Tumor-associated carcinoembryonic antigen (CEA) is a natural target for vaccines against colorectal cancers. Our previous experience with a DNA vaccine with scFv6.C4, a CEA surrogate, showed a CEA-specific immune response with 40% of tumor-free mice after challenge with B16F10-CEA and 47% with MC38-CEA cells. These percentages increased to 63% after using FrC as an adjuvant. To further enhance the vaccine efficacy, we tested GM-CSF and IFNγ as adjuvants. C57BL/6J-CEA2682 mice were immunized 4 times with uP-PS/scFv6.C4, uP-PS/scFv6.C4 + uP-IFNγ, or uP-PS/scFv6.C4 + uP-GMCSF. After one week, the mice were challenged with MC38-CEA, and tumor growth was monitored over 100 days. Immunization with scFv6.C4 and scFv6.C4 + GM-CSF resulted in a gradual increase in the anti-CEA antibody titer, while scFv6.C4 + IFNγ immunization led to a rapid and sustained increase in the titer. The addition of IFNγ also induced higher CD4 + and CD8 + responses. When challenged, almost 80% of the scFv6.C4 + IFNγ-vaccinated mice did not develop tumors, while the others had a significant tumor growth delay. The probability of being tumor-free was 2700% higher using scFv6.C4 + IFNγ than scFv6.C4. The addition of GM-CSF had no additional effect on tumor protection. DNA immunization with scFv6.C4 + IFNγ, but not GM-CSF, increased the antitumor effect via readily sustained specific humoral and cytotoxic responses to CEA.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7–30.
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.
Zanetti BF, Ferreira CP, de Vasconcelos JRC, Han SW. scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor. Gene Ther. 2019;26:441–54.
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. 2007;127:615–25.
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. 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. 1994;57:586–91.
Geethadevi A, Jadhav K, Kumar G, Parashar D scFv6.C4 DNA vaccine with fragment C of tetanus toxin increases protective immunity against CEA-expressing tumor. Gene Ther. 2020;26:441–54. https://doi.org/10.1038/s41434-020-0161-9.
Coleman DL, Chodakewitz JA, Bartiss AH, Mellors JW. Granulocyte-macrophage colony-stimulating factor enhances selective effector functions of tissue-derived macrophages. Blood. 1988;72:573–8.
Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992;176:1693–702.
Steinman RM, Witmer-Pack M, Inaba K. Dendritic cells: antigen presentation, accessory function and clinical relevance. Adv Exp Med Biol. 1993;329:1–9.
Clive KS, Tyler JA, Clifton GT, Holmes JP, Mittendorf EA, Ponniah S, et al. Use of GM-CSF as an adjuvant with cancer vaccines: beneficial or detrimental? Expert Rev Vaccines. 2010;9:519–25.
Kass E, Panicali DL, Mazzara G, Schlom J, Greiner JW Granulocyte / macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant granulocyte / macrophage-colony stimulating factor produced by recomb. Cancer Res. 2001;27:206–14.
Aarts WM, Schlom J, Hodge JW. Vector-based vaccine / cytokine combination therapy to enhance induction of immune responses to a self-antigen and antitumor activity. Am Assoc Cancer Res. 2002;62:5770–7.
Lima J, Jenkins C, Guerrero A, Triozzi PL, Shaw DR, Strong TV. A DNA vaccine encoding genetic fusions of carcinoembryonic antigen (CEA) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Vaccine. 2005;23:1273–83.
Hallermalm K, Johansson S, Bråve A, Ek M, Engström G, Boberg A, et al. Pre-clinical evaluation of a CEA DNA prime/protein boost vaccination strategy against colorectal cancer. Scand J Immunol. 2007;66:43–51.
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.
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.
Luo Y, O’Hagan D, Zhou H, Singh M, Ulmer J, Reisfeld RA, et al. Plasmid DNA encoding human carcinoembryonic antigen (CEA) adsorbed onto cationic microparticles induces protective immunity against colon cancer in CEA-transgenic mice. Vaccine. 2003;21:1938–47.
Samanci A, Yi Q, Fagerberg J, Strigard K, Smith G, Ruden U, et al. Pharmacological administration of granulocyte/macrophage-colony-stimulating factor is of significant importance for the induction of a strong humoral and cellular response in patients immunized with recombinant carcinoembryonic antigen. Cancer Immunol Immunother. 1998;47:131–42.
Ullenhag GJ, Frödin JE, Jeddi-Tehrani M, Strigård K, Eriksson E, Samanci A, et al. Durable carcinoembryonic antigen (CEA)-specific humoral and cellular immune responses in colorectal carcinoma patients vaccinated with recombinant CEA and granulocyte/macrophage colony-stimulating factor. Clin Cancer Res. 2004;10:3273–81.
Duggan MC, Jochems C, Donahue RN, Richards J, Karpa V, Foust E, et al. A phase I study of recombinant (r) vaccinia-CEA(6D)-TRICOM and rFowlpox-CEA(6D)-TRICOM vaccines with GM-CSF and IFN-α-2b in patients with CEA-expressing carcinomas. Cancer Immunol Immunother. 2016;65:1353–64.
