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. 2021 Sep;70(9):2467-2481.
doi: 10.1007/s00262-021-02860-4. Epub 2021 Feb 4.

IL-36γ-armed oncolytic virus exerts superior efficacy through induction of potent adaptive antitumor immunity

Min Yang #  1   2 , Esther Giehl #  1   3   4 , Chao Feng  1   2 , Mathilde Feist  1   3   5 , Hongqi Chen  1   3 , Enyong Dai  1   3 , Zuqiang Liu  1   3 , Congrong Ma  1   3 , Roshni Ravindranathan  1   3 , David L Bartlett  1   3   6 , Binfeng Lu  7   8 , Zong Sheng Guo  9   10
Affiliations

IL-36γ-armed oncolytic virus exerts superior efficacy through induction of potent adaptive antitumor immunity

Min Yang et al. Cancer Immunol Immunother. 2021 Sep.

Abstract

In this study, we aimed to apply the cytokine IL-36γ to cancer immunotherapy by constructing new oncolytic vaccinia viruses (OV) expressing interleukin-36γ (IL-36γ-OVs), leveraging unique synergism between OV and IL-36γ's ability to promote antitumor adaptive immunity and modulate tumor microenvironment (TME). IL-36γ-OV had dramatic therapeutic efficacies in multiple murine tumor models, frequently leading to complete cancer eradication in large fractions of mice. Mechanistically, IL-36-γ-armed OV induced infiltration of lymphocytes and dendritic cells, decreased myeloid-derived suppressor cells and M2-like tumor-associated macrophages, and T cell differentiation into effector cells. Further study showed that IL-36γ-OV increased the number of tumor antigen-specific CD4+ and CD8+ T cells and the therapeutic efficacy depended on both CD8+ and CD4+ T cells. These results demonstrate that these IL36γ-armed OVs exert potent therapeutic efficacy mainly though antitumor immunity and they may hold great potential to advance treatment in human cancer patients.

Keywords: CD4+ T cells; CD8+ T cells; Effector memory T cells; IL-36γ; Immunotherapy; Oncolytic virus.

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Conflict of interest statement

