arise from mutations in Cosmc. Cancer Res 68:1636–1646. doi:10.1158/0008-5472.CAN-07-2345

    Article  PubMed  CAS  Google Scholar 

  12. Freire T, Bay S, von Mensdorff-Pouilly S, Osinaga E (2005) Molecular basis of incomplete O-glycan synthesis in breast cancer cells: putative role of MUC6 in Tn antigen expression. Cancer Res 65:7880–7887. doi:10.1158/0008-5472.CAN-04-3746

    PubMed  CAS  Google Scholar 

  13. Springer GF (1984) T and Tn, general carcinoma autoantigens. Science 224:1198–1206. doi:10.1126/science.6729450

    Article  PubMed  CAS  Google Scholar 

  14. Cao Y, Merling A, Karsten U, Goletz S, Punzel M, Kraft R, Butschak G, Schwartz-Albiez R (2008) Expression of CD175 (Tn), CD175 s (sialosyl-Tn) and CD176 (Thomsen-Friedenreich antigen) on malignant human hematopoietic cells. Int J Cancer 123:89–99. doi:10.1002/ijc.23493

    Article  PubMed  CAS  Google Scholar 

  15. Itzkowitz SH, Bloom EJ, Lau TS, Kim YS (1992) Mucin associated Tn and sialosyl-Tn antigen expression in colorectal polyps. Gut 33:518–523. doi:10.136/gut.33.4.518

    Article  PubMed  CAS  Google Scholar 

  16. Babino A, Oppezzo P, Bianco S, Barrios E, Berois N, Navarrete H, Osinaga E (2000) Tn antigen is a pre-cancerous biomarker in breast tissue and serum in N-nitrosomethylurea-induced rat mammary carcinogenesis. Int J Cancer 86:753–759. doi:10.1002/(SICI)1097-0215(20000615)86:6<753::AID-IJC1>3.0.CO;2-#

    Article  PubMed  CAS  Google Scholar 

  17. Springer GF (1997) Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy. J Mol Med 75:594–602. doi:10.1007/s001090050144

    Article  PubMed  CAS  Google Scholar 

  18. Lo-Man R, Vichier-Guerre S, Perraut R, Dériaud E, Huteau V, BenMohamed L, Diop OM, Livingston PO, Bay S, Leclerc C (2004) A fully synthetic therapeutic vaccine candidate targeting carcinoma-associated Tn carbohydrate antigen induces tumor-specific antibodies in nonhuman primates. Cancer Res 64:4987–4994. doi:10.1158/0008-5472.CAN-04-0252

    Article  PubMed  CAS  Google Scholar 

  19. Tarp MA, Clausen H (2008) Mucin-type O-glycosylation and its potential use in drug and vaccine development. Biochim Biophys Acta 1780:546–563. doi:10.1016/j.bbagen.2007.09.010

    Article  PubMed  CAS  Google Scholar 

  20. Ju T, Otto VI, Cummings RD (2011) The Tn antigen—structural simplicity and biological complexity. Angew Chem Int Ed Engl 50:1770–1791. doi:10.1002/anie.201002313

    Article  PubMed  CAS  Google Scholar 

  21. Manimala JC, Li Z, Jain A, VedBrat S, Gildersleeve JC (2005) Carbohydrate array analysis of anti-Tn antibodies and lectins reveals unexpected specificities: implications for diagnostic and vaccine development. ChemBioChem 6:2229–2241

    Article  PubMed  CAS  Google Scholar 

  22. Huang J, Byrd JC, Siddiki B, Yuan M, Lau E, Kim YS (1992) Monoclonal antibodies against partially deglycosylated colon cancer mucin that recognize Tn antigen. Dis Markers 10:81–94

    PubMed  CAS  Google Scholar 

  23. Itzkowitz S, Kjeldsen T, Friera A, Hakomori S, Yang US, Kim YS (1991) Expression of Tn, sialosyl Tn, and T antigens in human pancreas. Gastroenterology 100:1691–1700

    PubMed  CAS  Google Scholar 

  24. Ching CK, Holmes SW, Holmes GK, Long RG (1994) Blood-group sialyl-Tn antigen is more specific than Tn as a tumor marker in the pancreas. Pancreas 9:698–702

    Article  PubMed  CAS  Google Scholar 

  25. Cao Y, Stosiek P, Springer GF, Karsten U (1996) Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study. Histochem Cell Biol 106:197–207. doi:10.1007/BF02484401

