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Intratumoral expression using a NFkB-based promoter enhances IL12 antitumor efficacy

Abstract

Interleukin 12 is a promising anti-cancer agent; however, IL12 systemic administration is hampered by side-effects. Although intratumoral administration of IL12 is giving promising results in clinical trials, only a small percentage of patients show a complete therapeutic response. This outcome could be improved by controlling the IL12 expression window. In this work we have tested the efficacy of a self-processing P2A and codon optimized murine IL12 (mIL12Pop) using inflammation-regulated lentivectors in a syngeneic tumor model. Our results show that implantation of cells expressing mIL12Pop employing either the strong constitutive SFFV promoter or a NFkB-based promoter reduced tumor growth, caused CD8+ T cell activation and increased IFNγ production. Importantly, the use of NFkBp-mIL12Pop increased the number of CD8+ TILs and improved the remission rate without increasing IL12-serum concentration. Further experiments suggest that there is a threshold intratumoral IL12 concentration that must be reached to trigger an efficient antitumor response and a limit that once surpassed causes detrimental systemic side effects. Altogether, these results demonstrate that using NFKBp-mIL12Pop significantly increases the overall survival of the mice. In summary, this new inflammation-regulated expression system might be useful for the development of new IL12 delivery systems with improved anti-tumor activity and limited toxicity.

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References

  1. 1.

    Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Invest. 2007;117:1137–46.

    CAS  Article  Google Scholar 

  2. 2.

    Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer. 2004;4:11–22.

    CAS  Article  Google Scholar 

  3. 3.

    Vignali DA, Kuchroo VK. IL-12 family cytokines: immunological playmakers. Nat Immunol. 2012;13:722–8.

    CAS  Article  Google Scholar 

  4. 4.

    Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev. 2002;13:155–68.

    CAS  Article  Google Scholar 

  5. 5.

    Germann T, Gately MK, Schoenhaut DS, Lohoff M, Mattner F, Fischer S, et al. Interleukin-12/T cell stimulating factor, a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells. Eur J Immunol. 1993;23:1762–70.

    CAS  Article  Google Scholar 

  6. 6.

    Voest EE, Kenyon BM, OʼReilly MS, Truitt G, DʼAmato RJ, Folkman J. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst. 1995;87:581–6.

    CAS  Article  Google Scholar 

  7. 7.

    Kodama T, Takeda K, Shimozato O, Hayakawa Y, Atsuta M, Kobayashi K, et al. Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12. Eur J Immunol. 1999;29:1390–6.

    CAS  Article  Google Scholar 

  8. 8.

    Gately MK, Gubler U, Brunda MJ, Nadeau RR, Anderson TD, Lipman JM, et al. Interleukin-12: a cytokine with therapeutic potential in oncology and infectious diseases. Ther Immunol. 1994;1:187–96.

    CAS  PubMed  Google Scholar 

  9. 9.

    Sarmiento UM, Riley JH, Knaack PA, Lipman JM, Becker JM, Gately MK, et al. Biologic effects of recombinant human interleukin-12 in squirrel monkeys (Sciureus saimiri). Lab Invest. 1994;71:862–73.

    CAS  PubMed  Google Scholar 

  10. 10.

    Mazzolini G, Prieto J, Melero I. Gene therapy of cancer with interleukin-12. Curr Pharm Des. 2003;9:1981–91.

    CAS  Article  Google Scholar 

  11. 11.

    Freytag SO, Barton KN, Zhang Y. Efficacy of oncolytic adenovirus expressing suicide genes and interleukin-12 in preclinical model of prostate cancer. Gene Ther. 2013;20:1131–9.

    CAS  Article  Google Scholar 

  12. 12.

    Paul D, Qazilbash MH, Song K, Xu H, Sinha BK, Liu J, et al. Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12). Cancer Gene Ther. 2000;7:308–15.

    CAS  Article  Google Scholar 

  13. 13.

    Wei LZ, Xu Y, Nelles EM, Furlonger C, Wang JC, Di Grappa MA, et al. Localized interleukin-12 delivery for immunotherapy of solid tumours. J Cell Mol Med. 2013;17:1465–74.

    CAS  Article  Google Scholar 

  14. 14.

