Armed Oncolytic Myxoma Virus Induces Systemic Antitumor Immunity Against Solid Tumors in Immunocompetent Mice

Technical Summary: Armed Oncolytic Myxoma Virus Induces Systemic Antitumor Immunity Against Solid Tumors in Immunocompetent Mice

Problem Statement
Oncolytic viruses (OVs) are promising cancer therapeutics that selectively infect and kill cancer cells while stimulating immunogenic cell death (ICD) to activate anti-tumor immunity. While "armed" OVs expressing immunostimulatory transgenes have shown superior efficacy compared to unmodified viruses in preclinical and clinical settings (e.g., T-VEC), the specific comparative efficacy of different cytokine arms in inducing systemic (abscopal) immunity remains to be fully characterized. Specifically, while IL-15 and LIGHT are known to activate lymphocytes and dendritic cells, their delivery via Myxoma virus (MYXV) in a solid tumor model requires direct comparison to determine which construct best remodels the tumor microenvironment (TME) and induces long-term survival. Previous studies indicated that simple IL-15 expression by MYXV had limited effects in immunocompetent mice, whereas fusion proteins (IL-15/IL-15Rα) and LIGHT showed promise in specific contexts, but a head-to-head evaluation of these variants for systemic anti-tumor immunity was lacking.

Methodology
The study employed a syngeneic mouse model using BALB/c mice and CT26 murine colorectal carcinoma cells to evaluate three recombinant MYXV variants:

1. vMyx-mIL15: Expressing murine IL-15.
2. vMyx-mIL15Rα: Expressing a murine IL-15/IL-15Rα fusion protein (superagonist).
3. vMyx-mLIGHT: Expressing murine LIGHT (TNFSF14).

These were compared against a wild-type MYXV (WT-MYXV) and a PBS control.

• In Vitro Characterization: Replication kinetics were assessed in murine (CT26) and human (HCT116, HT29, Colo205) colorectal cancer cell lines using single-step and multi-step viral replication assays. Cytotoxicity was measured via MTS assays at various multiplicities of infection (MOI). Protein expression and secretion of IL-15/IL-15Rα and LIGHT were quantified via ELISA in Vero and CT26 cells.
• In Vivo Therapeutic Model: Bilateral flank tumors were established in BALB/c mice. Once tumors reached 50–100 mm³, mice received four intratumoral (IT) injections over two weeks into the left flank tumor only. Tumor growth was monitored on both the injected and contralateral (uninjected) flanks to assess local and systemic (abscopal) effects. Survival was tracked until tumors reached endpoint criteria (1200–1500 mm³).
• Immunological Analysis:
  • Flow Cytometry: Tumor-infiltrating lymphocytes (TILs) were harvested from both treated and untreated tumors to quantify CD4+/CD8+ T cells, Natural Killer (NK) cells, Natural Killer T (NKT) cells, and Dendritic Cells (DCs). Activation markers (e.g., CD69) and memory phenotypes (Tem, Tcm) were analyzed.
  • Cytokine Profiling: Serum levels of 44 cytokines and chemokines were measured using a multiplex ELISA microarray to assess systemic immune modulation.
  • Histology: Immunohistochemical (IHC) staining was performed to visualize CD8+ T cell infiltration.

Key Results

• Viral Replication and Cytotoxicity: All armed MYXV variants replicated with kinetics and produced progeny titers comparable to WT-MYXV in both murine and human colorectal cancer cell lines, indicating that transgene expression did not impair viral fitness. In vitro cytotoxicity assays showed that at high MOI, all viruses reduced cell viability similarly. However, at low MOI (0.1), armed viruses demonstrated significantly enhanced cell killing compared to WT-MYXV, suggesting the transgenes contribute to anti-proliferative effects at lower infection densities.
• Protein Expression: Both mIL-15Rα/IL-15 and mLIGHT were successfully expressed and secreted by infected cells. The IL-15Rα/IL-15 fusion protein was detected as early as 2 hours post-infection, while mLIGHT secretion increased significantly by 24 hours.
• Therapeutic Efficacy and Abscopal Effect:
  • PBS Control: Tumors grew rapidly; all mice reached euthanasia criteria within 15 days.
  • WT-MYXV: Delayed tumor growth by approximately 3 days compared to PBS but failed to induce significant regression or long-term survival.
  • vMyx-mIL15: Reduced tumor burden on the injected side but failed to induce a significant abscopal effect on the contralateral tumor; survival was not significantly extended.
  • vMyx-mIL15Rα and vMyx-mLIGHT: Both variants induced significant regression in both injected and uninjected contralateral tumors. By day 30, two mice in each of these groups were tumor-free. Notably, vMyx-mIL15Rα treatment resulted in significantly longer overall survival compared to all other groups, including vMyx-mLIGHT.
• Immune Cell Infiltration:
  • vMyx-mIL15Rα: Significantly increased infiltration of effector memory CD8+ T cells, NKT cells, and NK cells in both treated and untreated tumors.
  • vMyx-mLIGHT: Enhanced infiltration of effector memory CD4+ T cells and Dendritic Cells (DCs).
  • Activation: Both armed treatments significantly increased the percentage of activated (CD69+) NK and NKT cells compared to controls.
• Cytokine Remodeling: Serum profiling revealed that armed MYXV treatments upregulated key Type I antitumor cytokines (e.g., IFNγ, IL-12, TNFα, IL-2) and downregulated protumor inflammatory cytokines and chemokines (e.g., IL-1β, IL-17, IL-20, MIP-2, MIP-3α). This shift suggests a systemic remodeling of the immune environment toward an anti-tumor state.

Significance and Claims
The authors claim that arming MYXV with immunostimulatory cytokines, specifically the IL-15/IL-15Rα fusion protein and LIGHT, significantly enhances both local and systemic anti-tumor activity against solid tumors. The study demonstrates that:

1. Systemic Immunity: Intratumoral delivery of these armed viruses can induce a potent abscopal effect, regressing untreated distal tumors, likely through the activation of systemic anti-tumor immunity.
2. Superiority of Fusion Protein: The IL-15/IL-15Rα fusion protein (vMyx-mIL15Rα) outperformed simple IL-15 expression and showed superior survival benefits compared to LIGHT, likely due to the enhanced bioavailability and stability of the fusion protein in circulation.
3. Mechanism of Action: The therapeutic efficacy is mediated by the recruitment and activation of specific immune subsets (CD8+ T cells, NKT cells, and DCs) and the remodeling of the TME from a protumor to an antitumor cytokine profile.
4. Clinical Relevance: These findings support the development of armed oncolytic MYXVs, particularly those expressing IL-15/IL-15Rα, as a strategy to overcome immunosuppressive TMEs in solid tumors and potentially treat metastatic disease. The authors suggest these viruses warrant further investigation in combination with immune checkpoint inhibitors for "cold" tumors.