scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor


The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10–29.

    Google Scholar 

  2. 2.

    Stewart BW, Wild CP. World cancer report 2014. World Health Organganization. Lyon, France: 2014.

  3. 3.

    Hammarström S. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol. 1999;9:67–81.

    PubMed  Google Scholar 

  4. 4.

    Thompson JA, Grunert F, Zimmermann W. Carcinoembryonic antigen gene family: molecular biology and clinical perspectives. J Clin Lab Anal U S. 1991;5:344–66.

    CAS  Google Scholar 

  5. 5.

    Ura Y, Ochi Y, Hamazu M, Ishida M, Nakajima K, Watanabe T. Studies on circulating antibody against carcinoembryonic antigen (CEA) and CEA-like antigen in cancer patients. Cancer Lett Neth; Jan De. 1985;25:283–95.

    CAS  Google Scholar 

  6. 6.

    Konstadoulakis MM, Syrigos KN, Albanopoulos C, Mayers G, Golematis B. The presence of anti-carcinoembryonic antigen (CEA) antibodies in the sera of patients with gastrointestinal malignancies. J Clin Immunol setembro De. 1994;14:310–3.

    CAS  Google Scholar 

  7. 7.

    Albanopoulos K, Armakolas A, Konstadoulakis MM, Leandros E, Tsiompanou E, Katsaragakis S, et al. Prognostic significance of circulating antibodies against carcinoembryonic antigen (anti-CEA) in patients with colon cancer. Am J Gastroenterol abril De. 2000;95:1056–61.

    CAS  Google Scholar 

  8. 8.

    Denapoli PMA, Zanetti BF, Dos Santos AA, de Moraes JZ, Han SW. Preventive DNA vaccination against CEA-expressing tumors with anti-idiotypic scFv6.C4 DNA in CEA-expressing transgenic mice. Cancer Immunol Immunother. 2016;66:333–42.

    PubMed  Google Scholar 

  9. 9.

    de Moraes JZ, Carneiro CR, Buchegger F, Mach JP, Lopes JD. Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system. J Cell Biochem novembro De. 1992;50:324–35.

    Google Scholar 

  10. 10.

    de Moraes JZ, Gesztesi JL, Westermann P, Le Doussal JM, Lopes JD, Mach JP. Anti-idiotypic monoclonal antibody AB3, reacting with the primary antigen (CEA), can localize in human colon-carcinoma xenografts as efficiently as AB1. Int J Cancer maio De. 1994;57:586–91.

    Google Scholar 

  11. 11.

    Pignatari GC, Takeshita D, Parise CB, Soares FA, de Moraes JZ, Han SW. Carcinoembryonic antigen (CEA) mimicry by an anti-idiotypic scFv isolated from anti-Id 6.C4 hybridoma. J Biotechnol Neth; Jan De. 2007;127:615–25.

    CAS  Google Scholar 

  12. 12.

    Foon KA, John WJ, Chakraborty M, Das R, Teitelbaum A, Garrison J, et al. Clinical and immune responses in resected colon cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen. J Clin Oncol. 1999;17:2889–95.

    CAS  PubMed  Google Scholar 

  13. 13.

    Foon KA, Chakraborty M, John WJ, Sherratt A, Kohler H, Bhattacharyachatterjee M. Immune-response to the carcinoembryonic antigen in patients treated with an antiidiotype antibody vaccine. J Clin Invest. 1995;96:334–42.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Chong G, Bhatnagar A, Cunningham D, Cosgriff TM, Harper PG, Steward W, et al. Phase III trial of 5-fluorouracil and leucovorin plus either 3H1 anti-idiotype monoclonal antibody or placebo in patients with advanced colorectal cancer. Ann Oncol. 2006;17:437–42.

    CAS  PubMed  Google Scholar 

  15. 15.

    Eades-Perner aM, van der Putten H, Hirth a, Thompson J, Neumaier M, von Kleist S, et al. Mice transgenic for the human carcinoembryonic antigen gene maintain its spatiotemporal expression pattern. Cancer Res. 1994;54:4169–76.

    CAS  PubMed  Google Scholar 

  16. 16.

    Rice J, Elliott T, Buchan S, Stevenson FK. DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines. J Immunol. 2001;167:1558–65.

    CAS  PubMed  Google Scholar 

  17. 17.