Marshall JL, Gulley JL, Arlen PM, Beetham PK, Tsang KY, Slack R, et al. Phase I study of sequential vaccinations with fowlpox-CEA(6D)-TRICOM alone and sequentially with vaccinia-CEA(6D)-TRICOM, with and without granulocyte-macrophage colony-stimulating factor, in patients with carcinoembryonic antigen-expressing carcinomas. J Clin Oncol. 2005;23:720–31.
Geynisman DM, Zha Y, Kunnavakkam R, Aklilu M, Catenacci DV, Polite BN, et al. A randomized pilot phase I study of modified carcinoembryonic antigen (CEA) peptide (CAP1-6D)/montanide/GM-CSF-vaccine in patients with pancreatic adenocarcinoma. J Immunother cancer. 2013;1:8.
Farrar M, Schreiber R. The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol. 1993;11:571–611.
Ikeda H, Old LJ, Schreiber RD. The roles of IFNγ in protection against tumor development and cancer immunoediting. Cytokine Growth Factor Rev. 2002;13:95–109.
Miller CHT, Maher SG, Young HA. Clinical use of Interferon- γ. Ann N Y Acad Sci. 2009;79:69–79.
Song K, Chang Y, Prud’homme GJ. Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer. Gene Ther. 2000;7:481–92.
Sacramento CB, Cantagalli VD, Grings M, Carvalho LP, Baptista-Silva JCC, Beutel A, et al. Granulocyte-macrophage colony-stimulating factor gene based therapy for acute limb ischemia in a mouse model. J Gene Med. 2009;11:345–53.
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. 1975;35:2434–9.
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. 1974;52:1101–10.
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.
Parise CB, Lisboa B, Takeshita D, Sacramento CB, de Moraes JZ, Han SW. Humoral immune response after genetic immunization is consistently improved by electroporation. Vaccine. 2008;26:3812–7.
Goldstein MJ, Mitchell EP. Carcinoembryonic antigen in the staging and follow-up of patients with colorectal cancer. Cancer Invest. England. 2005;23:338–51.
Duffy MJ. Carcinoembryonic antigen as a marker for colorectal cancer: is it clinically useful? Clin Chem. 2001;47:624–30.
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.
Finkelman FD, Katona IM, Mosmann TR, Coffman RL. IFN-gamma regulates the isotypes of Ig secreted during in vivo humoral immune responses. J Immunol. 1988;140:1022–7.
Kawano Y, Noma T, Yata J. Regulation of human IgG subclass production by cytokines. IFN-gamma and IL-6 act antagonistically in the induction of human IgG1 but additively in the induction of IgG2. J Immunol. 1994;153:4948–58.
Conaghan PJ, Ashraf SQ, Tytherleigh MG, Wilding JL, Tchilian E, Bicknell D, et al. Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen. Br J Cancer. 2008;98:1217–25.
Blumenthal RD, Osorio L, Hayes MK, Horak ID, Hansen HJ, Goldenberg DM. Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a human colonic carcinoma xenograft. Cancer Immunol Immunother. 2005;54:315–27.
Sharkey RM, Juweid M, Shevitz J, Behr T, Dunn R, Swayne LC, et al. Evaluation of a complementarity-determining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies. Cancer Res. 1995;55:5935s–5945s.
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.
Saha A, Chatterjee SK, Foon KA, Primus FJ, Sreedharan S, Mohanty K, et al. Dendritic cells pulsed with an anti-idiotype antibody mimicking carcinoembryonic antigen (CEA) can reverse immunological tolerance to CEA and induce antitumor immunity in CEA transgenic mice. Cancer Res. 2004;7:4995–5003.
Chiang CL-L, Benencia F, Coukos G. Whole tumor antigen vaccines. Semin Immunol. 2010;22:132–43.
Joshi VB, Geary SM, Gross BP, Wongrakpanich A, Norian LA, Salem AK. Tumor lysate-loaded biodegradable microparticles as cancer vaccines. Expert Rev Vaccines. 2014;13:9–15.
Tempero MA, Sivinski C, Steplewski Z, Harvey E, Klassen L, Kay HD. Phase II trial of interferon gamma and monoclonal antibody 17-1A in pancreatic cancer: biologic and clinical effects. J Clin Oncol. 1990;8:2019–26.
Wiesenfeld M, O’Connell MJ, Wieand HS, Gonchoroff NJ, Donohue JH, Fitzgibbons RJJ, et al. Controlled clinical trial of interferon-gamma as postoperative surgical adjuvant therapy for colon cancer. J Clin Oncol. 1995;13:2324–9.
This work was supported by the São Paulo Research Foundation (FAPESP; Grant Numbers: 2012/21861-1 and # 2013/17224-9). BFZ was a recipient of a FAPESP scholarship (2012/21861-1).
Conflict of interest
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The original online version of this article was revised: Fig. 2 has been corrected.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Zanetti, B.F., Ferreira, C.P., Vasconcelos, J.R.C. et al. Adjuvant properties of IFN-γ and GM-CSF in the scFv6.C4 DNA vaccine against CEA-expressing tumors. Gene Ther 30, 41–50 (2023). https://doi.org/10.1038/s41434-021-00270-w