Oncolytic adenoviruses synergistically enhance anti-PD-L1 and anti-CTLA-4 immunotherapy by modulating the tumour microenvironment in a 4T1 orthotopic mouse model

Effective therapeutic strategies for triple-negative breast cancer (TNBC) are still lacking. Clinical data suggest that a large number of TNBC patients cannot benefit from single immune checkpoint inhibitor (ICI) treatment due to the immunosuppressive tumour microenvironment (TME). Therefore, combination immunotherapy is an alternative approach to overcome this limitation. In this article, we combined two kinds of oncolytic adenoviruses with ICIs to treat TNBC in an orthotopic mouse model. Histopathological analysis and immunohistochemistry as well as multiplex immunofluorescence were used to analyse the TME. The immunophenotype of the peripheral blood and spleen was detected by using flow cytometry. Oncolytic adenovirus-mediated immune activity in a coculture system of lytic supernatant and splenocytes supported the study of the mechanism of combination therapy in vitro. Our results showed that the combination of oncolytic adenoviruses with anti-programmed cell death-ligand 1 (anti-PD-L1) and anti-cytotoxic T lymphocyte-associated antigen-4 (anti-CTLA-4) (aPC) can significantly inhibit tumour growth and prolong survival in a TNBC model. The combination therapy synergistically enhanced the antitumour effect by recruiting CD8(+) T and T memory cells, reducing the number of regulatory T cells and tumour-associated macrophages, and promoting the polarization of macrophages from the M2 to the M1 phenotype to regulate the TME. The rAd.GM regimen performed better than the rAd.Null treatment. Furthermore, aPC efficiently blocked oncolytic virus-induced upregulation of PD-L1 and CTLA-4. These findings indicate that oncolytic adenoviruses can reprogramme the immunosuppressive TME, while ICIs can prevent immune escape after oncolytic virus therapy by reducing the expression of immune checkpoint molecules. Our results provide a mutually reinforcing strategy for clinical combination immunotherapy.