CSPG4.CAR T-cells for Melanoma

The human melanoma WM115 cells were tagged with the fusion protein eGFP–firefly-luciferase. Human T cells were engineered with CSPG4.CAR as previously described^{41 (link)}. Here we used a CSPG4-specific CAR that encodes the CD28 endodomains with enhanced functionality. WM115 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco; Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U ml⁻¹ penicillin (Invitrogen) and 100 U ml⁻¹ streptomycin (Invitrogen). CAR-T cells were cultured in complete medium containing 45% RPMI 1640 and 45% Click’s medium (Irvine Scientific) with 10% FBS (HyClone), 2 mmoll⁻¹ GlutaMAX, human recombinant IL-7 (5 ng ml⁻¹, Pepro Tech) and human recombinant IL-15 (10 ng ml⁻¹, Pepro Tech). Cells were cultured in an incubator (Thermo Fisher Scientific) at 37 °C under an atmosphere of 5% CO₂ and 90% relative humidity. The cells were subcultivated approximately every 2–3 d at 80% confluence at a split ratio of 1:3.