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. 2020 Mar;8(1):e000710.
doi: 10.1136/jitc-2020-000710.

Oncolytic vaccinia virus delivering tethered IL-12 enhances antitumor effects with improved safety

Yan Ge #  1   2   3 , Haiyan Wang #  1   2   3 , Jinghua Ren  1   2   4 , Weilin Liu  1   2   5 , Lingjuan Chen  1   2   4 , Hongqi Chen  1   2   6 , Junjie Ye  1   2   7 , Enyong Dai  1   2   8 , Congrong Ma  1   2 , Songguang Ju  3 , Zong Sheng Guo  1   2 , Zuqiang Liu  9   2 , David L Bartlett  9   2
Affiliations

Oncolytic vaccinia virus delivering tethered IL-12 enhances antitumor effects with improved safety

Yan Ge et al. J Immunother Cancer. 2020 Mar.

Abstract

Immune checkpoint blockade is arguably the most effective current cancer therapy approach; however, its efficacy is limited to patients with "hot" tumors, warranting an effective approach to transform "cold" tumors. Oncolytic viruses (especially properly armed ones) have positive effects on almost every aspect of the cancer-immunity cycle and can change the cancer-immune set point of a tumor. Here, we tested whether oncolytic vaccinia virus delivering tethered interleukin 12 (IL-12) could turn a "cold" tumor into a "hot" tumor while avoiding IL-12's systemic toxicity. Our data demonstrated that tethered IL-12 could be maintained in the tumor without treatment-induced toxic side effects. Moreover, the treatment facilitated tumor infiltration of more activated CD4+ and CD8+ T cells and less Tregs, granulocytic myeloid-derivedsuppressor cells, and exhausted CD8+ T cells, with increased interferon γ and decreased transforming growth factor β, cyclooxygenase-2, and vascular endothelial growth factor expression, leading to transformed, immunogenic tumors and improved survival. Combined with programmed cell death 1 blockade, vaccinia virus expressing tethered IL-12 cured all mice with late-stage colon cancer, suggesting immediate translatability to the clinic.

Keywords: cytokines; immunotherapy; oncolytic virotherapy; oncolytic viruses.

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Conflict of interest statement

Competing interests: The University of Pittsburgh has filed a patent application directed to the subject matter of this manuscript.