    Passer BJ, Cheema T, Wu S, Wu CL, Rabkin SD, Martuza RL. Combination of vinblastine and oncolytic herpes simplex virus vector expressing IL-12 therapy increases antitumor and antiangiogenic effects in prostate cancer models. Cancer Gene Ther. 2013;20:17–24.

    CAS  Article  Google Scholar 

  15. 15.

    Tietje A, Li J, Yu X, Wei Y. MULT1E/mIL-12: a novel bifunctional protein for natural killer cell activation. Gene Ther. 2014;21:468–75.

    CAS  Article  Google Scholar 

  16. 16.

    Melero I, Quetglas JI, Reboredo M, Dubrot J, Rodriguez-Madoz JR, Mancheno U, et al. Strict requirement for vector-induced type I interferon in efficacious antitumor responses to virally encoded IL-12. Cancer Res. 2015;75:497–507.

    Article  Google Scholar 

  17. 17.

    Brown BD, Sitia G, Annoni A, Hauben E, Sergi LS, Zingale A, et al. In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance. Blood. 2007;109:2797–805.

    CAS  Article  Google Scholar 

  18. 18.

    Breckpot K, Aerts JL, Thielemans K. Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics. Gene Ther. 2007;14:847–62.

    CAS  Article  Google Scholar 

  19. 19.

    Dullaers M, Van Meirvenne S, Heirman C, Straetman L, Bonehill A, Aerts JL, et al. Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors. Gene Ther. 2006;13:630–40.

    CAS  Article  Google Scholar 

  20. 20.

    Rowe HM, Lopes L, Ikeda Y, Bailey R, Barde I, Zenke M, et al. Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene. Mol Ther. 2006;13:310–9.

    CAS  Article  Google Scholar 

  21. 21.

    Labbe A, Nelles M, Walia J, Jia L, Furlonger C, Nonaka T, et al. IL-12 immunotherapy of murine leukaemia: comparison of systemic versus gene modified cell therapy. J Cell Mol Med. 2009;13:1962–76.

    Article  Google Scholar 

  22. 22.

    Lorenzo C, Perez-Chacon G, Garaulet G, Mallorquin Z, Zapata JM, Rodriguez A. Efficient expression of bioactive murine IL12 as a self-processing P2A polypeptide driven by inflammation-regulated promoters in tumor cell lines. Cancer Gene Ther. 2015;22:542–51.

    CAS  Article  Google Scholar 

  23. 23.

    Garaulet G, Alfranca A, Torrente M, Escolano A, Lopez-Fontal R, Hortelano S, et al. IL10 released by a new inflammation-regulated lentiviral system efficiently attenuates zymosan-induced arthritis. Mol Ther. 2013;21:119–30.

    CAS  Article  Google Scholar 

  24. 24.

    Rodriguez A, Flemington EK. Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies. Anal Biochem. 1999;272:171–81.

    CAS  Article  Google Scholar 

  25. 25.

    Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol. 1997;15:871–5.

    CAS  Article  Google Scholar 

  26. 26.

    Scherr M, Battmer K, Blomer U, Ganser A, Grez M. Quantitative determination of lentiviral vector particle numbers by real-time PCR. Biotechniques. 2001;31:520–22. 524, passim.

    CAS  Article  Google Scholar 

  27. 27.

    Bishop CE. A miniaturised single-step method of cell cloning. J Immunol Methods. 1981;46:47–51.

    CAS  Article  Google Scholar 

  28. 28.

    Mukhopadhyay A, Wright J, Shirley S, Canton DA, Burkart C, Connolly RJ, et al. Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy. Gene Ther. 2018. https://doi.org/10.1038/s41434-018-0044-5. [Epub ahead of print].

    Article  Google Scholar 

  29. 29.

    Pavlin D, Cemazar M, Sersa G, Tozon N. IL-12 based gene therapy in veterinary medicine. J Transl Med. 2012;10:234–45.

    CAS  Article  Google Scholar 

  30. 30.

    Lasek W, Zagozdzon R, Jakobisiak M. Interleukin 12: still a promising candidate for tumor immunotherapy? Cancer Immunol Immunother. 2014;63:419–35.

    CAS  Article  Google Scholar 

  31. 31.

    Ruland J. Return to homeostasis: downregulation of NF-kappaB responses. Nat Immunol. 2011;12:709–14.