    Spellerberg MB, Zhu D, Thompsett A, King CA, Hamblin TJ, Stevenson FK. Promotion of anti-ldiotypic antibody responses induced by single chain Fv genes by fusion to tetanus toxin fragment C. J Immunol. 1997;159:1885–92

  18. 18.

    King CA, Spellerberg MB, Zhu D, Rice J, Sahota SS. Thompsett a R, et al. DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma. Nat Med. 1998;4:1281–6.

    CAS  PubMed  Google Scholar 

  19. 19.

    Smahel M, Polakova I, Duskova M, Ludvikova V, Kastankova I. The effect of helper epitopes and cellular localization of an antigen on the outcome of gene gun DNA immunization. Gene Ther Nat Publ Group. 2014;21:225–32.

    CAS  Google Scholar 

  20. 20.

    Lund LH, Andersson K, Zuber B, Karlsson A, Engström G, Hinkula J, et al. Signal sequence deletion and fusion to tetanus toxoid epitope augment antitumor immune responses to a human carcinoembryonic antigen (CEA) plasmid DNA vaccine in a murine test system. Cancer Gene Ther. 2003;10:365–76.

    CAS  PubMed  Google Scholar 

  21. 21.

    Facciabene A, Aurisicchio L, Elia L, Palombo F, Mennuni C, Ciliberto G, et al. DNA and adenoviral vectors encoding carcinoembryonic antigen fused to immunoenhancing sequences augment antigen-specific immune response and confer tumor protection. Hum Gene Ther. 2006;17:81–92.

    CAS  PubMed  Google Scholar 

  22. 22.

    Oosterhuis K, Hlschläger P, Van Den Berg JH, Toebes M, Gomez R, Schumacher TN, et al. Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7. Int J Cancer. 2011;129:397–406.

    CAS  PubMed  Google Scholar 

  23. 23.

    Panina-Bordignon P, Tan A, Termijtelen A, Corradin G, Lanzavecchia A. Universally immunogenic T cell epitopes:promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol. 1989;19:2237–42.

    CAS  PubMed  Google Scholar 

  24. 24.

    Demotz S, Lanzavecchia A, Eisel U, Niemann H, Widmann C, Corradin G. Delineation of several DR-restricted tetanus toxin T cell epitopes. J Immunol. 1989;142:394–402.

    CAS  PubMed  Google Scholar 

  25. 25.

    Helting TB, Zwisler O. Structure of Tetanus containing. J Biol Chem. 1977;252:194–8.

    CAS  PubMed  Google Scholar 

  26. 26.

    Chudley L, McCann K, Mander A, Tjelle T, Campos-Perez J, Godeseth R, et al. DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8+ T-cell responses and increases PSA doubling time. Cancer Immunol Immunother. 2012;61:2161–70.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    McCann KJ, Godeseth R, Chudley L, Mander A, Di Genova G, Lloyd-Evans P, et al. Idiotypic DNA vaccination for the treatment of multiple myeloma: Safety and immunogenicity in a phase I clinical study. Cancer Immunol Immunother Springe Berl Heidelb. 2015;64:1021–32.

    CAS  Google Scholar 

  28. 28.

    Aronovich EL, Bell JB, Belur LR, Gunther R, Koniar B, Erickson DCC, et al. Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: Implications for non-viral gene therapy of mucopolysaccharidoses. J Gene Med. 2007;9:403–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Tompkins WA, Watrach AM, Schmale JD, Schultz RM, Harris JA. Cultural and antigenic properties of newly established cell strains derived from adenocarcinomas of the human colon and rectum. J Natl Cancer Inst abril De. 1974;52:1101–10.

    CAS  Google Scholar 

  30. 30.

    Corbett TH, Griswold DPJ, Roberts BJ, Peckham JC, Schabel FMJ. Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with a note on carcinogen structure. Cancer Res setembro De. 1975;35:2434–9.

    CAS  Google Scholar 

  31. 31.

    Stilhano RS, Martin PKM, de Melo SM, Samoto VY, Peres GB, da Silva Michelacci YMC, et al. alpha- L-iduronidase gene-based therapy using the phiC31 system to treat mucopolysaccharidose type I mice. J Gene Med Engl. 2015;17:1–13.

    CAS  Google Scholar 

  32. 32.

