Intratumoral DNA-based delivery of checkpoint-inhibiting antibodies and interleukin 12 triggers T cell infiltration and anti-tumor response.

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 9432230 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1476-5500 (Electronic) Linking ISSN: 09291903 NLM ISO Abbreviation: Cancer Gene Ther Subsets: MEDLINE

Publication: <2002->: London : Nature Publishing Group
Original Publication: Norwalk, CT : Appleton & Lange, c1994-

CD8-Positive T-Lymphocytes* ; Immune Checkpoint Inhibitors*/administration & dosage ; Interleukin-12*/genetics ; Neoplasms*/drug therapy ; Neoplasms*/immunology; Animals ; Antibodies, Monoclonal/administration & dosage ; Cell Line, Tumor ; DNA ; Genetic Therapy ; Immunotherapy ; Mice

To improve the anti-tumor efficacy of immune checkpoint inhibitors, numerous combination therapies are under clinical evaluation, including with IL-12 gene therapy. The current study evaluated the simultaneous delivery of the cytokine and checkpoint-inhibiting antibodies by intratumoral DNA electroporation in mice. In the MC38 tumor model, combined administration of plasmids encoding IL-12 and an anti-PD-1 antibody induced significant anti-tumor responses, yet similar to the monotherapies. When treatment was expanded with a DNA-based anti-CTLA-4 antibody, this triple combination significantly delayed tumor growth compared to IL-12 alone and the combination of anti-PD-1 and anti-CTLA-4 antibodies. Despite low drug plasma concentrations, the triple combination enabled significant abscopal effects in contralateral tumors, which was not the case for the other treatments. The DNA-based immunotherapies increased T cell infiltration in electroporated tumors, especially of CD8 ⁺  T cells, and upregulated the expression of CD8 ⁺  effector markers. No general immune activation was detected in spleens following either intratumoral treatment. In B16F10 tumors, evaluation of the triple combination was hampered by a high sensitivity to control plasmids. In conclusion, intratumoral gene electrotransfer allowed effective combined delivery of multiple immunotherapeutics. This approach induced responses in treated and contralateral tumors, while limiting systemic drug exposure and potentially detrimental systemic immunological effects.
(© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359:1350–5. (PMID: 29567705739125910.1126/science.aar4060)
Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Lao CD, et al. Five-year survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl J Med. 2019;381:1535–46. (PMID: 3156279710.1056/NEJMoa1910836)
Kooshkaki O, Derakhshani A, Hosseinkhani N, Torabi M, Safaei S, Brunetti O, et al. Combination of ipilimumab and nivolumab in cancers: from clinical practice to ongoing clinical trials. Int J Mol Sci. 2020;21:4427. (PMID: 735297610.3390/ijms21124427)
Upadhaya S, Neftelino ST, Hodge JP, Oliva C, Campbell JR, Yu JX. Combinations take centre stage in PD1/PDL1 inhibitor clinical trials. Nat Rev Drug Disco. 2021;20:168–9. (PMID: 10.1038/d41573-020-00204-y)
Middleton MR, Hoeller C, Michielin O, Robert C, Caramella C, Öhrling K, et al. Intratumoural immunotherapies for unresectable and metastatic melanoma: current status and future perspectives. Br J Cancer. 2020;123:885–97. (PMID: 32713938749225210.1038/s41416-020-0994-4)
Algazi AP, Twitty CG, Tsai KK, Le M, Pierce R, Browning E, et al. Phase II trial of IL-12 plasmid transfection and PD-1 blockade in immunologically quiescent melanoma. Clin Cancer Res. 2020;26:2827–37. (PMID: 3237665510.1158/1078-0432.CCR-19-2217)
Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood. 1997;90:2541–8. (PMID: 9326219)
Algazi A, Bhatia S, Agarwala S, Molina M, Lewis K, Faries M, et al. Intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients. Ann Oncol. 2020;31:532–40. (PMID: 3214721310.1016/j.annonc.2019.12.008)
Bhatia S, Longino NV, Miller NJ, Kulikauskas R, Iyer JG, Ibrani D, et al. Intratumoral delivery of plasmid IL12 via electroporation leads to regression of injected and noninjected tumors in Merkel cell carcinoma. Clin Cancer Res. 2020;26:596–607. (PMID: 10.1158/1078-0432.CCR-19-0972)
