Figure 2.

Design of retroviral constructs. (A) In order to engineer an αvβ6-targted chimeric antigen receptors (CAR), the A20 peptide derived from the capsid protein VP1 from Foot and Mouth Disease Virus (serotype 01 BFS) was placed downstream of a CD124 signal peptide. A matched but scrambled peptide (named C20) was generated in which RGDL was replaced with AAAA. (B) The SFG retroviral vector was used to express CARs in human T-cells. LTR: long terminal repeat; S: signal peptide; T: CAR targeting peptide; M: human c-myc epitope tag, recognized by 9e10 antibody. In some constructs, equimolar co-expression of the chemokine receptor CXCR1 or CXCR2 was achieved using a Thosea Asigna (T)2A ribosomal skip peptide, placed downstream of a furin cleavage site, designed to remove peptide overhangs on the C-terminus of the upstream encoded polypeptide. (C) Representative example in which healthy donor T-cells were transduced with a retroviral vector encoding for CAR +/− chemokine receptor. After culture for 12 days in IL-2, cells were analyzed by flow cytometry for expression of the myc epitope-tagged CAR and indicated chemokine receptors by flow cytometry. SSC: side scatter. Quadrants were set using untransduced T-cells cultured in IL-2. Data are representative of replicate experiments undertaken with 7 independent donors.