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Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis

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Abstract

The transcription factor brachyury is a major driver of epithelial to mesenchymal transition in human carcinoma cells. It is overexpressed in several human tumor types versus normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and (e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury (native) devoid of the agonist epitope, the yeast-brachyury (agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury (agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (f) generation of genetically engineered targeted T cells.

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Abbreviations

ATCC:

American Type Culture Collection

BFA:

Brefeldin A

BLAST:

Basic local alignment search tool

CTL:

Cytotoxic T lymphocyte

DCs:

Dendritic cells

EMT:

Epithelial to mesenchymal transition

IHC:

Immunohistochemistry

MFI:

Mean fluorescence intensity

MHC:

Major histocompatibility complex

PBMCs:

Peripheral blood mononuclear cells

References

  1. Thiery JP (2002) Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer 2(6):442–454. doi:10.1038/nrc822

    Article  CAS  PubMed  Google Scholar 

  2. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117(7):927–939. doi:10.1016/j.cell.2004.06.006

    Article  CAS  PubMed  Google Scholar 

  3. Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, Portillo F, Nieto MA (2000) The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2(2):76–83. doi:10.1038/35000025

    Article  CAS  PubMed  Google Scholar 

  4. Hamilton DH, Litzinger MT, Fernando RI, Huang B, Palena C (2012) Cancer vaccines targeting the epithelial-mesenchymal transition: tissue distribution of brachyury and other drivers of the mesenchymal-like phenotype of carcinomas. Semin Oncol 39(3):358–366. doi:10.1053/j.seminoncol.2012.02.005

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Kispert A, Koschorz B, Herrmann BG (1995) The T protein encoded by brachyury is a tissue-specific transcription factor. EMBO J 14(19):4763–4772

    CAS  PubMed Central  PubMed  Google Scholar 

  6. Palena C, Polev DE, Tsang KY, Fernando RI, Litzinger M, Krukovskaya LL, Baranova AV, Kozlov AP, Schlom J (2007) The human T-box mesodermal transcription factor brachyury is a candidate target for T-cell-mediated cancer immunotherapy. Clin Cancer Res 13(8):2471–2478. doi:10.1158/1078-0432.CCR-06-2353

    Article  CAS  PubMed  Google Scholar 

  7. Fernando RI, Litzinger M, Trono P, Hamilton DH, Schlom J, Palena C (2010) The T-box transcription factor brachyury promotes epithelial-mesenchymal transition in human tumor cells. J Clin Invest 120(2):533–544. doi:10.1172/JCI38379

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Palena CSJ (2013) Target: brachyury, a master driver of epithelial-to-mesenchymal transition (EMT). In: Marshall J (ed) Cancer therapeutic targets: SpringerReference (www.springerreference.com). Springer, Berlin. doi:10.1007/SpringerReference_3676052014-03-1406:40:12UTC

  9. Roselli M, Fernando RI, Guadagni F, Spila A, Alessandroni J, Palmirotta R, Costarelli L, Litzinger M, Hamilton D, Huang B, Tucker J, Tsang KY, Schlom J, Palena C (2012) Brachyury, a driver of the epithelial-mesenchymal transition, is overexpressed in human lung tumors: an opportunity for novel interventions against lung cancer. Clin Cancer Res 18(14):3868–3879. doi:10.1158/1078-0432.CCR-11-3211

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Huang B, Cohen JR, Fernando RI, Hamilton DH, Litzinger MT, Hodge JW, Palena C (2013) The embryonic transcription factor brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies. Cell Death Dis 4:e682. doi:10.1038/cddis.2013.208

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Palena C, Roselli M, Litzinger MT, Ferroni P, Costarelli L, Spila A, Cavaliere F, Huang B, Fernando RI, Hamilton DH, Jochems C, Tsang KY, Cheng Q, Kim Lyerly H, Schlom J, Guadagni F (2014) Overexpression of the EMT driver brachyury in breast carcinomas: association with poor prognosis. J Natl Cancer Inst 106(5). doi:10.1093/jnci/dju054

  12. Haro A, Yano T, Kohno M, Yoshida T, Koga T, Okamoto T, Takenoyama M, Maehara Y (2013) Expression of Brachyury gene is a significant prognostic factor for primary lung carcinoma. Ann Surg Oncol 20(Suppl 3):S509–S516. doi:10.1245/s10434-013-2914-9

    Article  PubMed  Google Scholar 

  13. Pinto F, Pertega-Gomes N, Pereira MS, Vizcaino JR, Monteiro P, Henrique RM, Baltazar F, Andrade RP, Reis RM (2014) T-box transcription factor brachyury is associated with prostate cancer progression and aggressiveness. Clin Cancer Res. doi:10.1158/1078-0432.CCR-14-0421

  14. Kilic N, Feldhaus S, Kilic E, Tennstedt P, Wicklein D, Wasielewski R, Viebahn C, Kreipe H, Schumacher U (2011) Brachyury expression predicts poor prognosis at early stages of colorectal cancer. Eur J Cancer 47(7):1080–1085. doi:10.1016/j.ejca.2010.11.015

