(M1430-04-21) Anti-CD47 Peptide Combined with Oncolytic Vesiculovirus-Driven Intratumoral Immunotherapy for Colorectal Cancer

Purpose: In this study, we explored a new strategy for the treatment of colorectal cancer by combining a peptide that blocks 'Don't eat me signal' and oncolytic virotherapy.

Methods: Vesiculoviruses are one of the most attractive oncolytic virus platforms and have several advantages including its well-studied biology, ability to infect a wide range of laboratory cell lines and to produce very high virus yields, a small, easily manipulated genome, and lack of pre-existing immunity in humans. We developed earlier a synthetic chimeric virus (VMG) expressing the glycoprotein (G) from Morreton virus (MorV) and utilizing the remaining structural genes from the vesicular stomatitis virus (VSV). In this study, we engineered the VMG genome to express a proteolytic enzyme (ENZ) that breaks down cellular debris in the tumor microenvironment to help immune cells reach and identify cancer cells. Tumor cells have evolved to engage the immune checkpoints, e.g., CD47, and down-modulate the immune cells, thereby evading the antitumor immune response. The combination of oncolytic viruses and immune checkpoint inhibitors has been studied but mainly focused on the regulation of T cells. In a previous study, we discovered a 12-mer anti-CD47 peptide (GFM) that blocks ‘Don’t eat me signal’ and enhances the macrophage-mediated phagocytosis of tumor cells. We confirmed the in vitro cytotoxicity activity of VMG using proliferation and crystal violet assays. These assays were performed in LoVo human cell line. We studied the in vivo efficacy of combining GFM peptide with oncolytic virus VMG expressing ENZ  in the CT26 syngenic model.

Results: Using the MTS cell viability assay at 72 hours post-infection, we did not observe a significant reduction in the VMG-ENZ infectivity at multiplicities of infection (MOI) of 0.01, 0.1, 1, and 10. We found MC38, CT26 and LoVo cell lines showed susceptibility to oncolysis after 72 hours in culture. CT26 murine colorectal carcinoma cell line highly expresses CD47. The combination showed an additive effect to enhance the inhibition of tumor growth compared to GFM or VMG-ENZ alone. A combination of GFM and VMG-ENZ extended the survival of CT26 bearing mice compared with control mice (PBS). All mice in the control group died by day 39. In contrast, 70% of the mice in the combination group survived. We also studied the systemic toxicity of VMG and GFM in BALB/c mice.

Conclusion: Despite immune checkpoint inhibitors revolutionizing the cancer therapy field, it is largely ineffective in some patients. To address this limitation, oncolytic viruses in combination with anti-CD47 peptide can be a strategy to address the inefficacy of immune checkpoint inhibitors. We observed a synergistic effect of oncolytic virus VMG-ENZ and GFM peptide. A new strategy between oncolytic virus expressing a proteolytic enzyme and immune checkpoint inhibitor can be considered for the treatment of colorectal cancer.


In-vitro cytotoxicity activity of VMG-ENZ in LoVo cells. Data were collected from three replicates. Bars indicate mean ± SEM.



A) VMG-Hybrid Virus Construct. B) GFM peptide inhibiting ‘don’t eat me signal’ of CD47


A) Antitumor activity of GFM and VMG-ENZ combination in CT26 colon cancer mouse model. B) Survival study of GFM peptide and VMG-ENZ combination in CT26 model. C) Body weight changes of CT26-tumor bearing BALB/c mice.