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Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters

Abstract

The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines results in serious, potentially life-threatening, adverse effects. By contrast, nucleic acid transfer via electroporation (EP) is a safe and effective method of delivering plasmid-encoded cytokines to tumors. Intratumoral delivery of IL-12 plasmid DNA by electroporation (IT-pIL12-EP) produced objective response rates in Phase 2 clinical trials in metastatic melanoma. However, only 17.9% of patients receiving IT-pIL12-EP show a complete therapeutic response. Here, we sought to improve the antitumor efficacy of our clinical IT-pIL12-EP plasmid electroporation platform. We evaluated multiple plasmid designs for IL-12 expression. IL-12 expression from a plasmid incorporating a picornavirus-derived co-translational P2A site was the most effective in expressing IL-12p70. In addition, modifying the electroporation parameters improved transfection efficiency and expression of plasmid-derived IL-12p70, as well as its downstream effector IFN-γ in vivo. Finally, using a murine melanoma model that is representative of the intended target patient population, we show that combining modified electroporation conditions with the pIL12-P2A plasmid expression enhances the systemic antitumor response. These improvements to the IT-pIL12-EP platform may improve patient clinical response rates and survival when translated to clinical trials.

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References

  1. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1982;1:841–5.

    CAS  Article  Google Scholar 

  2. Yarmush ML, Golberg A, Sersa G, Kotnik T, Miklavcic D. Electroporation-based technologies for medicine: principles, applications, and challenges. Annu Rev Biomed Eng. 2014;16:295–320.

    CAS  Article  Google Scholar 

  3. Rosazza C, Meglic SH, Zumbusch A, Rols MP, Miklavcic D. Gene electrotransfer: a mechanistic perspective. Curr Gene Ther. 2016;16:98–129.

    CAS  Article  Google Scholar 

  4. Trinchieri G, Rengaraju M, D’Andrea A, Valiante NM, Kubin M, Aste M, et al. Producer cells of interleukin 12. Parasitol Today. 1993;9:97.

    CAS  Article  Google Scholar 

  5. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol. 2001;1:135–45.

    CAS  Article  Google Scholar 

  6. Presky DH, Yang H, Minetti LJ, Chua AO, Nabavi N, Wu CY, et al. A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits. Proc Natl Acad Sci USA. 1996;93:14002–7.

    CAS  Article  Google Scholar 

  7. Gately MK, Warrier RR, Honasoge S, Carvajal DM, Faherty DA, Connaughton SE, et al. Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo. Int Immunol. 1994;6:157–67.

    CAS  Article  Google Scholar 

  8. Trinchieri G. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv Immunol. 1998;70:83–243.

    CAS  Article  Google Scholar 

  9. Brown TJ, Lioubin MN, Marquardt H. Purification and characterization of cytostatic lymphokines produced by activated human T lymphocytes. Synergistic antiproliferative activity of transforming growth factor beta 1, interferon-gamma, and oncostatin M for human melanoma cells. J Immunol. 1987;139:2977–83.

    CAS  PubMed  Google Scholar 

  10. Coughlin CM, Salhany KE, Gee MS, LaTemple DC, Kotenko S, Ma X, et al. Tumor cell responses to IFNgamma affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity. 1998;9:25–34.

    CAS  Article  Google Scholar 

  11. 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 

  12. Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL. Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther. 1996;7:1995–2002.

    CAS  Article  Google Scholar 

  13. Shimizu T, Berhanu A, Redlinger RE,Jr., Watkins S, Lotze MT, Barksdale EM,Jr. Interleukin-12 transduced dendritic cells induce regression of established murine neuroblastoma. J Pediatr Surg. 2001;36:1285–92.

    CAS  Article  Google Scholar 

  14. Heinzerling L, Dummer R, Pavlovic J, Schultz J, Burg G, Moelling K. Tumor regression of human and murine melanoma after intratumoral injection of IL-12-encoding plasmid DNA in mice. Exp Dermatol. 2002;11:232–40.

    CAS  Article  Google Scholar 

  15. Lucas ML, Heller L, Coppola D, Heller R. IL-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous B16.F10 melanoma. Mol Ther. 2002;5:668–75.

    CAS  Article  Google Scholar 

  16. Lohr F, Lo DY, Zaharoff DA, Hu K, Zhang X, Li Y, et al. Effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation. Cancer Res. 2001;61:3281–4.

    CAS  PubMed  Google Scholar 

  17. Heller L, Merkler K, Westover J, Cruz Y, Coppola D, Benson K, et al. Evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a B16 mouse melanoma model. Clin Cancer Res. 2006;12:3177–83.

    CAS  Article  Google Scholar 

  18. 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 

  19. Cha E, Daud A. Plasmid IL-12 electroporation in melanoma. Hum Vaccin Immunother. 2012;8:1734–8.

    CAS  Article  Google Scholar 

  20. Cohen J. IL-12 deaths: explanation and a puzzle. Science. 1995;270:908.

    CAS  Article  Google Scholar 

  21. Trinchieri G, Pflanz S, Kastelein RA. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity. 2003;19:641–4.

    CAS  Article  Google Scholar 

  22. Harries M, Phillipps N, Anderson R, Prentice G, Collins M. Comparison of bicistronic retroviral vectors containing internal ribosome entry sites (IRES) using expression of human interleukin-12 (IL-12) as a readout. J Gene Med. 2000;2:243–9.