    Article  PubMed  Google Scholar 

  15. Vujovic S, Henderson S, Presneau N, Odell E, Jacques TS, Tirabosco R, Boshoff C, Flanagan AM (2006) Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas. J Pathol 209(2):157–165. doi:10.1002/path.1969

    Article  CAS  PubMed  Google Scholar 

  16. Ellis JM, Henson V, Slack R, Ng J, Hartzman RJ, Katovich Hurley C (2000) Frequencies of HLA-A2 alleles in five U.S. population groups. Predominance of A*02011 and identification of HLA-A*0231. Hum Immunol 61(3):334–340

  17. Hamilton DH, Litzinger MT, Jales A, Huang B, Fernando RI, Hodge JW, Ardiani A, Apelian D, Schlom J, Palena C (2013) Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury–yeast vaccine. Oncotarget 4(10):1777–1790

    PubMed Central  PubMed  Google Scholar 

  18. Heery CR, Singh H, Marte JL, Madan RA, O’Sullivan Coyne GH, Farsaci B, Rodell TC, Palena C, Schlom J, JL G (2014) NCI experience using yeast–brachyury vaccine (GI-6301) in patients (pts) with advanced chordoma. 2014 ASCO Annual Meeting J Clin Oncol 32:5 s (suppl; abstr 3081)

  19. Open label study to evaluate the safety and tolerability of GI-6301 (whole heat-killed recombinant yeast modified to express brachyury protein) in adults with solid tumors (NCT01519817) http://clinicaltrials.gov/show/NCT01519817

  20. Singh H, Heery CR, Marte JL, Farsaci B, Madan RA, O’Sullivan Coyne GH, Palena C, Rodell TC, Schlom J, Gulley JL (2014) A phase I study of a yeast-based therapeutic cancer vaccine, GI-6301, targeting brachyury in patients with metastatic carcinoma. J Clin Oncol 32 (suppl; abstr e14026)

  21. Grey HM, Ruppert J, Vitiello A, Sidney J, Kast WM, Kubo RT, Sette A (1995) Class I MHC-peptide interactions: structural requirements and functional implications. Cancer Surv 22:37–49

    CAS  PubMed  Google Scholar 

  22. Terasawa H, Tsang KY, Gulley J, Arlen P, Schlom J (2002) Identification and characterization of a human agonist cytotoxic T-lymphocyte epitope of human prostate-specific antigen. Clin Cancer Res 8(1):41–53

    CAS  PubMed  Google Scholar 

  23. Madan RA, Mohebtash M, Arlen PM, Vergati M, Rauckhorst M, Steinberg SM, Tsang KY, Poole DJ, Parnes HL, Wright JJ, Dahut WL, Schlom J, Gulley JL (2012) Ipilimumab and a poxviral vaccine targeting prostate-specific antigen in metastatic castration-resistant prostate cancer: a phase 1 dose-escalation trial. Lancet Oncol 13(5):501–508. doi:10.1016/S1470-2045(12)70006-2

    Article  CAS  PubMed  Google Scholar 

  24. Vaccine and antibody treatment of prostate cancer (NCT00113984). http://www.clinicaltrials.gov/ct2/show/NCT00113984?term=NCT00113984&rank=1

  25. Britten CM, Meyer RG, Kreer T, Drexler I, Wolfel T, Herr W (2002) The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8(+) T lymphocytes in IFN-gamma ELISPOT assays. J Immunol Methods 259(1–2):95–110

    Article  CAS  PubMed  Google Scholar 

  26. DeMars R, Rudersdorf R, Chang C, Petersen J, Strandtmann J, Korn N, Sidwell B, Orr HT (1985) Mutations that impair a posttranscriptional step in expression of HLA-A and -B antigens. Proc Natl Acad Sci USA 82(23):8183–8187

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Parker KC, Bednarek MA, Coligan JE (1994) Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 152(1):163–175

    CAS  PubMed  Google Scholar 

  28. Nijman HW, Houbiers JG, Vierboom MP, van der Burg SH, Drijfhout JW, D’Amaro J, Kenemans P, Melief CJ, Kast WM (1993) Identification of peptide sequences that potentially trigger HLA-A2.1-restricted cytotoxic T lymphocytes. Eur J Immunol 23(6):1215–1219. doi:10.1002/eji.1830230603

    Article  CAS  PubMed  Google Scholar 

  29. Cereda V, Poole D, Palena C, Das S, Bera T, Remondo C, Gulley J, Arlen P, Yokokawa J, Pastan I, Schlom J, Tsang K (2010) New gene expressed in prostate: a potential target for T cell-mediated prostate cancer immunotherapy. Cancer Immunol Immunother 59(1):63–71. doi:10.1007/s00262-009-0723-6

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Yokokawa J, Bera TK, Palena C, Cereda V, Remondo C, Gulley JL, Arlen PM, Pastan I, Schlom J, Tsang KY (2007) Identification of cytotoxic T-lymphocyte epitope(s) and its agonist epitope(s) of a novel target for vaccine therapy (PAGE4). Int J Cancer 121(3):595–605. doi:10.1002/ijc.22698