Figures

Figure 1
Figure 1
Tethered IL-12 variants show functional IL-12 membrane association and similar cytotoxicity. (A) Tumor cell MC38-luc (3×105 cells), B16 (2×105 cells), or AB12-luc (3×105 cells) were mock-infected or infected with vvDD, vvDD-IL-12, vvDD-IL-12-FG, or vvDD-IL-12-RG at an MOI of 1. The cell pellets were harvested to measure A34R or IL-12 expression at 24 hours using RT-qPCR. The culture supernatants were harvested to measure secreted IL-12 using ELISA (B) and the cell pellets were also harvested to measure membrane-bound IL-12 using flow cytometry (cell surface staining) (C) 24 hours post-infection. (D) MC38-luc (3×105 cells), B16 (2×105 cells), or AB12-luc (3×105 cells) were mock-infected or infected with vvDD, vvDD-IL-12, vvDD-IL-12-FG, or vvDD-IL-12-RG at MOIs of 0.1, 1, and 5. The cell pellets were harvested to measure membrane-bound IL-12 using ELISA after PI-PLC cleavage 24 hours post-infection. (E) Naïve B6 splenocytes were activated and stimulated with IL-12 variant-infected MC38 cells (Responder: stimulator=1:5) in the absence/presence of α-IL-12 Ab, and T cell proliferation was measured using MTT assay 48 hours after coculture. (F) MC38-luc (1×104 cells), B16 (5×103 cells), AB12-luc (5×103 cells), or CT26-luc (1×104 cells) were infected with IL-12 variants at indicated MOIs and cell viability was measured using Cell Counting Kit-8 or MTS assay 48 hours post-infection. Data represent two independent experiments. *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001. MOI, multiplicityof infection; NS, not significant; PI-PLC, phosphatidylinositol-specificphospholipase C.
Figure 2
Figure 2
vvDD-IL-12-FG treatment produces tethered IL-12 in tumors and is safe and effective in therapeutic tumor models. B6 mice were intraperitoneally inoculated with 5×105 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-12, vvDD-IL-12-FG, or vvDD-IL-12-RG at 1×109 PFU/mouse 9 days post-tumor inoculation. The mice treated above were sacrificed at day 5 after treatment. Tumor nodules were collected to measure the amount of IL-12 (A) and IFN-γ in tumor (B); the lungs and kidneys were collected to monitor pulmonary tissue edema (C and D); sera were collected to measure AST (E) and ALT (F) in sera. (G) B6 mice were intraperitoneally inoculated with 5×105 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-12, or vvDD-IL-12-FG at 2×108 PFU/mouse 5 days post-tumor inoculation (n≥8). The vvDD-IL-12-FG cured mice were subcutaneously re-challenged with MC38 (H) or LLC (I). B6 mice were intraperitoneally inoculated with 5×105 MC38-luc cells and treated with PBS or indicated viruses at 2×108 PFU/mouse 9 days post-tumor inoculation (n≥23). The survival curve was shown (J). Some of these treated mice were sacrificed and splenocytes were restimulated with mitomycin C-inactivated MC38 cells to monitor IFN-γ production (K). (L) BALB/c mice were intraperitoneally inoculated with 4×105 AB12-luc cells and treated with PBS or indicated viruses at 2×108 PFU/mouse 9 days post-tumor inoculation (n≥10). A log-rank (Mantel–Cox) test was used to compare survival rates. *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001. ALT, alanine transaminase; AST, aspartate transaminase; IFN-γ, interferon γ; IL-12, interleukin 12; NS, not significant; PBS, phosphate-buffered saline; PFU, plaque-forming units.
Figure 3
Figure 3
IL-12-variant treatments modify the tumor microenvironment and improve the therapeutic effect with PD-1 blockade. B6 mice were intraperitoneally inoculated with 5×105 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-12, or vvDD-IL-12-FG at 2×108 PFU/mouse 9 days post-tumor inoculation. Tumor-bearing mice were sacrificed 5 days post-treatment and primary tumors were collected and analyzed using flow cytometry to determine CD4+Foxp3− (A) and CD8+ T cells (B), IFN-γ+CD8+ (C), IFN-γ+CD4+ (D), exhausted CD8+ T cell (E–H), G-MDSCs (I), or regulatory T cells (CD4+Foxp3+) (J), TGF-β+Treg (N), TGF-β+CD45− (O), using RT-qPCR to determine TGF-β, COX-2, and VEGF (K–M). In a separate experiment, B6 mice were intraperitoneally inoculated with 5×105 MC38-luc cells and treated with vvDD-IL-12-FG or PBS 9 days post-tumor inoculation. Anti-CD8 Ab (250 µg/injection), α-CD4 Ab (150 µg/injection), PK136 (300 µg/injection), α-IFN-γ Ab (200 µg/injection), or α-PD-1 Ab (200 µg/injection), (n≥7) were intraperitoneally injected into mice to deplete CD8+ T cells, CD4+ T cells, or NK1.1+ cells, neutralize circulating IFN-γ, or enhance virotherapy with α-PD-1 Ab (P), and a log-rank (Mantel–Cox) test was used to compare survival rates (Q and R), respectively. *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001. COX-2, cyclooxygenase-2; IFN-γ, interferon γ; IL-12, interleukin 12; G-MDSC, granulocytic myeloid-derived suppressor cells; NS, not significant; PBS, phosphate-buffered saline; PFU, plaque-forming units; TGF-β, transforming growth factor β; VEGF, vascular endothelial growth factor.
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