    Article  PubMed  CAS  Google Scholar 

  26. Kawaguchi T, Takazawa H, Imai S, Morimoto J, Watanabe T, Kanno M, Igarashi S (2006) Expression of Vicia villosa agglutinin (VVA)-binding glycoprotein in primary breast cancer cells in relation to lymphatic metastasis: is atypical MUC1 bearing Tn antigen a receptor of VVA? Breast Cancer Res Treat 98:31–43. doi:10.1007/s10549-005-9115-6

    Article  PubMed  CAS  Google Scholar 

  27. Grinstead JS, Koganty RR, Krantz MJ, Longenecker BM, Campbell AP (2002) Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions. Biochemistry 41:9946–9961. doi:10.1021/bi012176z

    Article  PubMed  CAS  Google Scholar 

  28. Medeiros A, Bianchi S, Calvete JJ, Balter H, Bay S, Robles A, Cantacuzène D, Nimtz M, Alzari PM, Osinaga E (2000) Biochemical and functional characterization of the Tn-specific lectin from Salvia sclarea seeds. Eur J Biochem 267:1434–1440. doi:10.1046/j.1432-1327.2000.01141.x

    Article  PubMed  CAS  Google Scholar 

  29. Osinaga E, Bay S, Tello D, Babino A, Pritsch O, Assemat K, Cantacuzene D, Nakada H, Alzari P (2000) Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectrospcopy. FEBS Lett 469:24–28. doi:10.1016/S0014-5793(00)01248-5

    Article  PubMed  CAS  Google Scholar 

  30. Nakada H, Inoue M, Numata Y, Tanaka N, Funakoshi I, Fukui S, Mellors A, Yamashina I (1993) Epitopic structure of Tn glycophorin A for an anti-Tn antibody (MLS 128). Proc Natl Acad Sci USA 90:2495–2499. doi:10.1073/pnas.90.6.2495

    Article  PubMed  CAS  Google Scholar 

  31. Reis CA, Sorensen T, Mandel U, David L, Mirgorodskaya E, Roepstorff P, Kihlberg J, Hansen JE, Clausen H (1998) Development and characterization of an antibody directed to an alpha-N-acetyl-D-galactosamine glycosylated MUC2 peptide. Glycoconj J 15:51–62. doi:10.1023/A:1006939432665

    Article  PubMed  CAS  Google Scholar 

  32. Kuduk S, Schwarz J, Chen XT, Glunz P, Sames D, Ragupathi G, Livingston PO, Danishefsky S (1998) Synthetic and immunological studies of clustered modes of mucin-related Tn and TF O-linked antigens: the preparation of a glycopeptide-based vaccines for clinical trials against prostate cancer. J Am Chem Soc 120:12474–12485

    Article  CAS  Google Scholar 

  33. Bay S, Lo-Man R, Osinaga E, Nakada H, Leclerc C, Cantacuzène D (1997) Preparation of a multiple antigen glycopeptide (MAG) carrying the Tn antigen. A possible approach to a synthetic carbohydrate vaccine. J Peptide Res 49:620–625

    Article  CAS  Google Scholar 

  34. Lo-Man R, Bay S, Vichier-Guerre S, Deriaud E, Cantacuzene D, Leclerc C (1999) A fully synthetic immunogen carrying a carcinoma-associated carbohydrate for active specific immunotherapy. Cancer Res 59:1520–1524

    PubMed  CAS  Google Scholar 

  35. Lo-Man R, Vichier-Guerre S, Bay S, Deriaud E, Cantacuzene D, Leclerc C (2001) Anti-tumor immunity provided by a synthetic multiple antigenic glycopeptide displaying a tri-Tn glycotope. J Immunol 166:2849–2854

    PubMed  CAS  Google Scholar 

  36. Pancino G, Osinaga E, Vorauher W, Kakouche A, Mistro D, Charpin C, Roseto A (1990) Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method. Hybridoma 9:389–395

    Article  PubMed  CAS  Google Scholar 

  37. Vichier-Guerre S, Lo-Man R, Bay S, Deriaud E, Nakada H, Leclerc C, Cantacuzène D (2000) Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen. J Peptide Res 55:173–180. doi:10.1034/j.1399-3011.2000.00167.x

