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.
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Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10–29.
Stewart BW, Wild CP. World cancer report 2014. World Health Organganization. Lyon, France: 2014.
Hammarström S. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol. 1999;9:67–81.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
Helting TB, Zwisler O. Structure of Tetanus containing. J Biol Chem. 1977;252:194–8.
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.
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.
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.
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.
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.
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.
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.
MacCallum RM, Martin AC, Thornton JM. Antibody-antigen interactions: contact analysis and binding site topography. J Mol Biol outubro De. 1996;262:732–45.
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.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol outubro De. 1990;215:403–10.
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.
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.
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.
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.
Larsen JEP, Lund O, Nielsen M. Improved method for predicting linear B-cell epitopes. Immunome Res. 2006;2:2.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Toivonen JM, Oliván S, Osta R. Tetanus toxin c-fragment: The courier and the cure? Toxins (Basel). 2010;2:2622–44.
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).
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.
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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). https://doi.org/10.1038/s41434-019-0062-y