Heller R, Heller LC. Gene electrotransfer clinical trials. Adv Genet. 2015;89:235–62. (PMID: 2562001310.1016/bs.adgen.2014.10.006)
Jacobs L, De Smidt E, Geukens N, Declerck P, Hollevoet K. Electroporation outperforms in vivo-jetPEI for intratumoral DNA-based reporter gene transfer. Sci Rep. 2020;10:19532. (PMID: 33177564765931710.1038/s41598-020-75206-2)
Hollevoet K, Declerck PJ. State of play and clinical prospects of antibody gene transfer. J Transl Med. 2017;15:131. (PMID: 28592330546333910.1186/s12967-017-1234-4)
Vermeire G, De Smidt E, Casteels P, Geukens N, Declerck P, Hollevoet K. DNA-based delivery of anti-DR5 nanobodies improves exposure and anti-tumor efficacy over protein-based administration. Cancer Gene Ther. 2021;28:828–38. (PMID: 3273305510.1038/s41417-020-0204-9)
Jacobs L, De Smidt E, Geukens N, Declerck P, Hollevoet K. DNA-based delivery of checkpoint inhibitors in muscle and tumor enables long-term responses with distinct exposure. Mol Ther. 2020;28:1068–77. (PMID: 32101701713261910.1016/j.ymthe.2020.02.007)
Hollevoet K, De Smidt E, Geukens N, Declerck P. Prolonged in vivo expression and anti-tumor response of DNA-based anti-HER2 antibodies. Oncotarget. 2018;9:13623–36. (PMID: 29568382586260310.18632/oncotarget.24426)
Hollevoet K, De Vleeschauwer S, De Smidt E, Vermeire G, Geukens N, Declerck P. Bridging the clinical gap for DNA-based antibody therapy through translational studies in sheep. Hum Gene Ther. 2019;30:1431–43. (PMID: 3138277710.1089/hum.2019.128)
Vermeire G, De Smidt E, Geukens N, Williams JA, Declerck P, Hollevoet K. Improved potency and safety of DNA-encoded antibody therapeutics through plasmid backbone and expression cassette engineering. Hum Gene Ther. 2021;32:1200–9. (PMID: 3448275710.1089/hum.2021.105)
Campbell J, Canton DA, Pierce RH. Plasmid constructs for heterologous protein expression and methods of use. Patent US20190153469A1; 2019.
Roca CP, Burton OT, Gergelits V, Prezzemolo T, Whyte CE, Halpert R, et al. AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data. Nat Commun. 2021;12:2890. (PMID: 34001872812907110.1038/s41467-021-23126-8)
Garris CS, Arlauckas SP, Kohler RH, Trefny MP, Garren S, Piot C, et al. Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12. Immunity. 2018;49:1148–1161.e7. (PMID: 30552023630109210.1016/j.immuni.2018.09.024)
Hewitt SL, Bailey D, Zielinski J, Apte A, Musenge F, Karp R, et al. Intratumoral IL12 mRNA therapy promotes TH1 transformation of the tumor microenvironment. Clin Cancer Res. 2020;26:6284–98. (PMID: 3281707610.1158/1078-0432.CCR-20-0472)
Wei SC, Anang NAS, Sharma R, Andrews MC, Reuben A, Levine JH, et al. Combination anti-CTLA-4 plus anti-PD-1 checkpoint blockade utilizes cellular mechanisms partially distinct from monotherapies. Proc Natl Acad Sci USA. 2019;116:22699–709. (PMID: 31636208684262410.1073/pnas.1821218116)
Ishihara J, Fukunaga K, Ishihara A, Larsson HM, Potin L, Hosseinchi P, et al. Matrix-binding checkpoint immunotherapies enhance antitumor efficacy and reduce adverse events. Sci Transl Med. 2017;9:eaan040. (PMID: 10.1126/scitranslmed.aan0401)
Pai CS, Simons DM, Lu X, Evans M, Wei J, Wang YH, et al. Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity. J Clin Invest. 2019;129:349–63. (PMID: 3053099110.1172/JCI123391)
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. (PMID: 2952387810.1038/s41434-018-0006-y)
Momin N, Mehta NK, Bennett NR, Ma L, Palmeri JR, Chinn MM, et al. Anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy. Sci Transl Med. 2019;11:eaaw2614. (PMID: 31243150781180310.1126/scitranslmed.aaw2614)
Selby MJ, Engelhardt JJ, Johnston RJ, Lu LS, Han M, Thudium K, et al. Preclinical development of ipilimumab and nivolumab combination immunotherapy: mouse tumor models, in vitro functional studies, and cynomolgus macaque toxicology. PLoS One. 2016;11:e0161779. (PMID: 27610613501774710.1371/journal.pone.0161779)
Quetglas JI, Labiano S, Aznar M, Bolaños E, Azpilikueta A, Rodriguez I, et al. Virotherapy with a Semliki Forest virus-based vector encoding IL12 synergizes with PD-1/PD-L1 blockade. Cancer Immunol Res. 2015;3:449–54. (PMID: 2569132610.1158/2326-6066.CIR-14-0216)
