Generation of huEGFRt expressing construct. (A) Molecular model of human EGFR versus EGFRt proteins. Models were created using Accelrys Discovery Studio, Version 2.0 software and are based on the human EGFR crystal structure files 1YY9 (http://www.pdb.org/pdb/explore/explore.do?structureId = 1YY9), which provides the structure of the 4 extracellular domains (red, green, teal, and purple), and 3GOP (http://www.pdb.org/pdb/explore/explore.do?structureId = 3GOP), which provides the crystal structure of the intracellular juxtamembrane domain (brown) and the tyrosine kinase domain (blue). The transmembrane helix (magenta) is modeled on known helix structure. The structure of the last approximately 190 amino acids of the C-terminal intracellular domain is not known, so the placeholder for this is indicated (gray). (B) Schematic of the huEGFRt amino acid sequence. Amino acid ranges that were used from the GM-CSF receptor-α chain signal sequence (GMCSFRss, which directs surface expression) and the huEGFR sequence (domains III, IV and the transmembrane domain) are indicated. (C) Schematic of the CD19CAR-T2A-EGFRt construct contained in the lentiviral vector. Codon optimized sequence portions of the CD19-specific, CD28 costimulatory CAR (CD19CAR), followed by the self-cleavable T2A, and huEGFRt genes are indicated, along with the Elongation Factor 1 promoter sequences (EF-1p), the GM-CSF receptor-α chain signal sequences (GMCSFRss), and the 3 nucleotide stop codon.