Granulocyte-macrophage colony-stimulating factor-armed oncolytic measles virus is an effective therapeutic cancer vaccine

Abstract

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased antitumor activity, and are currently under investigation in clinical phase 1 trials. Approaches with other viral vectors have shown that insertion of immunomodulatory transgenes enhances the therapeutic potency. In this study, we engineered MV for expression of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). For the first time, therapeutic efficacy and adaptive immune response in the context of MV oncolysis could be evaluated in the previously established immunocompetent murine colon adenocarcinoma model MC38cea. MC38cea cells express the human carcinoembryonic antigen (CEA), allowing for infection with retargeted MV. Intratumoral application of MV-GMCSF significantly delayed tumor progression and prolonged median overall survival compared with control virus-treated mice. Importantly, more than one-third of mice treated with MV-GMCSF showed complete tumor remission and rejected successive tumor reengraftment, demonstrating robust long-term protection. An enhanced cell-mediated tumor-specific immune response could be detected by lactate dehydrogenase assay and interferon-γ enzyme-linked immunospot assay. Furthermore, MV-GMCSF treatment correlated with increased abundance of tumor-infiltrating CD3(+) lymphocytes analyzed by quantitative microscopy of tumor sections. These findings underline the potential of oncolytic, GM-CSF-expressing MV as an effective therapeutic cancer vaccine actively recruiting adaptive immune responses for enhanced therapeutic impact and tumor elimination. Thus, the treatment benefit of this combined immunovirotherapy approach has direct implications for future clinical trials.

FIG. 1.

Recombinant measles virus, transgene expression, and virus kinetics. (A) Schematic representation of vectors harboring either unmodified hemagglutinin (H) or fully retargeted H against carcinoembryonic antigen (CEA) (listed at bottom, middle) and encoding transgene X, either granulocyte-macrophage colony-stimulating factor (GM-CSF) or enhanced green fluorescent protein (EGFP), in an additional transcription unit (bottom left) upstream of the nucleocapsid protein (N) gene. Corresponding names of viruses are listed at bottom right. (B) MV producer cell lines Vero-αHis (left) and murine MC38cea (right) were infected in triplicate with MV-EGFP-antiCEA (circles) or MV-GMCSF-antiCEA (squares) at a multiplicity of infection (MOI) of 1. GM-CSF concentrations in supernatants were determined at the indicated time points. (C) One-step growth curves of either the parental MV-EGFP (solid circles) or the targeted MV-EGFP-antiCEA (open symbols) and MV-GMCSF-antiCEA (solid symbols). Vero-αHis cells (left) and MC38cea cells (right) were infected at an MOI of 3. At the designated time points progeny viral particles were determined in octuplicate by serial dilution titration assay on Vero-αHis cells. The detection limit of progeny particles lies below 12.5 cell infectious units (CIU)/ml.

FIG. 2.

In vivo antitumor activity of MV-GMCSF-antiCEA in an immunocompetent murine colon carcinoma model. MC38cea cells (10⁶) were implanted subcutaneously into the right flank of 6- to 8-week-old C57BL/6 mice. When tumor volumes reached 30–50 μl, mice were treated intratumorally with carrier fluid (mock; n=10), 10⁶ particles of UV-inactivated MV-GMCSF-antiCEA (UV-MV; n=10), 10⁶ particles of MV-EGFP-antiCEA (n=10), or 10⁶ particles of MV-GMCSF-antiCEA (n=9), each on four consecutive days (days 5, 6, 7, and 8 postimplantation). Tumor volumes were measured every third day. (A) Blood was collected at the indicated time points after tumor implantation and an ELISA was performed to evaluate GM-CSF serum levels. (B) Tumor volume was measured every third day. (C) Distribution of tumor volumes on day 16 postimplantation. Each dot represents one mouse. (D) Kaplan–Meier plot documenting survival effects of MV-GMCSF-antiCEA and MV-EGFP-antiCEA compared with mock- or UV-MV-treated mice. Mice were sacrificed when tumors reached a volume of 1500 μl or when severe ulceration occurred. Circles, mock treatment; asterisks, UV-MV; triangles, MV-EGFP-antiCEA; squares, MV-GMCSF-antiCEA.

FIG. 3.

Induction of an antitumor immune response. MC38cea cells (10⁶) were engrafted subcutaneously. When tumor volumes reached 30–50 μl, mice were treated intratumorally on four consecutive days with carrier fluid (mock), 10⁶ particles of UV-inactivated MV-GMCSF-antiCEA (UV-MV), 10⁶ particles of MV-EGFP-antiCEA, or 10⁶ particles of MV-GMCSF-antiCEA per day. Spleens were harvested aseptically 15 days after tumor implantation. (A) Lactate dehydrogenase (LDH) assay. MC38cea cells were cocultivated with prestimulated splenocytes obtained from mock-treated mice and mice treated with UV-MV, MV-EGFP-antiCEA, or MV-GMCSF-antiCEA. Mean values of n=7 (mock), n=8 (MV-EGFP-antiCEA), n=9 (MV-GMCSF-antiCEA), and n=4 (UV-MV) mice are shown, and the standard deviations. (B) ELISPOT assay for tumor-specific interferon (IFN)-γ secretion. Splenocytes isolated from treated mice were cocultivated with MC38cea tumor cells for 24 hr. Spots of IFN-γ-secreting cells were counted with an automated colony-counting device. (C) Virus-specific IFN-γ secretion. Splenocytes were cocultivated with MV-GMCSF-antiCEA or MV-EGFP-antiCEA at an MOI of 0.5 for 24 hr. Circles, mock treatment; asterisks, UV-MV; triangles, MV-EGFP-antiCEA; squares, MV-GMCSF-antiCEA. **p≤0.01; n.s., not significant.

FIG. 4.

Immunohistological staining of CD3⁺ lymphocytes. Remaining tumors were isolated 7 days after the last virus application. The numbers of stained immune cells in tumors and at the invasive margin of (A) mock-treated, (B) MV-EGFP-antiCEA-treated, and (C) MV-GMCSF-antiCEA-treated mice were quantified automatically. Scale bars: 100 μm. (D) The average total number of CD3⁺ cells per tumor and invasive margin, respectively, in each group.

FIG. 5.

Memory immune response. (A) Percentage of naive and MV-GMCSF-antiCEA-pretreated mice, rechallenged with MC38cea cells, that demonstrate tumor engraftment. (B) Mice that demonstrated complete tumor remission were sacrificed 6 months after the initial experiment. Spleens were isolated and an antitumor-specific LDH assay was performed. LDH release of MC38cea cells cocultivated with prestimulated splenocytes was measured and calculated as the percentage of dying MC38cea cells. Circles, mock; squares, MV-GMCSF-antiCEA.

FIG. 6.

Secondary engraftment of MC38cea tumors. Tumor cells (10⁶) were implanted subcutaneously into the right flank of C57BL/6 mice (primary engraftment) and treated intratumorally with Opti-MEM (n=6), MV-EGFP-antiCEA (n=5), or MV-GMCSF-antiCEA (n=6) on four consecutive days as soon as tumors reached a volume of approximately 30 μl. Two days after the last virus treatment, 10⁶ MC38cea cells were injected into the left flank (secondary engraftment). Tumor volume development of each individual is shown after primary engraftment (right) and secondary engraftment (left) of groups treated with carrier fluid, MV-EGFP-antiCEA, or MV-GMCSF-antiCEA. Two of six mice treated with MV-GMCSF-antiCEA demonstrated complete tumor remission of the primary engraftment. Arrows indicate the time point of secondary tumor implantation.