    Article  CAS  Google Scholar 

  38. Kononen J, Bubendorf L, Kallioniemi A, Bärlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP (1998) Tissue microarrays for high-throughput profiling of tumor specimens. Nat Med 4:844–847. doi:10.1038/nm0798-844

    Article  PubMed  CAS  Google Scholar 

  39. Camp RL, Charette LA, Rimm DL (2000) Validation of tissue microarray technology in breast carcinoma. Lab Invest 80:1943–1949. doi:10.1038/labinvest.3780204

    Article  PubMed  CAS  Google Scholar 

  40. Li Q, Anver MR, Butcher DO, Gildersleeve JC (2009) Resolving conflicting data on expression of the Tn antigen and implications for clinical trials with cancer vaccines. Mol Cancer Ther 8:971–979. doi:10.1158/1535-7163.MCT-08-0934

    Article  PubMed  CAS  Google Scholar 

  41. Reddish MA, Jackson L, Koganty RR, Qiu D, Hong W, Longenecker BM (1997) Specificities of anti-sialyl-Tn and anti-Tn monoclonal antibodies generated using novel clustered synthetic glycopeptide epitopes. Glycoconj J 14:549–560

    Article  PubMed  CAS  Google Scholar 

  42. Corzana F, Busto JH, Jiménez-Osés G, García de Luis M, Asensio JL, Jiménez-Barbero J, Peregrina JM, Avenoza A (2007) Serine versus threonine glycosylation: the methyl group causes a drastic alteration on the carbohydrate orientation and on the surrounding water shell. J Am Chem Soc 129:9458–9467. doi:10.1021/ja072181b

    Article  PubMed  CAS  Google Scholar 

  43. Corzana F, Busto JH, García de Luis M, Jiménez-Barbero J, Avenoza A, Peregrina JM (2009) The nature and sequence of the amino acid aglycone strongly modulates the conformation and dynamics effects of Tn antigen’s clusters. Chemistry 15:3863–3874. doi:10.1002/chem.200801777

    Article  PubMed  CAS  Google Scholar 

  44. Tachibana Y, Fletcher GL, Fujitani N, Tsuda S, Monde K, Nishimura SI (2004) Antifreeze glycoproteins: elucidation of the structural motifs that are essential for antifreeze activity. Angew Chem Int Ed 43:856–862. doi:10.1002/anie.200353110

    Article  CAS  Google Scholar 

  45. Blixt O, Lavrova OI, Mazurov DV, Cló E, Kracun SK, Bovin NV, Filatov AV (2012) Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells. Glycobiology 22:529–542. doi:10.1093/glycob/cwr178

    Article  PubMed  CAS  Google Scholar 

  46. Thanka Christlet TH, Veluraja K (2001) Database analysis of O-glycosylation sites in proteins. Biophys J 80:952–960

    Article  PubMed  CAS  Google Scholar 

  47. Elhammer AP, Kézdy FJ, Kurosaka A (1999) The acceptor specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycoconj J 16:171–180. doi:10.1023/A:1026465232149

    Article  PubMed  CAS  Google Scholar 

  48. Von Mensdorff-Pouilly S, Petrakou E, Kenemans P, Van Uffelen K, Verstraeten AA, Snijdewint FG, Van Kamp GJ, Schol DJ, Reis CA, Price MR, Livingston PO, Hilgers J (2000) Reactivity of natural and induced human antibodies to MUC1 mucin with MUC1 peptides and N-acetylgalactosamine (GalNAc) peptides. Int J Cancer 86:702–712. doi:10.1002/(SICI)1097-0215(20000601)86:5<702:AID-IJC16>3.0.CO;2-1

    Article  Google Scholar 

  49. Smorodin EP, Kurtenkov OA, Sergeyev BL, Kodar KE, Chuzmarov VI, Afanasyev VP (2008) Postoperative change of anti-Thomsen-Friedenreich and Tn IgG level: the follow-up study of gastrointestinal cancer patients. World J Gastroenterol 14:4352–4358. doi:10.3748/wjg.14.4352

    Article  PubMed  Google Scholar 

  50. Oyelaran O, Li Q, Farnsworth D, Gildersleeve JC (2009) Microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies. J Proteome Res 8:3529–3538. doi:10.1021/pr9002245

