Recombinant human her2 fc chimera

Enzyme-linked immunosorbent assay (ELISA) plates (Corning) were coated overnight with 2 μg/mL recombinant human HER2 Fc chimera (R&D Systems). The following day, 100 µl culture supernatant of either vector control or LM008–HER2Ab cells were applied and incubated for 2 h at RT. Increasing concentrations of trastuzumab (0 to 200 ng/mL) were also incubated and used as a positive control. Nonspecific binding was eliminated by vigorous washes with Tris-buffered saline (TBS)-Tween (Boston Bioproducts). The plates were then incubated with secondary antibody, anti-human IgG (Fab specific)-alkaline phosphatase conjugate (Sigma) for 2 h. After washing with TBS-Tween, plates were then incubated with para-Nitrophenyl Phosphate (pNPP) substrate (Sigma) for 5 to 15 min, and the optical density was measured at 405 nM.

Both murine and human CAR-T cells were stimulated using plate-bound recombinant human HER2-Fc chimera (1,000 ng/ml in phosphate-buffered saline; R&D Systems, Minneapolis, MN) or vehicle control for 4 hours at 37 °C in the presence of GolgiPlug protein transport inhibitor (BD Pharmingen, San Jose, CA). Following stimulation, cells were resuspended in 5% FBS (in phosphate-buffered saline) and stored at 4 °C overnight.