A patent partly based on this work has been filed by DLB, BL and ZSG. All other authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
IL-36γ-armed VVs are OVs and produce the recombinant cytokine in infected cancer cells in vitro. a Three IL-36γ–armed oncolytic VVs containing various backbones with deletional mutations of viral genes. b Production and secretion of IL-36γ from infected HeLa cells. HeLa cells in six-well plate were mock-infected or infected with vvTK- or vvTK-IL-36γ at MOI of ~ 1.0. At 48 h post-infection, conditioned media were collected and subjected to western blot analysis. M: protein markers; lanes 1, 2: vvTK-IL-36γ; lane 3: vvTK-; lane 4: mock-infected. Lanes 5 & 6: B16-IL-36γ cells. c Viral replication in MC38 cancer cells. Harvested cells were lysed and the cell lysate was tittered using viral plaque assay. d MC38-luc cells were infected with OVs at MOI of 0.5 then harvested at varying time points. Cell suspensions were stained with 0.4% trypan blue solution and then viable cells were counted under visible light microscopy. e and f Oncolysis of virus-infected human cancer cells (HepG2 cells and MDA-MB-468 cells). Cancer cells in 96-well culture plates were infected with viruses at MOI of 1.0, then cell viability was assessed at 24, 36, 48, and 72 h after infection using MTS assays. These data are representatives of two or more independent experiments. For oncolysis in MDA-MB-468 cells, when comparing the two pairs of OVs (vvTK- and vvTL-IL36, versus vvTD and vvTD-IL36), p < 0.01. However, p > 0.05 when the 2 OVs in the same pair was compared
Fig. 2
Fig. 2
IL-36γ-armed OVs displayed potent antitumor effects in four murine tumor models. Aa–c B6 mice were inoculated i.p. with 5.0 × 105 MC38-luc cells. On day 5, mice were imaged by bioluminescence to exclude mice with no tumor, and remaining mice were randomly divided into three groups and treated with PBS (n = 8), vvTK- (n = 10), or vvTK-IL-36γ (n = 10) at the dose of 1.0e8 pfu/mouse. a Tumor burden of mice on days 5 (the day of treatment) and 23 were determined via bioluminescence imaging. b Long term survival of MC38-luc tumor-bearing mice as Kaplan–Meier survival curves. *** p < 0.001 between PBS vs vvTK; p = 0.029 between vvTK vs vvTK-IL-36γ groups. c On day 140, the previously cured mice with vvTK- or vvTK-IL-36γ, along with a group of naïve mice, were re-challenged with cells of MC38-luc tumor (5.0e5 tumor cells) on the right flank and Lewis lung carcinoma (LLC, 5.0e5 cells) on the left flank. Tumor formation was observed twice a week until day 40. d Tumor curves of subcutaneous MC38-luc tumors treated with IL-36γ-OVs or PBS. e Long-term survival analysis of subcutaneous MC38-luc tumor-bearing mice after indicated treatments. Data were representative of two independent experiments. f Long-term survival of panc02-luc pancreatic tumor-bearing mice treated with indicated reagents was analyzed. p = 0.017, vvTD vs vvTD-IL-36γ; p ≤ 0.001 between PBS vs OV-treated groups. g Long-term survival of B16 melanoma-bearing mice treated with indicated reagents was analyzed. p = 0.024, vvTK- vs vvTK-IL-36γ; p = 0.002 vvTD vs vvTD-IL-36γ. Data were representative of two independent experiments. n = 6–10 for each group
Fig. 3
Fig. 3
Treatment with IL-36γ-armed OV increased the number of T cells in MC38 solid colon tumor tissue. B6 mice were subcutaneously inoculated with 5.0e5 MC38 cancer cells. When the tumor size reached ~ 5 mm in diameter, PBS, vvDD, vvDD-IL-36γ (1.0e8 pfu per tumor) was injected intratumorally (n = 6 ~ 8/group). a Ten days post-treatment, tumor tissues were collected, fixed, and stained for CD3, CD4, CD8, and DAPI. Representative images from each group were presented. b Statistics of the percentages of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells per area, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Fig. 4
Fig. 4
Therapeutic efficacy of IL-36γ-armed OV depends on multiple types of immune cells. Peritoneal MC38 tumor-bearing mice were imaged, randomized, and injected i.p. with PBS or 1.0e8 pfu of vvTK-IL-36γ. The mice treated with vvTK-IL-36γ were divided into four groups (n = 7–8) and further treated with PBS, anti-CD4 ab, anti-CD8 Abs, or anti-NK1.1 Abs as described in Methods. Mouse survival was monitored and Kaplan Meier analysis was performed. Statistical analyses: p = 0.025 for vvTK-IL-36γ versus vvTK-IL-36γ + anti-CD4; p < 0.01 for vvTK-IL-36γ versus vvTK-IL-36γ plus anti-CD8; p = 0.06 for vvTK-IL-36γ versus vvTK-IL-36γ plus anti-NK mAb treatment
Fig. 5
Fig. 5
IL-36γ-armed OV promoted antitumor immunity via changing the TME. Mice were inoculated with 5.0e5 MC38-luc cells i. p., and seven days later, mice with similar sizes of tumor burden were randomly divided into three groups and treated with PBS or OVs (n = 6 for each group). Six days later, lavaged cells were analyzed using FACS. a Lymphocytes were gated based on the CD45 expressions in addition to Forward and Side scatters. Percentage of CD45+ lymphocytes out of total live cells. b Percentage of IFNγ+CD8+ T cells out of CD8+ T cells. c Percentage of Treg out of CD4+ T cells. d Percentage of CD11b+GR-1hi g-MDSCs out of CD45+ population. e Percentage of CD11b+GR-1int m-MDSCs out of CD45+ population. f Percentage of TAMs (CD24−F4/80+) out of CD11b+GR-1−MHCII+ population. g Percentage of CD206+ TAMs out of CD11b+GR-1−MHCII+CD24−F4/80+ population. h Percentage of DCs (CD24+F4/80−) out of CD11b+GR-1−MHCII+ population. i Percentage of Naïve CD8+ T cells gated as CD44− CD62L+CD8+ out of total CD8+ T cells. j Effector memory-phenotype CD8+ T cells gated as CD44+ CD62L−CD8+ out of total CD8+ T cells. ** p < 0.01. *** p < 0.001. **** p < 0.0001
Fig. 6
Fig. 6
vvTD-IL-36γ enhanced recognition of murine MC38 colon adenocarcinoma cells by intraperitoneal 4-1BB+ CD8+ T cells on day 6 after oncolytic virotherapy. a Representative image of IFNγ ELISpot assay of 2.0e4 CD90.2+ T cells isolated from lavage specimens on day 6 post-oncolytic virotherapy and co-cultured 1:1 with specific (MC38) and unspecific (medium, ID8, splenocytes) target cells and analysis of ImmunoSpot™ counted spots. b 4-1BB+ CD44+ CD8+ T cells showed enhanced MC38-specific activation assessed by 4-1BB surface expression after co-culture assay with MC38 and unspecific target cells (medium, unloaded splenocytes, ID8 cells). c Percentage of 4-1BB+CD44+ CD8+ T cells, following co-culture assay with p15E604–611 and B8R20–27 loaded splenocytes and OVA257–264 and unloaded splenocytes as unspecific targets, revealed augmented 4-1BB dependent activation by retroviral peptide p15E604–611 and B8R20–27. d Percentage of OX40+ CD4+ T cells per total CD4+ T cells following in vitro co-culture assay with MC38 and unspecific targets as negative controls. ** p < 0.01; *** p < 0.001; **** p < 0.0001. The data were representatives of two or three independent experiments
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