    Article  PubMed  CAS  Google Scholar 

  51. Matsukita S, Nomoto M, Kitajima S, Tanaka S, Goto M, Irimura T, Kim YS, Sato E, Yonezawa S (2003) Expression of mucins (MUC1, MUC2, MUC5AC and MUC6) in mucinous carcinoma of the breast: comparison with invasive ductal carcinoma. Histopathology 42:26–36. doi:10.1046/j.1365-2559.2003.01530.x

    Article  PubMed  CAS  Google Scholar 

  52. Pereira MB, Dias AJ, Reis CA, Schmitt FC (2001) Immunohistochemical study of the expression of MUC5AC and MUC6 in breast carcinomas and adjacent breast tissues. J Clin Pathol 54:210–213. doi:10.1136/jcp.54.3.210

    Article  PubMed  CAS  Google Scholar 

  53. Berois N, Varangot M, Sóñora C, Zarantonelli L, Pressa C, Laviña R, Porchet N, Aubert JP, Osinaga E (2003) Detection of bone marrow-disseminated breast cancer cells using a nested RT-PCR assay of MUC5B mRNA. Int J Cancer 103:550–555. doi:10.1002/ijc.10853

    Article  PubMed  CAS  Google Scholar 

  54. de Bolòs C, Gumà M, Barranco C, Garrido M, Kim YS, Real FX (1998) MUC6 expression in breast tissues and cultured cells: abnormal expression in tumors and regulation by steroid hormones. Int J Cancer 77:193–199. doi:10.1002/(SICI)1097-0215(19980717)77:2<193:AID-IJC4>3.0.CO;2-L

    Article  PubMed  Google Scholar 

  55. Byrd JC, Bresalier RS (2004) Mucins and mucin binding proteins in colorectal cancer. Cancer Metastasis Rev 23:77–99. doi:10.1023/A:1025815113599

    Article  PubMed  CAS  Google Scholar 

  56. Welinder C, Baldetorp B, Borrebaeck C, Fredlund BM, Jansson B (2011) A new murine IgG1 anti-Tn monoclonal antibody with in vivo anti-tumor activity. Glycobiology 21:1097–1107. doi:10.1093/glycob/cwr048

    Article  PubMed  CAS  Google Scholar 

  57. Ando H, Matsushita T, Wakitani M, Sato T, Kodama-Nishida S, Shibata K, Shitara K, Ohta S (2008) Mouse-human chimeric anti-Tn IgG1 induced anti-tumor activity against Jurkat cells in vitro and in vivo. Biol Pharm Bull 31:1739–1744. doi:10.1248/bpb.31.1739

    Google Scholar 

  58. Hubert P, Heitzmann A, Viel S, Nicolas A, Sastre-Garau X, Oppezzo P, Pritsch O, Osinaga E, Amigorena S (2011) Antibody-dependent cell cytotoxicity synapses form in mice during tumor-specific antibody immunotherapy. Cancer Res 71:5134–5143. doi:10.1158/0008-5472.CAN-10-4222

    Article  PubMed  CAS  Google Scholar 

  59. Lavrsen K, Madsen CB, Rasch MG, Woetmann A, Odum N, Mandel U, Clausen H, Pedersen AE, Wandall HH (2012) Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity. Glycoconj J. doi:10.1007/s10719-012-9437-7

    PubMed  Google Scholar 

  60. Danussi C, Coslovi A, Campa C, Mucignat MT, Spessotto P, Uggeri F, Paoletti S, Colombatti A (2009) A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction. Glycobiology 19:1056–1067. doi:10.1093/glycob/cwp085

    Article  PubMed  CAS  Google Scholar 

  61. Morita N, Yajima Y, Asanuma H, Nakada H, Fujita-Yamaguchi Y (2009) Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Biosci Trends 3:32–37

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by grants from Programmes Transversaux de Recherche (PTR, Institut Pasteur, Paris, France) and ECOS France-Uruguay Program to Eduardo Osinaga, Sylvie Bay and Claude Leclerc and from the Ligue Nationale Contre le Cancer (Equipe Labellisée 2011) and Banque Privée Européenne to Claude Leclerc, and Programa Grupos de Investigación (CSIC, Universidad de la República, Uruguay) to Eduardo Osinaga and Otto Pritsch.

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Mazal, D., Lo-Man, R., Bay, S. et al. Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer. Cancer Immunol Immunother 62, 1107–1122 (2013). https://doi.org/10.1007/s00262-013-1425-7

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