The sequence of tmTNF-α CAR was synthesized by TSINGKE Biological Technology (Beijing, China). It comprised a human GM-CSF signal peptide, scFv of anti-tmTNF-α antibody, human CD8α hinge and transmembrane domains, and 4-1BB and CD3ζ cytoplasmic domains. The tmTNF-α CAR gene was cloned into the pHAGE-CMV-MCS-PGK puro vector at the BamH I and Xho I sites following PCR amplification. The construct was verified by DNA sequencing (TSINGKE Biological Technology). To produce lentiviral particles, the HEK 293 T cells were transfected with the tmTNF-α CAR lentiviral expression vector, a packaging vector psPAX2, and an envelope vector pMD2G11 (link) using polyethylenimine (PEI) Max, linear, MW 40 000 (PolyScience, Illinois, USA) according to the manufacturer’s instructions. The viral supernatants were collected 72 hours after transfection, filtered through a 0.45 µm filter (Millipore, Darmstadt, Germany), and concentrated by ultracentrifugation (Beckman Coulter, Miami, Florida, USA) through a 20% sucrose cushion at 25 000 rpm 4°C for 2 hours. Viral titers were determined in the HEK 293 T cells after infection with serial dilutions of virus by flow cytometry using an APC-conjugated anti-F(ab’)2 fragment of murine IgG (Jackson ImmunoResearch, West Grove, Pennsylvania, USA).