The combination of immunosuppression and carrier cells significantly enhances the efficacy of oncolytic poxvirus in the pre-immunized host

Abstract

Pre-existing antipoxvirus immunity in cancer patients presents a severe barrier to poxvirus-mediated oncolytic virotherapy. We have explored strategies of immunosuppression (IS) and/or immune evasion for efficient delivery of an oncolytic double-deleted vaccinia virus (vvDD) to tumors in the pre-immunized mice. Transient IS using immunosuppressive drugs, including tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate, have been used successfully in organ transplantation. This drug cocktail alone did not enhance viral recovery from subcutaneous tumor after systemic viral delivery. Using B-cell knockout mice, we confirmed that the neutralizing antibodies had a significant role in preventing poxvirus infection. Using a MC38 peritoneal carcinomatosis model, we found that the combination of IS and tumor cells as carriers led to the most effective viral delivery, viral replication and viral spread inside the tumor mass. We found that our immunosuppressive drug cocktail facilitated recruitment of tumor-associated macrophages and conversion into an immunosuppressive M2 phenotype (interleukin (IL)-10(hi)/IL-12(low)) in the tumor microenvironment. A combination of IS and carrier cells led to significantly prolonged survival in the tumor model. These results showed the feasibility of treating pre-vaccinated patients with peritoneal carcinomatosis using an oncolytic poxvirus and a combined immune intervention strategy.

Figure 1. Generation of anti-poxviral immunity by vaccination and vaccinia virus replication in tumors of naïve, but not pre-immunized B6 mice

(a). Oncolytic virus vvDD was recovered from tumors in naïve, but not pre-immunized mice. One group of B6 mice (n = 5) were injected i.p. with 4.0 × 10⁶ pfu of vvDD and housed for one month before further experiment. Subcutaneous MC38 tumors were subsequently established. When tumors reached ∼5 × 5 mm in size, 1.0 × 10⁸ pfu of vvDD was injected i.p. Tumor tissues were harvested on days 4 and 8 after viral administration, and viral titers were quantified by plaque assays. Data are presented as median values (p < 0.0001). N.D.: No virus detected (in tumors from the pre-immunized mice). (b). Presence of anti-poxvirus neutralizing antibodies in the sera from the immunized B6 mice. B6 mice were mock-vaccinated or vaccinated with 4.0 × 10⁶ pfu of the virus vvDD i. p. Thirty days later sera were collected. vvDD was incubated with the collected sera at the indicated dilutions and the incubated mixtures were used to infect A2780 human cancer cells as described. Cytopathic effect was observed. Representative data from two experiments are presented as mean ± s.d.

Figure 2. Imaging and viral recovery from tumor in B cell KO mice with or without IS drugs treatment

Viral recovery from tumor tissues in B cell knockout (KO) versus wild type (WT) B6 mice. Mice were vaccinated with 4.0 × 10⁶ pfu per mouse of vvDD i.p. Thirty days later (day 0), mice were inoculated with 2 × 10⁵ MC38 cells s.c., and injected with vvDD (5.0 × 10⁷ pfu) i.p. on day 7 with or without the administration of the three IS drug cocktail. (a). Whole animal imaging was conducted on day 4 after viral administration. The intensity of light indicated the level of luciferase expression from virus-infected cells. Typical results of B cell KO mice with treatment of IS drugs (A) or without IS drugs (B). (b). Tumor tissues were harvested on days 4 and 8 after virus administration (n = 5) and assessed for viral recovery by plaque assays in CV-1 cells. All values are expressed as medians.

Figure 3. The safety of the virus-infected MC38 cancer cells as carrier cells

The MC38-luc (MC38 cells tagged with the firefly luciferase gene from a lentivirus vector) cancer cells were infected with a vvDD virus at MOI of 0, 1 and 10, as described in the Materials and methods. Following infection, 1.0 × 10⁶ MC38-luc cancer cells were injected i.p. into B6 mice. The light images were taken on days 7 and 36. Mice (n = 5 per group) were injected with mock-infected MC38-luc cells (a), MC38-luc cells infected with vvDD at MOI of 1.0 (b), or MC38-luc cells infected with vvDD at MOI of 10 (c). All mice with mock-infected MC38-luc tumor died around 20 days, thus no images on day 36 could be obtained.

Figure 4. The combined effects of IS drugs and carrier cells on viral replication and viral gene expression (firefly luciferase as viral marker gene) in mice bearing intraperitoneal MC38 colorectal carcinomatosis

B6 mice were pre-vaccinated with vvDD at 4.0E6 pfu/mouse on day -30. The mice were inoculated with MC38 cancer cells (2 × 10⁵) i.p (as day 0). On day 7, mice were treated i.p. with either vvDD alone (5.0E7 pfu/mouse) or with 5.0E6 MC38 cells infected ex vivo with vvDD at MOI = 5.0, as described in Materials and methods (n = 15/group). (a). The viral recovery from cancer cells in the peritoneal cavity at days 4 and 8 after viral delivery. IS, animals with IS; NS: non-immunosuppressed. ND: not detected. The p values are ** p < 0.032; * p < 0.05. (b). live whole animal imaging after virus delivery. The pre-immunized and tumor-bearing mice were injected i.p. with 1.0E8 pfu of vv.luc per mouse. The imaging was performed on day 4 after viral injection as described.. The representative images were from mice with MC38 i.p. tumors with and treated with either vv.luc alone (A), IS and vv.luc (B), or IS and vv.luc delivered via MC38 carrier cells (C).

Figure 5. Analyses of the tumor-infiltrated leukocytes in the tumor of pre-immunized hosts mock-treated or treated with immunosuppressive drugs on day 4 after viral administration

(a). Relative quantities of major classes of leukocytes as analyzed by immunostaining of cell surface markers and then flow cytometry. The antibodies are against cell surface proteins of CD4 (CD4⁺ T cells), CD8 (CD8⁺ T cells), CD11c (dendritic cells), NK1.1 (NK cells), Ly6G (granulocytes and neutrophils) and MAC-3 (macrophages). (b). Examples of flow cytometric analysis of F4/80⁺/MAC-3⁺ dual positive TAMs in the tumors of pre-immunized mice with or without treatment with IS drugs. (c). Expression of two key cytokines in F4/80+/MAC-3+ dual positive TAMs from tumors in either IS drug-treated or untreated mice, and naïve and activated pMACs serve as controls. Total RNAs were purified by standard procedure as described. The expression of murine IL-10 and IL-12/P40 mRNA (left and right graphs) was quantified by real-time RT-PCR. The copy number of target mRNA was normalized to that of the housekeeping gene GAPDH. Data represent mean +/- SEM.

Figure 6. The combined effects of immunosuppressive drugs and carrier cells on the efficacy of vvDD oncolytic virotherpy in pre-immunized mice bearing intraperitoneal MC38 colorectal carcinomatosis

B6 were pre-immunized for one month before inoculation of MC38 tumor cells for i.p. colorectal carcinomatosis. Ten days after tumor cell inoculation, IS was initiated in all mice (n = 15 per group), and then treated with either HBSS saline (CTL), vvDD (vvDD naked), or vvDD delivered via MC38 carrier cells (vvDD with carriers). The survival and health of these mice were closely monitored. Survival data were plotted on a Kaplan-Maier curve. Statistical comparisons were performed using a log-rank test. The p values: p < 0.0001 (control versus naked); p < 0.00001 (control versus vvDD with carriers); p ≤ 0.017 (vvDD [naked] vs vvDD with carriers).