Scott T. R. Walsh

Hinge modifications do not improve antigen specificity of CD123-targeted chimeric IL7Rα. A, Amino acid sequences of hinge regions used in functional studies. B, Transduction efficiency measured using his-tagged rhCD123 binding of engineered receptors and flow cytometry. n = 3 unique T-cell donors. C, Quantification of phosphorylated STAT5 percentage in CCR-T cells measured with and without activation on the immobilized rhCD123 target. n = 3 unique T-cell donors, comparison between pSTAT5% in cytokine-starved cells with or without rhCD123 activation; *, P < 0.05. D, Representative Western blot testing IgG4 vs. IH STAT5 activation with and without immobilized rhCD123. NT, nontransduced.

T cells with IL7 pathway activation expand and circulate in vivo. A, Schematic of experimental design. NSG mice were injected on day 0 with 107 T cells expressing a ffLuc reporter. BLI was conducted biweekly, and peripheral blood collected on days 15 and 30. One group of mice was treated with ffLuc-only–modified T cells and intraperitoneal injections of IL7 daily from day 1 to 21. B, BLI monitoring T-cell expansion. C, Quantified radiance per mouse. Thick solid line, median BLI; dotted lines, individual values per mouse. D, Calculated doubling time of T-cell products with 95% confidence interval represented. E, T cells (CD3+GFP+) detected in peripheral blood on days 15 and 30 after injection. Thick solid line, median; dotted lines, individual values per mouse. Comparison between treatment cohorts on day 30 vs. no treatment: *, P < 0.05; ***, P < 0.001. F, Concentration of human IL7 in murine plasma detected with ELISA. For the intraperitoneally injected mice on day 15, blood was collected 1 hour after IL7 injection. (n = 5 mice per group). hIL7, human IL7; NSG, NOD/SCIDγ; NT, nontransduced; PB, peripheral blood.

Chimeric antigen receptor (CAR) T cells can effectively treat leukemias, but sustained antitumor responses can be hindered by a lack of CAR T-cell persistence. Cytotoxic effector T cells are short-lived, and establishment of CAR-T cells with memory to ensure immune surveillance is important. Memory T cells depend on cytokine support, with IL7 activation of the IL7 receptor (IL7R) being critical. However, IL7R surface expression is negatively regulated by exposure to IL7. We aimed to support CAR T-cell persistence by equipping CAR-T cells with a sustained IL7Rα signal. We engineered T cells to constitutively secrete IL7 or to express an anti–acute myeloid leukemia–targeted IL7Rα–chimeric cytokine receptor (CCR) and characterized the phenotype of these cell types. Canonical downstream signaling was activated in CCR-T cells with IL7R activation. When coexpressed with a cytotoxic CAR, functionality of both the CCR and CAR was maintained. We designed hybrid CAR–CCR and noted membrane proximity of the intracellular domains as vital for signaling. These data show cell-intrinsic cytokine support with canonical signaling, and functionality can be provided via expression of an IL7Rα domain whether independently expressed or incorporated into a cytotoxic CAR for use in anticancer therapy.Significance:To improve the phenotype of tumor-directed T-cell therapy, we show that provision of cell-intrinsic IL7R-mediated signaling is preferable to activation of cells with exogenous IL7. We engineer this signaling via independent receptor engineering and incorporation into a CAR and validate maintained antigen-specific cytotoxic activity.

CCR expression does not diminish CAR T-cell antitumor activity. A, Schematic of the xenograft model. On day 0, NSG mice were injected via tail vein with 1 × 106 CD123+ MV-4-11.ffLuc cells. In treatment groups, 2 × 106 T cells were administered on day 7. Cohorts: unmodified (NT), CD28.ζ CAR, and CD28.ζ CAR + IL7Rα CCR-T cells. B, Leukemia proliferation was monitored with BLI, and whole-animal radiance was recorded (photons/second/cm2/sr). Comparison of radiance in each group was performed following log transformation of each measured value. C, Kaplan–Meier survival analysis of MV-4-11 xenografts (n = 9 mice per condition; two independent experiments; median survival: NT: 45.5 days; CAR: 93.5 days; CAR + CCR: undefined; ***, P < 0.001; ****, P < 0.0001). NSG, NOD/SCIDγ; NT, nontransduced.