Intratumoral expression of interleukin 23 variants using oncolytic vaccinia virus elicit potent antitumor effects on multiple tumor models via tumor microenvironment modulation

Abstract

Background: Newly emerging cancer immunotherapy has led to significant progress in cancer treatment; however, its efficacy is limited in solid tumors since the majority of them are "cold" tumors. Oncolytic viruses, especially when properly armed, can directly target tumor cells and indirectly modulate the tumor microenvironment (TME), resulting in "hot" tumors. These viruses can be applied as a cancer immunotherapy approach either alone or in combination with other cancer immunotherapies. Cytokines are good candidates to arm oncolytic viruses. IL-23, an IL-12 cytokine family member, plays many roles in cancer immunity. Here, we used oncolytic vaccinia viruses to deliver IL-23 variants into the tumor bed and explored their activity in cancer treatment on multiple tumor models. Methods: Oncolytic vaccinia viruses expressing IL-23 variants were generated by homologue recombination. The characteristics of these viruses were in vitro evaluated by RT-qPCR, ELISA, flow cytometry and cytotoxicity assay. The antitumor effects of these viruses were evaluated on multiple tumor models in vivo and the mechanisms were investigated by RT-qPCR and flow cytometry. Results: IL-23 prolonged viral persistence, probably mediated by up-regulated IL-10. The sustainable IL-23 expression and viral oncolysis elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, TNF-α, Perforin, IL-2, Granzyme B and activated T cells in the TME, transforming the TME to be more conducive to antitumor immunity. This leads to a systemic antitumor effect which is dependent on CD8⁺ and CD4⁺ T cells and IFN-γ. Oncolytic vaccinia viruses could not deliver stable IL-23A to the tumor, attributed to the elevated tristetraprolin which can destabilize the IL-23A mRNA after the viral treatment; whereas vaccinia viruses could deliver membrane-bound IL-23 to elicit a potent antitumor effect which might avoid the possible toxicity normally associated with systemic cytokine exposure. Conclusion: Either secreted or membrane-bound IL-23-armed vaccinia virus can induce potent antitumor effects and IL-23 is a candidate cytokine to arm oncolytic viruses for cancer immunotherapy.

Keywords: IL-23; cancer immunotherapy; efficacy.; oncolytic virus; tumor microenvironment.

Figure 1

vvDD-IL-23 infection shows significantly higher IL-23 secretion and similar replication and cytotoxicity in vitro. (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 or vvDD-IL-23 at an MOI of 1. The cell pellets were harvested to measure A34R or IL-23 expression 24 h after infection using RT-qPCR. (B) MC38-luc (3×10⁵ cells), B16 (2×10⁵ cells) or AB12-luc (3×10⁵ cells) were mock-infected or infected with vvDD or vvDD-IL-23 at MOIs of 0.1, 1, and 5. The supernatants were harvested to measure IL-23 using ELISA 24 h after infection. (C) MC38-luc (1×10⁴ cells), B16 (8×10³ cells) or LLC (8×10³ cells) were infected with vvDD-IL-23 or vvDD at indicated MOIs and cell viability was measured 48 h after infection.

Figure 2

vvDD-IL-23 treatment elicits potent therapeutic effects in multiple tumor models. (A) B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with PBS, vvDD, or vvDD-IL-23 at 2×10⁸ PFU/mouse five days after tumor inoculation. The Kaplan Meier survival curve is shown. BalB/c mice were s.c. inoculated with 1×10⁶ CT26 or B6 mice were s.c. inoculated with 2×10⁵ B16 or 5×10⁵ LLC in the right flanks, and were i.t. treated with 60 µL PBS or 5×10⁷ PFU/60 µL virus per mouse at day 6, 10 or 7 after tumor cell inoculation, respectively. Tumor growth curves are shown in (B), (C) and (D), respectively. The endpoints were determined by natural death or tumor size over 2 cm. B6 mice were s.c. inoculated with 1×10⁶ LLC in the right flanks and sacrificed at the first mouse with a tumor size over 2 cm. The lung metastatic tumor nodules were counted (E). The individual draining inguinal lymph nodes were collected, weighed (F) and photographed (G). A log-rank (Mantel-Cox) test was used to compare survival rates. A two-way ANOVA test was used to compare tumor growth cures. **: P<0.01; ***: P<0.001; and **** P<0.0001. ns: not significant.

Figure 3

vvDD-IL-23 treatment prolongs viral persistence. B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with PBS, vvDD, or vvDD-IL-23 at 2×10⁸ PFU/mouse five days after tumor inoculation. Tumor-bearing mice were sacrificed five or nine days post-treatment and primary tumors were collected and analyzed using RT-qPCR to determine the expression of A34R (A), IL12p40 (B), IL-10 (C) and IL-23R (D). The IL-23R expression on CD45^- or CD45⁺ cells were determined by flow cytometry (E, F). MC38-luc (3×10⁵ cells), CT26 (3×10⁵ cells), B16 (2×10⁵ cells), LLC (3×10⁵ cells), EMT (3×10⁵ cells), or AB12-luc (3×10⁵ cells) tumor cells were mock-infected or infected with vvDD at an MOI of 1. The cell pellets were harvested to measure IL-23R expression at 24 h after infection using flow cytometry (G). MC38-luc-bearing mice treated as above were also sacrifice at D11, D13 and D17 to monitor viral persistence in tumors using RT-qPCR (H). *: P<0.05; **: P<0.01; and ****: P<0.0001. ns: not significant.

