Abstract Novel strategies to reprogram tumor-infiltrating myeloid cells for cancer immunotherapy are urgently needed, given that the primary and acquired resistance to immune checkpoint blockade (ICB) therapy has hindered the overall success of immunotherapy. Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus and an approved vaccine against smallpox and monkeypox. Here we report rational engineering of recombinant MVA, MQ833, by removing three immune suppressive genes, E5R, E3L, and WR199, from the MVA genome and inserting three transgenes encoding Flt3L, OX40L, and IL-12. Intratumoral (IT) delivery of MQ833 generates potent antitumor responses dependent on CD8 + T cells, neutrophils, and M1-like macrophages, the nucleic acid-sensing pathways mediated by MDA5/STING, and interferon feedback loop. IT MQ833 promotes the recruitment and activation of neutrophils and inflammatory monocytes into the injected tumors, depletion of M2-like macrophages, and expansion of M1-like macrophages, generating potent antitumor immunity against tumors resistant to ICB.

IL-12-expressing highly immunogenic recombinant modified vaccinia virus Ankara reprograms tumor-infiltrating myeloid cells to overcome immune resistance