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Quantification and functional evaluation of CD40L production from the adenovirus vector ONCOS-401


Adaptive immunity involves activation of T cells via antigen presentation by antigen presenting cells (APCs) along with the action of co-stimulatory molecules and pattern recognition receptors. Cluster of differentiation 40 (CD40) is one such costimulatory molecule that is expressed on APCs that binds to CD40 ligand (CD40L) on T helper cells and activates a signaling cascade, subsequently resulting in a wide range of immune and inflammatory responses. Considering its important role in regulation of immune response, CD40/40 L has been used for developing antitumor vaccines. In this study, we developed methods for evaluating and quantifying the activity of CD40L expressed from an adenovirus vector ONCOS-401. Our results show that the ONCOS-401 vector produces functional CD40L, which can bind and activate a NF-κB-dependent signaling cascade, leading to secreted embryonic alkaline phosphatase reporter production in HEK293-BLUE cells. In addition, quantification of CD40L production using enzyme-linked immunosorbent assay and HEK-293 BLUE reporter cells showed reproducibly higher recovery of CD40L from ONCOS-401 than from the negative control vector or uninfected cells with consistent inter and intra-assay precision. Thus, a rapid and easy method for quantifying and assessing CD40L production and activity from adenovirus vectors would support the assessment of efficacy of the vector for gene therapy - this was the objective of our study.

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  1. 1.

    Kambayashi T, Laufer TM. Atypical MHC class II-expressing antigen-presenting cells: can anything replace a dendritic cell? Nat Rev Immunol. 2014;14:719–730.

    CAS  Article  Google Scholar 

  2. 2.

    Mann ER, Li X. Intestinal antigen-presenting cells in mucosal immune homeostasis: crosstalk between dendritic cells, macrophages and B-cells. World J Gastroenterol. 2014;20:9653–9664.

    Article  Google Scholar 

  3. 3.

    Grewal IS, Flavell RA. CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol. 1998;16:111–135.

    CAS  Article  Google Scholar 

  4. 4.

    McWhirter SM, Pullen SS, Holton JM, Crute JJ, Kehry MR, Alber T. Crystallographic analysis of CD40 recognition and signaling by human TRAF2. Proc Natl Acad Sci USA. 1999;96:8408–8413.

    CAS  Article  Google Scholar 

  5. 5.

    Tsukamoto N, Kobayashi N, Azuma S, Yamamoto T, Inoue J. Two differently regulated nuclear factor kappaB activation pathways triggered by the cytoplasmic tail of CD40. Proc Natl Acad Sci USA. 1999;96:1234–1239.

    CAS  Article  Google Scholar 

  6. 6.

    Malinin NL, Boldin MP, Kovalenko AV, Wallach D. MAP3K-related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1. Nature. 1997;385:540–544.

    CAS  Article  Google Scholar 

  7. 7.

    Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC. The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J. 1997;16:6914–6925.

    CAS  Article  Google Scholar 

  8. 8.

    Ishida TK, Tojo T, Aoki T, Kobayashi N, Ohishi T, Watanabe T, et al. TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling. Proc Natl Acad Sci USA. 1996;93:9437–9442.

    CAS  Article  Google Scholar 

  9. 9.

    Kawabe T, Naka T, Yoshida K, Tanaka T, Fujiwara H, Suematsu S, et al. The immune responses in CD40-deficient mice: Impaired immunoglobulin class switching and germinal center formation. Immunity. 1994;1:167–178.

    CAS  Article  Google Scholar 

  10. 10.

    Chatzigeorgiou A, Lyberi M, Chatzilymperis G, Nezos A, Kamper E. CD40/CD40L signaling and its implication in health and disease. Biofactors. 1994;35:474–483.

    Article  Google Scholar 

  11. 11.

    Carlring J, Altaher HM, Clark S, Chen X, Latimer SL, Jenner T, et al. CD154-CD40 interactions in the control of murine B cell hematopoiesis. J Leukoc Biol. 2011;89:697–706.

    CAS  Article  Google Scholar 

  12. 12.

    Parameswaran S, Khalil M, Ahmed KA, Sharma RK, Xiang J. Enhanced protective immunity derived from dendritic cells with phagocytosis of CD40 ligand transgene-engineered apoptotic tumor cells via increased dendritic cell maturation. Tumori J. 2015;101:637–643.

    CAS  Article  Google Scholar 

  13. 13.

    Hanyu K, Iida T, Shiba H, Ohashi T, Eto Y, Yanaga K. Immunogene therapy by adenovirus vector expressing CD40 ligand for metastatic liver cancer in rats. Anticancer Res. 2008;28:2785–2789.

    PubMed  Google Scholar 

  14. 14.

    Vardouli L, Lindqvist C, Vlahou K, Loskog AS, Eliopoulos AG. Adenovirus delivery of human CD40 ligand gene confers direct therapeutic effects on carcinomas. Cancer Gene Ther. 2009;16:848–860.

    CAS  Article  Google Scholar 

  15. 15.

    Iida T, Shiba H, Misawa T, Ohashi T, Eto Y, Yanaga K. Immunogene therapy against colon cancer metastasis using an adenovirus vector expressingCD40 ligand. Surgery. 2010;148:925–935.

    Article  Google Scholar 

  16. 16.

    Dzojic H, Loskog A, Tötterman TH, Essand M. Adenovirus-mediated CD40 ligand therapy induces tumor cell apoptosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model. Prostate. 2006;66:831–838.

    CAS  Article  Google Scholar 

  17. 17.

    Malmström PU, Loskog AS, Lindqvist CA, Mangsbo SM, Fransson M, Wanders A, et al. AdCD40L immunogene therapy for bladder carcinoma--the first phase I/IIa trial. Clin Cancer Res. 2010;16:3279–3287.

    Article  Google Scholar 

  18. 18.

    Belousova B, Korokhov N, Krendelshchikova V, Simonenko V, Mikheeva G, Triozzi PL, et al. Genetically targeted adenovirus vector directed to CD40-expressing cells. J Virol. 2003;77:11367–11377.

    CAS  Article  Google Scholar 

  19. 19.

    Kuryk L, Haavisto E, Garofalo M, Capasso C, Hirvinen M, Pesonen S, et al. Synergistic anti-tumor efficacy of immunogenic adenovirus ONCOS-102 (Ad5/3-D24-GM-CSF) and standard of care chemotherapy in preclinical mesothelioma model. Int J Cancer. 2016;139:1883–1893.

    CAS  Article  Google Scholar 

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We thank Antti Vuolanto for the help and assistance with the method development.

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Correspondence to Lukasz Kuryk.

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LK, ASM, MJ are employees and/or shareholders in Targovax Oy in Finland and Targovax ASA in Norway.

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Kuryk, L., Møller, AS.W. & Jaderberg, M. Quantification and functional evaluation of CD40L production from the adenovirus vector ONCOS-401. Cancer Gene Ther 26, 26–31 (2019).

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