Figure 4

vvDD-IL-23 treatment transforms TME. B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with PBS, vvDD, or vvDD-IL-23 at 2×10⁸ PFU/mouse five days after tumor inoculation. Tumor-bearing mice were sacrificed five or nine days after treatment and primary tumors were collected and analyzed using RT-qPCR to determine the expression of Th1 chemokines (A), antitumor immunity mediators (B) and immune checkpoints (C) in the TME. The percentages of G-MDSC (CD45⁺CD11b⁺Ly-6G⁺Ly-6C^low) and M-MDSC (CD45⁺CD11b⁺Ly-6G^-Ly-6C^hi) in tumor CD45⁺CD11b⁺ cells were determined by flow cytometry (D). *: P<0.05; **: P<0.01; and ***: P<0.001. ns: not significant.

Figure 5

vvDD-IL-23 treatment elicits potent therapeutic effects in late-stage tumor models. B6 mice were i.p. inoculated with 5×10⁵ MC38-luc or 3.5×10⁶ ID8-luc cells and treated with PBS, vvDD, or vvDD-IL-23 at 2×10⁸ PFU/mouse nine days after tumor inoculation, and the survival curves are shown in (A) and (B), respectively. (C) Naïve B6 mice or MC38-luc-tumor-bearing B6 mice treated with vvDD-IL-23, which had survived for more than 120 days, were s.c. challenged with 1×10⁶ MC38 cells. The tumor growth curve is shown. B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with PBS, vvDD, or vvDD-IL-23 at 2×10⁸ PFU/mouse nine days after tumor inoculation. Tumor-bearing mice were sacrificed five days after treatment and primary tumors were collected and analyzed using flow cytometry to determine CD4⁺Foxp3^- (D), CD4⁺IFN-γ⁺ (E), CD8⁺ (F), CD8⁺IFN-γ⁺ (G), CD8⁺TNF-α⁺(H) T cells in tumors and CD8⁺/Treg ratio (I). In a separate experiment, B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with vvDD-IL-23 nine days after tumor inoculation. α-CD8 Ab (250 µg per injection), α-CD4 Ab (150 µg per injection), or α-IFN-γ Ab (200 µg per injection) were i.p. injected into mice to deplete CD8⁺ T cells, CD4⁺ T cells, or neutralize circulating IFN-γ, respectively (J), and a log-rank (Mantel-Cox) test was used to compare survival rates (K). *: P<0.05; **: P<0.01. ns: not significant.

Figure 6

vvDD-IL-23A treatment cannot modulate therapeutic effects in vivo. (A) MC38-luc (3×10⁵ cells), AB12-luc (3×10⁵ cells) or B16 (2×10⁵ cells) tumor cells were infected with vvDD-IL-23 or vvDD-IL-23A at an MOI of 1. The cell pellets were harvested to measure A34R or IL-23 expression at 24 h using RT-qPCR. (B) MC38-luc (3×10⁵ cells) tumor cells were infected with vvDD-IL-23 or vvDD-IL-23A at an MOI of 1. The cell pellets were harvested to determine TTP expression at 24 h using RT-qPCR. (C) B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with vvDD-IL-23 or vvDD-IL-23A at 2×10⁸ PFU/mouse five days after tumor inoculation. Tumor-bearing mice were sacrificed nine days after treatment and primary tumors were collected and analyzed using RT-qPCR to determine TTP expression using RT-qPCR. (D) B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with PBS, vvDD, vvDD-IL-23 or vvDD-IL-23A at 2×10⁸ PFU/mouse five days after tumor inoculation, and a log-rank (Mantel-Cox) test was used to compare survival rates. *: P<0.05; **: P<0.01; and ****: P<0.0001. ns: not significant.

Figure 7

vvDD-IL-23-FG treatment elicits therapeutic effects in vivo. MC38-luc (3×10⁵ cells), AB12-luc (3×10⁵ cells) or B16 (2×10⁵ cells) tumor cells were infected with vvDD-IL-23 or vvDD-IL-23A at an MOI of 1. Twenty-four h after infection, the supernatants were harvested to measure IL-23 using ELISA (A) and the cell pellets were harvested to measure membrane-bound IL-23 using flow cytometry (B). (C) B6 mice were i.p. inoculated with 5×10⁵ MC38-luc cells and treated with PBS, vvDD, vvDD-IL-23 or vvDD-IL-23-FG at 2×10⁸ PFU/mouse five days after tumor inoculation, and a log-rank (Mantel-Cox) test was used to compare survival rates. B6 mice were s.c. inoculated with 1×10⁶ LLC or 2×10⁵ B16 in the right flanks, and were i.t. treated with 60 µL PBS or 5×10⁷ PFU/60 µL virus per mouse when tumor volume reached 50 mm³ or 140 mm³, and tumor growth curves are shown in (D) and (E), respectively. The endpoints were determined by natural death or tumor size over 2 cm. *: P<0.05; ****: P<0.0001. ns: not significant.