    Article  CAS  PubMed  Google Scholar 

  31. Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J (1995) Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 87(13):982–990

    Article  CAS  PubMed  Google Scholar 

  32. Jochems C, Tucker JA, Vergati M, Boyerinas B, Gulley JL, Schlom J, Tsang KY (2014) Identification and characterization of agonist epitopes of the MUC1-C oncoprotein. Cancer Immunol Immunother 63(2):161–174. doi:10.1007/s00262-013-1494-7

    Article  CAS  PubMed  Google Scholar 

  33. Remondo C, Cereda V, Mostbock S, Sabzevari H, Franzusoff A, Schlom J, Tsang KY (2009) Human dendritic cell maturation and activation by a heat-killed recombinant yeast (Saccharomyces cerevisiae) vector encoding carcinoembryonic antigen. Vaccine 27(7):987–994. doi:10.1016/j.vaccine.2008.12.002

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Yokokawa J, Palena C, Arlen P, Hassan R, Ho M, Pastan I, Schlom J, Tsang KY (2005) Identification of novel human CTL epitopes and their agonist epitopes of mesothelin. Clin Cancer Res 11(17):6342–6351. doi:10.1158/1078-0432.ccr-05-0596

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Vitiello A, Marchesini D, Furze J, Sherman LA, Chesnut RW (1991) Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex. J Exp Med 173(4):1007–1015

    Article  CAS  PubMed  Google Scholar 

  36. Irwin MJ, Heath WR, Sherman LA (1989) Species-restricted interactions between CD8 and the alpha 3 domain of class I influence the magnitude of the xenogeneic response. J Exp Med 170(4):1091–1101

    Article  CAS  PubMed  Google Scholar 

  37. Hamann D, Kostense S, Wolthers KC, Otto SA, Baars PA, Miedema F, van Lier RA (1999) Evidence that human CD8 + CD45RA + CD27- cells are induced by antigen and evolve through extensive rounds of division. Int Immunol 11(7):1027–1033

    Article  CAS  PubMed  Google Scholar 

  38. Plunkett FJ, Franzese O, Finney HM, Fletcher JM, Belaramani LL, Salmon M, Dokal I, Webster D, Lawson AD, Akbar AN (2007) The loss of telomerase activity in highly differentiated CD8 + CD28-CD27-T cells is associated with decreased Akt (Ser473) phosphorylation. J Immunol 178(12):7710–7719

    Article  CAS  PubMed  Google Scholar 

  39. Hamilton DH, Huang B, Fernando RI, Tsang KY, Palena C (2014) WEE1 inhibition alleviates resistance to immune attack of tumor cells undergoing epithelial-mesenchymal transition. Cancer Res 74(9):2510–2519. doi:10.1158/0008-5472.CAN-13-1894

    Article  CAS  PubMed  Google Scholar 

  40. Bilusic M, Heery CR, Arlen PM, Rauckhorst M, Apelian D, Tsang KY, Tucker JA, Jochems C, Schlom J, Gulley JL, Madan RA (2014) Phase I trial of a recombinant yeast-CEA vaccine (GI-6207) in adults with metastatic CEA-expressing carcinoma. Cancer Immunol Immunother 63(3):225–234. doi:10.1007/s00262-013-1505-8

    Article  CAS  PubMed  Google Scholar 

  41. Bernstein MB, Chakraborty M, Wansley EK, Guo Z, Franzusoff A, Mostbock S, Sabzevari H, Schlom J, Hodge JW (2008) Recombinant Saccharomyces cerevisiae (yeast-CEA) as a potent activator of murine dendritic cells. Vaccine 26(4):509–521. doi:10.1016/j.vaccine.2007.11.033

    Article  CAS  PubMed  Google Scholar 

  42. Wansley EK, Chakraborty M, Hance KW, Bernstein MB, Boehm AL, Guo Z, Quick D, Franzusoff A, Greiner JW, Schlom J, Hodge JW (2008) Vaccination with a recombinant Saccharomyces cerevisiae expressing a tumor antigen breaks immune tolerance and elicits therapeutic antitumor responses. Clin Cancer Res 14(13):4316–4325. doi:10.1158/1078-0432.CCR-08-0393

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. A phase 2 study of GI-6207 in patients with recurrent medullary thyroid cancer (NCT01856920). http://clinicaltrials.gov/ct2/show/NCT01856920?term=NCT01856920&rank=1

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Acknowledgments

Grant support was provided by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, and a Cooperative Research and Development Agreement between the National Cancer Institute and GlobeImmune, Inc. The authors thank Diane J. Poole, Garland Davis and Curtis Randolph for technical assistance and Debra Weingarten for editorial assistance in the preparation of this manuscript.

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The authors declare that they have no conflicts of interest.

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Correspondence to Jeffrey Schlom.

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Jo A. Tucker and Caroline Jochems have contributed equally to this paper as primary authors. Jeffrey Schlom and Kwong-Yok Tsang have contributed equally to this paper as senior authors.

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Tucker, J.A., Jochems, C., Boyerinas, B. et al. Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis. Cancer Immunol Immunother 63, 1307–1317 (2014). https://doi.org/10.1007/s00262-014-1603-2

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