    Martin PKM, Stilhano RS, Samoto VY, Takiya CM, Peres GB, da Silva Michelacci YMC, et al. Mesenchymal stem cells do not prevent antibody responses against human alpha-L-iduronidase when used to treat mucopolysaccharidosis type I. PLoS One. 2014;9:e92420.

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    MacCallum RM, Martin AC, Thornton JM. Antibody-antigen interactions: contact analysis and binding site topography. J Mol Biol outubro De. 1996;262:732–45.

    CAS  Google Scholar 

  34. 34.

    Ye J, Ma N, Madden TL, Ostell JM. IgBLAST: an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res Engl; julho De. 2013;41:W34–40.

    Google Scholar 

  35. 35.

    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol outubro De. 1990;215:403–10.

    CAS  Google Scholar 

  36. 36.

    McWilliam H, Li W, Uludag M, Squizzato S, Park YM, Buso N, et al. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res. 2013;41(Web Server issue):W597–600.

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, et al. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci. 2003;12:1007–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Moutaftsi M, Peters B, Pasquetto V, Tscharke DC, Sidney J, Bui H-H, et al. A consensus epitope prediction approach identifies the breadth of murine T(CD8+ )-cell responses to vaccinia virus. Nat Biotechnol. 2006;24:817–9.

    CAS  PubMed  Google Scholar 

  39. 39.

    Kotturi MF, Peters B, Buendia-Laysa FJ, Sidney J, Oseroff C, Botten J, et al. The CD8+T-cell response to lymphocytic choriomeningitis virus involves the L antigen: uncovering new tricks for an old virus. J Virol. 2007;81:4928–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Larsen JEP, Lund O, Nielsen M. Improved method for predicting linear B-cell epitopes. Immunome Res. 2006;2:2.

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Parise CB, Lisboa B, Takeshita D, Sacramento CB, de Moraes JZ, Han SW. Humoral immune response after genetic immunization is consistently improved by electroporation. Vaccines. 2008;26:3812–7.

    CAS  Google Scholar 

  42. 42.

    Hammarstrom S, Shively JE, Paxton RJ, Beatty BG, Larsson a, Ghosh R, et al. Antigenic sites in carcinoembryonic antigen. Cancer Res. 1989;49:4852–8.

    CAS  PubMed  Google Scholar 

  43. 43.

    Bjerner J, Lebedin Y, Bellanger L, Kuroki M, Shively JE, Varaas T, et al. Protein epitopes in carcinoembryonic antigen. Tumor Biol. 2002;23:249–62.

    CAS  Google Scholar 

  44. 44.

    Murakami M, Kuroki M, Arakawa F, Kuwahara M, Oikawa S, Nakazato H, et al. A reference of the GOLD classification of monoclonal antibodies against carcinoembryonic antigen to the domain structure of the carcinoembryonic antigen molecule. Hybridoma. 1995;14:19–28.

    CAS  PubMed  Google Scholar 

  45. 45.

    Sharma A, Kuzu OF, Nguyen FD, Sharma A, Noory M. Current state of animal (mouse) modeling in melanoma research. Cancer Growth Metastas-. 2015;8:81.

    Google Scholar 

  46. 46.

    Pervin S, Chakraborty M, Bhattacharya-Chatterjee M, Zeytin H, Foon KA, Chatterjee SK. Induction of antitumor immunity by an anti-idiotype antibody mimicking carcinoembryonic antigen. Cancer Res. 1997;57:728–34.

    CAS  PubMed  Google Scholar 

  47. 47.

    Saha A, Chatterjee SK, Foon KA, Bhattacharya-Chatterjee M. Anti-idiotype antibody induced cellular immunity in mice transgenic for human carcinoembryonic antigen. Immunology. 2006;118:483–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Saha A, Chatterjee SK, Foon KA, Celis E, Bhattacharya-Chatterjee M. Therapy of established tumors in a novel murine model transgenic for human carcinoembryonic antigen and HLA-A2 with a combination of anti-idiotype vaccine and CTL peptides of carcinoembryonic antigen. Cancer Res. 2007;67:2881–92.

    CAS  PubMed  Google Scholar 

  49. 49.

    Schwegler C, Dorn-Beineke A, Nittka S, Stocking C, Neumaier M. Monoclonal anti-idiotype antibody 6G6.C4 fused to GM-CSF is capable of breaking tolerance to carcinoembryonic antigen (CEA) in CEA-transgenic mice. Cancer Res. 2005;65:1925–33.

