Anti cd3 anti cd28 antibody coated beads

A 32A9-based second generation 4-1BB CAR fragment was synthesized (GenScript, Nanjing, Jiangsu). To detect the expression of CAR, an EGFP coding sequence was added upstream of the 32A9-CAR fragment separated by an F2A sequence. The whole sequence was subcloned into the pLVX lentiviral vector. The same version of FMC63 4-1BB CAR targeting CD19 was used as a negative control [38 (link)]. To produce viral supernatant, HEK293T cells were cotransfected with CAR lentiviral vector, packaging plasmid and envelope plasmid using Lipofectamine 2000 according to the manufacturer’s protocol. The supernatant was collected and concentrated 72 h later. PBMCs were stimulated for 24 h with anti-CD3/anti-CD28 antibody-coated beads (Invitrogen, Carlsbad, CA) at a 2:1 bead-to-T cell ratio in growth medium supplemented with 50 U/ml IL-2 (GenScript, Nanjing, Jiangsu). Activated T cells were then transduced with the lentivirus expressing CARs (MOI = 10). Cells were counted and fed fresh growth medium every other day. The transduction efficiency was detected by GFP fluorescence with flow cytometry.

We generated the PD-L1-target, shark V_NAR-based, CAR-T cell lentiviral vector following the design principle of a CAR construct published in our previous study.¹⁰ (link) Briefly, the V_NAR fragment of B2 was subcloned into a CAR construct (pMH330). The CAR-expressing lentivirus was produced as described previously.¹⁰ (link) Whole blood was collected from healthy donors under the Oklahoma Blood Institute Institutional Review Board approval. Human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were stimulated for 24 h using anti-CD3/anti-CD28 antibody-coated beads (Invitrogen) at a bead:cell ratio of 2:1 according to the manufacturer’s instructions in the presence of IL-2.

The CT3 variable regions were cloned using 5′RACE with modified primers and conducted as described previously.⁵⁰ (link)^,51 (link) The single-chain variable fragment (scFv) of CT3 was subcloned into a 4-1BB-based CAR construct (pMH303). The hEGFRt was included for cell tracking and ablation. CT3 CAR lentiviruses were produced by co-transfecting with packaging plasmid psPAX2 and envelope plasmid pMD2.G into HEK293T cells using Calfectin (SignaGen) as described previously.¹⁹ (link) PBMCs from healthy donors were stimulated for 24h using anti-CD3/anti-CD28 antibody-coated beads (Invitrogen) at a bead: cell ratio of 2:1 according to manufacturer’s instructions in the presence of IL-2. To track T cell numbers over time, viable cells were counted using trypan blue.