Natalie Jordan Holl

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.

Background: Natural Killer (NK) cells have intrinsic anticancer activity that can be redirected toward acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) engineering. Here, we study the functional consequences of CAR binding affinity and targeted epitope on CAR-NK cell activation, cytolytic synapse formation, and antitumor activity. Methods: We characterized NK-92 and primary NK cell populations expressing variant affinity AML-specific CARs containing single-chain variable fragments (scFvs, 26292 or 7G3) targeting two epitopes on CD123. 26292 affinity variants were discovered through directed evolution of an error-prone mutagenic library, while 7G3 affinity variants were previously reported. The resulting CAR-NK cell panel was studied with in vitro binding, activation, and cytotoxicity studies and in mouse xenograft models. Results: 26292 and 7G3 CARs of variable CD123 binding affinities were highly expressed in NK cells and conferred antigen-specific activation in vitro. High-resolution imaging demonstrated greater clustering of high-affinity 7G3 CAR-NK cells and consequent AML target cell death in a short-term time lapse. Low-affinity 7G3 CAR-NK cells exhibited enhanced antigen density discrimination with greater membrane-proximal signaling, cytokine production, and cytotoxicity. In longer-term assays, low-affinity 7G3 CAR-NK cells demonstrated more sustained killing of AML cells. In vivo testing highlighted greater expansion of low-affinity 7G3 CAR-NK cells in two xenograft models. Conclusions: Expression of 26292 and 7G3 CARs with a range of CD123 binding affinities in NK cells leads to antigen-specific activation and cytotoxicity against AML. Affinity-based differences in functional activation and antitumor activity are dependent on time course and are scFv/epitope specific.