    CAS  Article  Google Scholar 

  32. 32.

    Ben-Neriah Y, Karin M. Inflammation meets cancer, with NF-kappaB as the matchmaker. Nat Immunol. 2011;12:715–23.

    CAS  Article  Google Scholar 

  33. 33.

    Hess SD, Egilmez NK, Bailey N, Anderson TM, Mathiowitz E, Bernstein SH, et al. Human CD4+T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma. J Immunol. 2003;170:400–12.

    CAS  Article  Google Scholar 

  34. 34.

    Yang L, Zaharoff DA. Role of chitosan co-formulation in enhancing interleukin-12 delivery and antitumor activity. Biomaterials. 2013;34:3828–36.

    CAS  Article  Google Scholar 

  35. 35.

    Mendiratta SK, Quezada A, Matar M, Wang J, Hebel HL, Long S, et al. Intratumoral delivery of IL-12 gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity. Gene Ther. 1999;6:833–9.

    CAS  Article  Google Scholar 

  36. 36.

    Goyvaerts C, Broos K, Escors D, Heirman C, Raes G, De Baetselier P, et al. The transduction pattern of IL-12-encoding lentiviral vectors shapes the immunological outcome. Eur J Immunol. 2015;45:3351–61.

    CAS  Article  Google Scholar 

  37. 37.

    Reed JM, Branigan PJ, Bamezai A. Interferon gamma enhances clonal expansion and survival of CD4+T cells. J Interferon Cytokine Res. 2008;28:611–22.

    CAS  Article  Google Scholar 

  38. 38.

    Lucas ML, Heller R. IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma. DNA Cell Biol. 2003;22:755–63.

    CAS  Article  Google Scholar 

  39. 39.

    Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, et al. Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol. 2008;26:5896–903.

    CAS  Article  Google Scholar 

  40. 40.

    Burkart C, Mukhopadhyay A, Shirley SA, Connolly RJ, Wright JH, Bahrami A, et al. Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters. Gene Ther. 2018;25:93–103.

    CAS  Article  Google Scholar 

  41. 41.

    Mahvi DM, Henry MB, Albertini MR, Weber S, Meredith K, Schalch H, et al. Intratumoral injection of IL-12 plasmid DNA–results of a phase I/IB clinical trial. Cancer Gene Ther. 2007;14:717–23.

    CAS  Article  Google Scholar 

  42. 42.

    Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. Non-viral vectors for gene-based therapy. Nat Rev Genet. 2014;15:541–55.

    CAS  Article  Google Scholar 

  43. 43.

    Komita H, Zhao X, Katakam AK, Kumar P, Kawabe M, Okada H, et al. Conditional interleukin-12 gene therapy promotes safe and effective antitumor immunity. Cancer Gene Ther. 2009;16:883–91.

    CAS  Article  Google Scholar 

  44. 44.

    Barrett JA, Cai H, Miao J, Khare PD, Gonzalez P, Dalsing-Hernandez J, et al. Regulated intratumoral expression of IL-12 using a RheoSwitch Therapeutic System® (RTS®) gene switch as gene therapy for the treatment of glioma. Cancer Gene Ther. 2018;25:106–16.

    CAS  Article  Google Scholar 

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Acknowledgements

We thank Dr. Filip Lim for critical reading of the manuscript and helpful discussions. We thankfully acknowledge the technical assistance at the CNIO Histopathology Core Unit and Animal Facility.

Funding

AR is supported by the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2012-32166) and the Comunidad Autonoma de Madrid, Spain (S2010/BMD-2312). FM is supported by the Comunidad Autonoma de Madrid, Spain (S2017/BMD-3867) and co-financed by European Structural and Investment Funds. AA and JMZ are supported by the Instituto de Salud Carlos III, Spain (PI15/01491 and PI16/00895, respectively). HA holds a research fellowship from Spanish Ministry of Education, Culture and Sports (FPU14/04726).

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Correspondence to Antonio Rodríguez.

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Garaulet, G., Pérez-Chacon, G., Alarcón, H. et al. Intratumoral expression using a NFkB-based promoter enhances IL12 antitumor efficacy. Cancer Gene Ther 26, 216–233 (2019). https://doi.org/10.1038/s41417-018-0076-4

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