Figure 2.

Sequence and expression of candidate MET-specific CARs in human T-cells. (A) Targeting moieties were derived from the NK1 splice variant of HGF and contained the indicated mutations. Both M2.2 and cM2.2 contain a D127N revertant mutation which restores the naturally occurring sequence found in human NK1. (B) Cartoon structure of CARs. The horizontal line indicates the transmembrane domain. (C) Representative examples of cell surface expression of the indicated CARs. Detection was performed by flow cytometry after incubation with the anti-Myc 9e10 antibody. Percentage positivity has been calculated with respect to staining by secondary antibody alone. Data are representative of 10 independent replicates. (D) Expression of CARs in human T-cells was also detected by western blotting, performed under reducing conditions and probed with an anti-CD3ζ antibody. Arrowed CAR bands are of the predicted molecular mass while the endogenous T-cell receptor-associated CD3ζ band (predicted molecular mass 18 kDa) serves as a loading control.