    CAS  Article  Google Scholar 

  23. Jiang C, Magee DM, Cox RA. Construction of a single-chain interleukin-12-expressing retroviral vector and its application in cytokine gene therapy against experimental coccidioidomycosis. Infect Immun. 1999;67:2996–3001.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lorenzo C, Perez-Chacon G, Garaulet G, Mallorquin Z, Zapata JM, Rodriguez A. Efficient expression of bioactive murine IL-12 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 

  25. Canton DA, Shirley S, Wright J, Connolly R, Burkart C, Mukhopadhyay A, et al. Melanoma treatment with intratumoral electroporation of tavokinogene telseplasmid (pIL-12, tavokinogene telseplasmid). Immunotherapy. 2017;9:1309–21.

    CAS  Article  Google Scholar 

  26. de Felipe P. Polycistronic viral vectors. Curr Gene Ther. 2002;2:355–78.

    Article  Google Scholar 

  27. Weaver JC, Smith KC, Esser AT, Son RS, Gowrishankar TR. A brief overview of electroporation pulse strength-duration space: a region where additional intracellular effects are expected. Bioelectrochemistry. 2012;87:236–43.

    CAS  Article  Google Scholar 

  28. Daud AI, Loo K, Pauli ML, Sanchez-Rodriguez R, Sandoval PM, Taravati K, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. J Clin Invest. 2016;126:3447–52.

    Article  Google Scholar 

  29. Shindo G, Endo T, Onda M, Goto S, Miyamoto Y, Kaneko T. Is the CD4/CD8 ratio an effective indicator for clinical estimation of adoptive immunotherapy for cancer treatment? J Cancer Ther. 2013; 04:No.08: 9.

  30. Sin JI, Park JB, Lee IH, Park D, Choi YS, Choe J, et al. Intratumoral electroporation of IL-12 cDNA eradicates established melanomas by Trp2(180-188)-specific CD8+ CTLs in a perforin/granzyme-mediated and IFN-gamma-dependent manner: application of Trp2(180-188) peptides. Cancer Immunol Immunother. 2012;61:1671–82.

    CAS  Article  Google Scholar 

  31. Pierce RH, Campbell JS, Pai SI, Brody JD, Kohrt HE. In-situ tumor vaccination: bringing the fight to the tumor. Hum Vaccin Immunother. 2015;11:1901–9.

    Article  Google Scholar 

  32. Pestova TV, Kolupaeva VG, Lomakin IB, Pilipenko EV, Shatsky IN, Agol VI, et al. Molecular mechanisms of translation initiation in eukaryotes. Proc Natl Acad Sci USA. 2001;98:7029–36.

    CAS  Article  Google Scholar 

  33. Mizuguchi H, Xu Z, Ishii-Watabe A, Uchida E, Hayakawa T. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther. 2000;1:376–82.

    CAS  Article  Google Scholar 

  34. Shikama Y, Hu H, Ohno M, Matsuoka I, Shichishima T, Kimura J. Transcripts expressed using a bicistronic vector pIREShyg2 are sensitized to nonsense-mediated mRNA decay. BMC Mol Biol. 2010;11:42.

    Article  Google Scholar 

  35. Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol. 2004;22:589–94.

    CAS  Article  Google Scholar 

  36. Kuzmich AI, Vvedenskii AV, Kopantsev EP, Vinogradova TV. [Quantitative comparison of expression for genes linked in bicistronic vectors via ires or 2A-peptide of porcine teschovirus-1 sequence]. Bioorg Khim. 2013;39:454–65.

    CAS  PubMed  Google Scholar 

  37. Gillessen S, Carvajal D, Ling P, Podlaski FJ, Stremlo DL, Familletti PC, et al. Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur J Immunol. 1995;25:200–6.

    CAS  Article  Google Scholar 

  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. Horikoshi T, Naganuma H, Ohashi Y, Ueno T, Nukui H. Enhancing effect of electric stimulation on cytotoxicity of anticancer agents against rat and human glioma cells. Brain Res Bull. 2000;51:371–8.

    CAS  Article  Google Scholar 

  40. Ogihara M, Yamaguchi O. Potentiation of effects of anticancer agents by local electric pulses in murine bladder cancer. Urol Res. 2000;28:391–7.

    CAS  Article  Google Scholar 

  41. Cemazar M, Miklavcic D, Sersa G. Intrinsic sensitivity of tumor cells to bleomycin as an indicator of tumor response to electrochemotherapy. Jpn J Cancer Res. 1998;89:328–33.

    CAS  Article  Google Scholar 

  42. Cemazar M, Pipan Z, Grabner S, Bukovec N, Sersa G. Cytotoxicity of different platinum (II) analogues to human tumour cell lines in vitro and murine tumour in vivo alone or combined with electroporation. Anticancer Res. 2006;26:1997–2002.

    CAS  PubMed  Google Scholar 

  43. Miklavcic D, Mali B, Kos B, Heller R, Sersa G. Electrochemotherapy: from the drawing board into medical practice. Biomed Eng Online. 2014;13:29.

    Article  Google Scholar 

  44. Currier MA, Adams LC, Mahller YY, Cripe TP. Widespread intratumoral virus distribution with fractionated injection enables local control of large human rhabdomyosarcoma xenografts by oncolytic herpes simplex viruses. Cancer Gene Ther. 2005;12:407–16.

    CAS  Article  Google Scholar 

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Correspondence to D. A. Canton.

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All authors are past or present employees of Oncosec Medical Inc. Dr. Canton, Dr. Connolly, Dr. Campbell, Dr. Bahrami, and Dr. Pierce are inventors of patents, pending and published.

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Burkart, C., Mukhopadhyay, A., Shirley, S.A. et al. Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters. Gene Ther 25, 93–103 (2018). https://doi.org/10.1038/s41434-018-0006-y

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  • DOI: https://doi.org/10.1038/s41434-018-0006-y

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