Combination of immunogenic oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab exhibits synergistic antitumor effect in humanized A2058 melanoma huNOG mouse model

Lukasz Kuryk, Anne-Sophie W Møller, Magnus Jaderberg, Lukasz Kuryk, Anne-Sophie W Møller, Magnus Jaderberg

Abstract

Malignant melanoma is an aggressive type of skin cancer whose incidence is increasing globally. Although surgery is effective in early stage melanoma, patients with advanced melanoma only have a 20% 5-year survival rate. Hence, combinations of existing and new immunotherapy technologies and immunotherapeutic agents are being evaluated. ONCOS-102 is an oncolytic adenovirus armed with human GM-CSF and an Ad5/3 chimeric capsid. It has shown to be well tolerated in phase I study (NCT01598129) wherein it induced antitumor immunity, infiltration of CD8 + T cells to tumors, and up-regulation of PD-L1. We propose that ONCOS-102 could serve as an immunosensitizer in combination therapies with checkpoint inhibitors. In this preclinical study, we investigated the cytotoxicity of ONCOS-102 and pembrolizumab, an anti-PD-1 antibody, in four human melanoma cell lines, A375, A2058, SK-Mel-2 and SK-Mel-28. Humanized mice engrafted with A2058 melanoma cells showed significant tumor volume reduction after ONCOS-102 treatment. Combination of pembrolizumab with ONCOS-102 reduced tumor volume to an even greater extent, while pembrolizumab (200 µg, or 400 µg) did not show any therapeutic benefit by itself. Body weight loss, and metastasis were not significantly affected by any treatment. These data support the scientific rationale for the ongoing clinical study of combination therapy of ONCOS-102 and pembrolizumab for the treatment of melanoma (NCT03003676).

Keywords: NOG; ONCOS-102; PD-1 inhibitor; Pembrolizumab; antitumor; cancer vaccine; immunogenic cell death; melanoma; oncolytic adenovirus.

Figures

Figure 1.
Figure 1.
Expression of CAR, CD46, Desmoglein-2 and PD-L1 in human melanoma cells measured by flow cytometry (at least 104 cells/events were analyzed by flow cytometry in one replicate experiment). Data are expressed as percentage of cells positive for the marker.
Figure 2.
Figure 2.
Effects of ONCOS-102 and pembrolizumab on annexin V (A) and propidium iodide staining (B) in human melanoma cell lines after 72 h (A375, SK-Mel-2) and 96 h (A2058, SK-Mel-28). Data are expressed as percentage of cells staining positive. Analyses were performed in one replicate experiment.
Figure 3.
Figure 3.
Effects of ONCOS-102 and pembrolizumab treatment on three indicators of immunogenic cell death in human melanoma cell lines after 72 treatment. A. Calreticulin exposure. B. ATP release in human melanoma cell lines after 72 treatment. C. Effects of ONCOS-102 and pembrolizumab on HMGB1 release in human melanoma cell lines after 72 treatment. Data are expressed as percentage of cells staining positive for specific marker. Analyses were performed in two replicates experiment.
Figure 4.
Figure 4.
Effects of ONCOS-102 and pembrolizumab treatments on tumor volume of A2058 engrafted hu-mice. A. Tumor volume on day 26 for right and left tumor. B. Tumor volume on day 40 for right and left tumor. C. Tumor volume throughout the treatment (pooled for right and left tumors). D. Tumour volume on day 40 (pooled for right and left tumors). Data represent mean ± SEM. *P < 0.05, **P < 0.01 vs vehicle. Groups: 1–6 had 8 animals/group, groups: 7–8 had 6 animals/group.
Figure 5.
Figure 5.
Effects of ONCOS-102 and pembrolizumab on number of liver nodules in each mouse at time of sacrifice (day 40). Data represent mean ± SEM. Groups: 1–6 had 8 animals/group, groups: 7–8 had 6 animals/group.
Figure 6.
Figure 6.
Effects of ONCOS-102 and pembrolizumab on number of hCD45+ leukocytes. A. Number of hCD45+ leukocytes per gram of tumor on left and right flank. B. Percentage of hCD3+ leukocytes among hCD45+ leukocytes in tumors on left and right flank. The populations were gated with forward and side scattering (FSC-A/SSC-A dot plot) in leukocytic regions (at least 104 cells/events were analyzed by flow cytometry, 6–8 tumors/experimental group have been analysed in one experimental replicate). Flow cytometry analysis was performed on FlowJo v10 software. Data represent mean ± SEM. n = 2–6, P* < 0.05, P** < 0.01 vs vehicle.
Figure 7.
Figure 7.
Effects of ONCOS-102 and pembrolizumab on subsets of leukocytes in tumors on left and right flank. A. Percentage of hCD8+ leukocytes among hCD3+ leukocytes. B. percentage of hCD4+ leukocytes among hCD3+ leukocytes. C. Percentage of PD-L1+ leukocytes in tumors. The populations were gated with forward and side scattering (FSC-A/SSC-A dot plot) in leukocytic regions (at least 104 cells/events were analyzed by flow cytometry, 6–8 tumors/experimental group have been analysed in one experimental replicate). Flow cytometry analysis was performed on FlowJo v10 software. Data represent mean ± SEM.

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