Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.

Pancreatic ductal adenocarcinoma (PDAC) has been increasing by 0.5% per year in the United States. PDAC portends a dismal prognosis and novel therapies are needed. This study describes the generation and characterization of a novel oncolytic chimeric orthopoxvirus for the treatment of pancreatic cancer. After chimerization and high-throughput screening, CF33 was chosen from 100 new chimeric orthopoxvirus isolates for its ability to kill pancreatic cancer cells. In vitro cytotoxicity was assayed in six pancreatic cancer cell lines. In vivo efficacy and toxicity were evaluated in PANC-1 and MIA PaCa-2 xenograft models. CF33 caused rapid killing of six pancreatic cancer cells lines in vitro, releasing damage-associated molecular patterns, and regression of PANC-1 injected and non-injected distant xenografts in vivo after a single low intratumoral dose of 103 plaque-forming units. Using luciferase imaging, CF33 was noted to preferentially replicate in tumors which corresponds to the low viral titers found in solid organs. The low dose of CF33 required to treat pancreatic cancer in this preclinical study may ease the manufacturing and dosing challenges currently facing oncolytic viral therapy.

Peritoneal carcinomatosis (PC) from pancreatic ductal adenocarcinoma (PDAC) is fatal. Our preclinical study presents an effective treatment against PDAC PC using a novel oncolytic viral agent, CF33-hNIS-antiPDL1. CF33-hNIS-antiPDL1 is a genetically engineered chimeric orthopoxvirus, CF33, armed with the human Sodium Iodide Symporter (hNIS) and anti-PD-L1 antibody (anti-PD-L1). The in vitro cytotoxic ability of this virus against 5 PDAC cell lines was tested at various doses (multiplicity of infection [MOI] = 0.01, 0.1, 1, 10). Production and blockade function of virus-encoded anti-PD-L1 antibody were verified using immunoblot, immunoprecipitation, and PD-1/PD-L1 bioassay. In vivo mouse models of PC, with or without subcutaneous (SC) tumors, created by injecting AsPC-1-ffluc cells into nude mice, were treated with PBS or a single dose (1×10⁵ plaque-forming units) of either intraperitoneal (IP) or IV injection of CF33-hNIS-antiPDL1. Mice with PC tumors were treated on days 0, 2, or 14 after tumor implantation. CF33-hNIS-antiPDL1 killed PDAC cells in a dose-dependent manner, achieving >90% cell killing by day 8. Cells infected with CF33-hNIS-antiPDL1 produced bioactive anti-PD-L1 antibody, which blocked PD-1/PD-L1 interaction. In vivo, a single dose of virus reduced tumor burden and prolonged survival of treated mice. It was observed that IP administration of CF33-hNIS-antiPDL1 was more effective than IV administration. CF33-hNIS-antiPDL1 virus is effective in infecting and killing human PDACs and producing functional anti-PD-L1 antibody. Intraperitoneal delivery of CF33-hNIS-antiPDL1 effectively reduces peritoneal tumor burden and improves survival after only 1 dose and is superior to IV delivery.