Abstract Oncolytic viruses can be engineered to become multifunctional cancer immunotherapeutic agents, used either as monotherapy or in combination with immune checkpoint blockade for antitumor effects. Intratumoral (IT) delivery of oncolytic viruses expressing immunomodulatory agents can alter the tumor immunosuppressive microenvironment and facilitate the proliferation and activation of antitumor effector and memory T cells. Although anti-cytotoxic T lymphocyte protein 4 (CTLA-4) antibody has been approved for the treatment of advanced melanoma, its toxicity profile and modest efficacy as a single agent have limited its use in the clinic. Poxviruses are large cytoplasmic DNA viruses and vaccinia virus is a prototypic poxvirus that has been investigated intensively as an oncolytic virus. Its 200-kb genome size allows large insertions of multiple foreign genes. Vaccinia virus has been used in humans extensively in the past during smallpox vaccination. In this study, we engineered a vaccinia (Western Reserve)-based oncolytic virus that expresses anti-muCTLA-4 antibody and human FMS-like tyrosine kinase 3 ligand (Flt3L) for the treatment of murine implantable melanoma via intratumoral injection. We used a mutant vaccinia virus E3LΔ83N as the parental virus. This virus has a deletion of the Z-DNA-binding domain of E3, a key virulence factor, which results in 1000-fold attenuation of the virus. Through homologous recombination at the thymidine kinase (TK) locus of vaccinia E3LΔ83N virus, we successfully generated an attenuated recombinant virus (E3LΔ83N-TK--hFlt3L-anti-muCTLA-4) with a deletion of TK and an insertion of a cassette that allows the expression of both hFlt3L and anti-muCTLA-4 antibody under the vaccinia synthetic early and late promoter. This virus replicates in murine and human tumor cell lines and expresses desired anti-muCTLA-4 antibody and hFlt3L in murine and human melanoma cell lines. IT delivery of this recombinant vaccinia virus is more efficacious compared with E3LΔ83N-TK- (used either alone or in combination of systemic delivery of anti-CTLA-4 antibody), in eradicating or delaying the growth of both injected tumors and non-injected distant tumors, as well as in prolonging the survival of mice in a murine bilateral B16-F10 melanoma tumor implantation model. Furthermore, immunological analyses of tumor-infiltrating lymphocytes, as well as antitumor T cells in spleens, showed highest numbers of activated Granzyme B+ CD4+ and CD8+ T cells in the non-injected distant tumors and highest numbers of antitumor CD8+ T cells in the spleens of mice treated with IT delivery of E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 compared with those treated with E3LΔ83N-TK--hFlt3L or E3LΔ83N-TK-. No toxicities related to IT E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 have been observed. Taken together, our results demonstrate that IT delivery of oncolytic vaccinia virus expressing anti-CTLA-4 and hFlt3L is a safe and effective strategy to enhance antitumor immunity. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Wei Yan, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-306.