    CAS  PubMed  Google Scholar 

  50. 50.

    Kass E, Schlom J, Thompson J, Guadagni F, Graziano P, Greiner JW. Induction of protective host immunity to carcinoembryonic antigen (CEA), a self-antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus. Cancer Res. 1999;59:676–83.

    CAS  PubMed  Google Scholar 

  51. 51.

    Facciabene A, Aurisicchio L, Elia L, Palombo F, Mennuni C, Ciliberto G, et al. Vectors encoding carcinoembryonic antigen fused to the B subunit of heat-labile enterotoxin elicit antigen-specific immune responses and antitumor effects. Vaccine. 2007;26:47–58.

    CAS  PubMed  Google Scholar 

  52. 52.

    Kass E, Panicali DL, Mazzara G, Schlom J, Greiner JW. Granulocyte/macrophage-colony stimul factor prod recomb avian poxviruses enriches reg lymph nodes antigen-present cells acts immunoadjuvant. Cancer Res. 2001;27:206–14.

    Google Scholar 

  53. 53.

    Lurquin C, Lethe B, De Plaen E, Corbiere V, Theate I, van Baren N, et al. Contrasting frequencies of antitumor and anti-vaccine T cells in metastases of a melanoma patient vaccinated with a MAGE tumor antigen. J Exp Med. 2005;201:249–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Disis ML, Shiota FM, Cheever MA. Human HER-2/neu protein immunization circumvents tolerance to rat neu: a vaccine strategy for “self” tumour antigens. Immunology. 1998;93:192–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Chatterjee SK, Tripathi PK, Chakraborty M, Yannelli J, Wang H, Foon KA, et al. Molecular mimicry of carcinoembryonic antigen by peptides derived from the structure of an anti-idiotype antibody. Cancer Res. 1998;1217–25.

  56. 56.

    Gaida FJ, Pieper D, Roder UW, Shively JE, Wagener C, Neumaier M. Molecular characterization of a cloned idiotypic cascade containing a network antigenic determinant specific for the human carcinoembryonic antigen. J Biol Chem. 1993;268:14138–45.

    CAS  PubMed  Google Scholar 

  57. 57.

    Greiner JW, Zeytin H, Anver MR, Schlom J. Vaccine-based therapy directed against carcinoembryonic antigen demonstrates antitumor activity on spontaneous intestinal tumors in the absence of autoimmunity. Cancer Res. 2002;62:6944–51.

    CAS  PubMed  Google Scholar 

  58. 58.

    Hodge JW, Poole DJ, Aarts WM, Yafal AG, Gritz L, Schlom J. Modified vaccinia virus ankara recombinants are as potent as vaccinia recombinants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses. Cancer Res. 2003;63:7942–9.

    CAS  PubMed  Google Scholar 

  59. 59.

    Saha A, Chatterjee SK, Foon KA, Primus FJ, Sreedharan S, Mohanty K, et al. Dendritic cells pulse anti-idiot antib mimicking carcinoembryonic antigen (cea) can reverse immunol toler cea induce antitumor immun CEA transgenic mice. Cancer Res. 2004;7:4995–5003.

  60. 60.

    Toivonen JM, Oliván S, Osta R. Tetanus toxin c-fragment: The courier and the cure? Toxins (Basel). 2010;2:2622–44.

    CAS  Google Scholar 

Download references


This work was supported by São Paulo Research Foundation (FAPESP; Grant Numbers: 2012/21861-1 and # 2013/17224-9). BFZ was a recipient of FAPESP scholarship (2012/21861-1).

Authors’ contributions

BFZ: conception and design of the study, acquisition of data, analysis and interpretation of the data, drafting of the manuscript; CPF: acquisition of data, analysis and interpretation of data; JRCV: analysis and interpretation of the data; SWH: conception and design of the study, drafting of the manuscript, critical review of the manuscript for important intellectual content, and final approval of the version to be submitted.

Author information



Corresponding author

Correspondence to Sang Won Han.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zanetti, B.F., Ferreira, C.P., de Vasconcelos, J.R.C. et al. scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor. Gene Ther 26, 441–454 (2019).

Download citation

Further reading