Oncolytic virotherapy is a clinically validated class of immune modulatory drugs with activity in solid cancer. The mode of action of armed oncolytic Vaccinia viruses (oVV) includes cell death induction following selective replication in tumor cells, local inflammation and priming of adaptive immune responses, and intra-tumoral delivery of therapeutic modalities e.g. antibodies, cytokines, enzymes and engineered receptor ligands. BioInvent and Transgene are collaborating in the development of a next generation armed oncolytic viruses, bringing respectively their n-CoDeR F.I.R.S.T antibody (mAb) discovery and Invir.IO oVV platforms. The first product, named BT-001, consists of the Copenhagen oVV strain deleted in J2R and I4L viral genes allowing restricted replication in proliferating cells, and the human IgG1 4-E03 recognizing human CTLA4. 4-E03 shows improved Treg-depleting activity compared with ipilimumab. BT-001 also encodes GM-CSF, the cytokine expressed in clinically advanced products like T-Vec (Imlygic) or Pexa-Vec. A surrogate mAb with similar pronounced Treg-depleting properties to 4-E03 was vectorized into the same oVV (mBT-1) allowing for functional and mechanistic in vivo studies. Since virus replication and expression of recombinant transgenes are tightly linked, the armed oVV is an interesting design for intra-tumoral delivery of therapeutic proteins exhibiting toxicological limitation (e.g. anti-CTLA4) and/or pharmacokinetic issues (e.g. GM-CSF). Oncoviral tumor-localized delivery of anti-CTLA4 may also allow better tolerated and more effective combination therapy with approved antibodies targeting the PD-1/PDL1 axis. Our studies demonstrate that 4-E03 and GM-CSF were expressed at high level as functional molecules after infection by BT-001 of several human tumoral cell lines in vitro. Moreover, following intratumoral administration in immune competent and immune deficient mice transplanted with mouse or human tumors, transgene expression was sustained at levels associated with receptor saturation for days to weeks. In contrast, and supporting the tumor-selective nature of oVV, blood concentrations of anti-CTLA4 mAb were lower compared to those observed following I.V. administration of therapeutic doses (3 mg/kg) of mAb. The in vivo anti-tumor activity of mBT-1 was assessed in multiple syngeneic mouse tumor models including CT26, EMT6, A20, and C38. mBT-1 conferred cures in the majority of challenged mice irrespective of tumor origin. The excellent anti-tumoral profile depends on anti-CTLA4 expression and could be boosted by co-administration of anti-PD-1 mAb. A clinical batch of BT-001 has been produced and toxicological evaluation is ongoing. Transgene and BioInvent are preparing CTA and IND filings for a multicentre clinical trial targeting injectable superficial tumors. Citation Format: Jean-Baptiste Marchand, Monika Semmrich, Laetitia Fend, Matilda Rehn, Nathalie Silvestre, Ingrid Teige, Johann Foloppe, Linda Martensson, Eric Quemeneur, Bjorn Frendeus. BT-001, an oncolytic vaccinia virus armed with a Treg-depletion-optimized recombinant human anti-CTLA4 antibody and GM-CSF to target the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5602.