De Lucia M, Cotugno G, Bignone V, Garzia I, Nocchi L, Langone F, et al. Retargeted and multi-cytokine-armed herpes virus is a potent cancer endovaccine for local and systemic anti-tumor treatment. Mol Ther Oncolytics. 2020;19:253–64. (PMID: 33209980765857810.1016/j.omto.2020.10.006)
Ge Y, Wang H, Ren J, Liu W, Chen L, Chen H, et al. Oncolytic vaccinia virus delivering tethered IL-12 enhances antitumor effects with improved safety. J Immunother Cancer. 2020;8:e000710. (PMID: 32209602710380110.1136/jitc-2020-000710)
Wei SC, Levine JH, Cogdill AP, Zhao Y, Anang NAS, Andrews MC, et al. Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpoint blockade. Cell. 2017;170:1120–1133.e17. (PMID: 28803728559107210.1016/j.cell.2017.07.024)
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-γ-dependent manner: application of Trp2(180–188) peptides. Cancer Immunol Immunother. 2012;61:1671–82. (PMID: 2238236110.1007/s00262-012-1214-8)
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. 2019;26:1–15. (PMID: 3032335210.1038/s41434-018-0044-5)
Shi G, Edelblute C, Arpag S, Lundberg C, Heller R. IL-12 gene electrotransfer triggers a change in immune response within mouse tumors. Cancers. 2018;10:498. (PMID: 631580810.3390/cancers10120498)
Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci USA. 2010;107:4275–80. (PMID: 20160101284009310.1073/pnas.0915174107)
Gao X, Wang X, Yang Q, Zhao X, Wen W, Li G, et al. Tumoral expression of IL-33 inhibits tumor growth and modifies the tumor microenvironment through CD8+ T and NK cells. J Immunol. 2015;194:438–45. (PMID: 2542907110.4049/jimmunol.1401344)
Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, et al. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res. 2013;1:32–42. (PMID: 2477724810.1158/2326-6066.CIR-13-0013)
Zhang P, Lee JS, Gartlan KH, Schuster IS, Comerford I, Varelias A, et al. Eomesodermin promotes the development of type 1 regulatory T (T R 1) cells. Sci Immunol. 2017;2:eaah7152. (PMID: 28738016571429410.1126/sciimmunol.aah7152)
Mazzoni A, Maggi L, Siracusa F, Ramazzotti M, Rossi MC, Santarlasci V, et al. Eomes controls the development of Th17-derived (non-classic) Th1 cells during chronic inflammation. Eur J Immunol. 2019;49:79–95. (PMID: 3014403010.1002/eji.201847677)
Roessner PM, Llaó Cid L, Lupar E, Roider T, Bordas M, Schifflers C, et al. EOMES and IL-10 regulate antitumor activity of T regulatory type 1 CD4 ⁺ T cells in chronic lymphocytic leukemia. Leukemia. 2021;35:2311–24. (PMID: 33526861832447910.1038/s41375-021-01136-1)
Liu J, Blake SJ, Harjunpää H, Fairfax KA, Yong MC, Allen S, et al. Assessing immune-related adverse events of efficacious combination immunotherapies in preclinical models of cancer. Cancer Res. 2016;76:5288–301. (PMID: 2750392510.1158/0008-5472.CAN-16-0194)
Adam K, Iuga A, Tocheva AS, Mor A. A novel mouse model for checkpoint inhibitor-induced adverse events. PLoS One. 2021;16:e0246168. (PMID: 33571254787761310.1371/journal.pone.0246168)
Zhong W, Myers JS, Wang F, Wang K, Lucas J, Rosfjord E, et al. Comparison of the molecular and cellular phenotypes of common mouse syngeneic models with human tumors. BMC Genomics. 2020;21:2. (PMID: 31898484694126110.1186/s12864-019-6344-3)
Bosnjak M, Jesenko T, Kamensek U, Sersa G, Lavrencak J, Heller L, et al. Electrotransfer of different control plasmids elicits different antitumor effectiveness in B16.F10 melanoma. Cancers. 2018;10:37. (PMID: 583606910.3390/cancers10020037)
Marrero B, Shirley S, Heller R. Delivery of interleukin-15 to B16 melanoma by electroporation leads to tumor regression and long-term survival. Technol Cancer Res Treat. 2014;13:551–60. (PMID: 24000979)
Heller LC, Coppola D. Electrically mediated delivery of vector plasmid DNA elicits an antitumor effect. Gene Ther. 2002;9:1321–5. (PMID: 1222401510.1038/sj.gt.3301802)

ClinicalTrials.gov NCT02493361; NCT03132675; NCT03567720; NCT04526730

0 (Antibodies, Monoclonal)
0 (Immune Checkpoint Inhibitors)
187348-17-0 (Interleukin-12)
9007-49-2 (DNA)

Date Created: 20211110 Date Completed: 20220720 Latest Revision: 20230207

20231215

10.1038/s41417-021-00403-8

34754076