Figure 1.

Generation of Reporter Expressing pan-ErbB Family CAR T Cells

(A) Scheme of the construct platform developed for combined gene transfer of CAR, reporter, and auxiliary genes. Expression is driven from the spleen focus-forming virus promoter (P_SFFV). Selective expansion of clones is enabled by 4αβ. The functional CAR is T1E28z while the truncated CAR T1E28Δz is functionally deficient. The radionuclide reporter NIS is fused to the fluorescent protein mTagRFP (TRFP) in these preclinical cassettes; for clinical applications, the fluorescent protein can be omitted. Proteins are separated by self-cleaving peptide sequences (T2A), whereby trimming of the 4αβ to remove T2A remnants was enabled via an additional furin site. (B) Exposure with corresponding viruses resulted in transduction of primary human T cells as assessed by flow cytometry. Selection of positively transduced clones using IL-4 permitted significant enrichment of CAR-T cells without the need of FACS sorting. (C and D) Phenotypes of transduced T4NT CAR T cells after selection demonstrating purity of T cell features (CD3) and the ratio of CD4⁺ and CD8⁺ T cells. CD4⁺:CD8⁺ dependent on donors (see Figure S2 for examples). Typical data are shown in (B)–(D).