4 1bb cd3ζ expression cassette

The chimeric GD2/CAR is composed of GD2 scFv and a 4-1BB-CD3ζ expression cassette that was designed and synthesized by the GeneChem Biotechnology Company (Shanghai, China), as shown in Fig. 3a. The GD2 scFv was derived from a high-affinity 14.G2a monoclonal antibody. The 4-1BB-CD3ζ expression cassette contains the hinge and transmembrane (TM) region of CD8α. GD2 scFv and 4-1BB-CD3ζ were connected in-frame by overlap PCR. The generated GD2/CAR was verified by DNA sequencing and cloned into the BamHI sites of a lentiviral vector (Genechem Biotechnology, China); the resultant product was named GD2.BBζ CAR. The specific structure of the viral vector is shown in Additional file 1: Figure S1. The intracellular domain of the CARs has the self-cleaving 2A peptide connected to an EGFP green fluorescent label. The sequences of all PCR primers are available upon request.

The chimeric CD166/CAR is composed of CD166 scFv and a 4-1BB-CD3ζ expression cassette that was designed and synthesized by the GeneChem Biotechnology Company (Shanghai, China), as is shown in Fig. 2a. The CD166 scFv was derived from a high-affinity monoclonal antibody. The 4-1BB-CD3ζ expression cassette contains the hinge and transmembrane (TM) region of CD8α. CD166 scFv and 4-1BB-CD3ζ were connected in-frame by overlap PCR. The generated CD166/CAR was verified by DNA sequencing and cloned into the BamHI sites of a lentiviral vector (Genechem Biotechnology, China); the resultant product was named CD166.BBζ CAR. The intracellular domain of the CARs has the self-cleaving 2A peptide connected to a GFP green fluorescent label. The sequences of all PCR primers are available upon request.