William-Ray Vista

On the basis that T-cell acute lymphoblastic leukemia (T-ALL) cells overexpress IL7 receptor (IL7R), which promotes resistance to chemotherapy and disease relapse, here we develop IL7Rα (CD127)-targeted chimeric antigen receptor (CAR) T cells with low- and high-affinity single-chain variable fragments. We establish the antitumor efficacy of CD127 CAR against T-ALL cells in vitro, in female mouse models of T-ALL, and against blasts from patients with T-ALL using the patients' own T cells transduced with CD127 CAR. Antitumor efficacy is higher with low-affinity CAR T cells than high-affinity CAR T cells, albeit with fratricide of CAR T cells following eradication of CD127-overexpressing blasts. CRISPR-Cas9 knockout of CD127 eliminates fratricide at the risk of prolonged lymphopenia in vivo. To overcome fratricide, we investigate short-term ( < 7 days) co-culture with or without dasatinib, a tyrosine kinase inhibitor, versus a natural selection method (10 days) and demonstrate that co-culturing with dasatinib facilitates higher CAR T-cell yield, improved fitness, and preserved functionality. In vivo, dasatinib can be used to temporarily and reversibly suppress CAR T-cell activity. With this supporting translational data, we are initiating a trial with low-affinity CD127 CAR T cells for adult and pediatric patients with relapsed or refractory T-ALL.

Supplemental Figure 2. Estimation of cell death within 3-dimensional hydrogel tissue mimic following treatment with IRE and sIRE.

Supplemental Table 3. Pulse parameters used for conventional IRE and sIRE.

Supplemental Figure 3. Fluorescent imaging of IRE and sIRE treatment effects on (A) MSTO-GM, (B) untransduced, and (C) CAR T cells embedded in 3D collagen mimics that were treated with various parameters of sIRE or IRE 24 hours post-treatment in vitro. 4x magnification images were scanned and stitched to illustrate PEF treatment area of effect within the whole well of a 24-well using a 2-pin probe electrodes with 4 mm spacing. Dead cells were identified by propidium iodide (red) and live cells identified with Calcein AM (green). (D–F) Area of cell death were quantified using script on FIJI ImageJ. Scale bar = 4 mm.

Supplemental Figure 4. Intratumoral T cell viability after sIRE or IRE in an immunocompetent syngeneic flank tumor model.

Supplemental Figure 7. sIRE promotes infiltration of CAR T cells with minimal killing of preexisting intratumoral T cells.

Supplemental Figure 5. Transwell migration assay of CAR T cells when exposed to sIRE and IRE conditioned media in vitro.

Supplemental Figure 1. Mesothelioma and T cell viability at 24 hours post-IRE in vitro. Mesothelioma cell lines.

Supplemental Table 2. Cell viability material properties.

Purpose:One recurring challenge in cell therapy for solid tumors is poor tumor infiltration of adoptively transferred T cells. We previously showed that a subablative dose of tumor-targeted radiation generates a chemokine gradient that promotes infiltration, proliferation, and a memory phenotype of chimeric antigen receptor (CAR) T cells in solid tumors. However, radiation is cytotoxic to infiltrating CAR T cells, limiting its repeated use.Experimental Design:We hypothesized that irreversible electroporation could generate a chemokine gradient that promotes CAR T-cell infiltration into solid tumors and that selective irreversible electroporation (sIRE) tuned for selective cancer cell lysis (thus sparing infiltrating CAR T cells) can be used in a repeated fashion. Using experimental screening and simulation models, we optimized sIRE parameters to kill cancer cells while sparing T cells.Results:Using 3D tumor mimics and mouse models of malignant pleural mesothelioma, we confirmed the therapeutic benefit of repeated sIRE. Chemokine secretion by cancer cells injured by sIRE promoted migration and tumor infiltration of systemically administered CAR T cells and facilitated sustained immunity in a tumor-rechallenge model.Conclusions:By leveraging a dual-purpose translational strategy—through direct cancer cell–targeted cytotoxicity and augmented CAR T-cell infiltration—sIRE can reduce cancer burden while preserving and enhancing CAR T-cell function.