AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells

Abstract

The receptor tyrosine kinase AXL is a member of the TAM (Tyro3, AXL, and proto-oncogene tyrosine-protein kinase Mer) family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CART)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CART (CART19)-cell functions. Our results demonstrate that T cells and CART cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CART cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CART cells, Th2 cytokines, reversal of CART-cell inhibition, and promotion of CART-cell effector functions. AXL inhibition is a novel strategy to enhance CART-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CART-cell inhibition through selective targeting of M2-polarized macrophages.

Optimized CART Cell Therapy for Metastatic Aggressive Thyroid Cancer

“…Cytotoxicity assays were performed as previously described. (50,53–56) Luciferase + TSHR + K562 or TSHR + FTC133 cells were used as target cells. UTD or TSHR-CART cells were co-cultured with target cells at various effector: target (E:T) ratios in T cell medium.…”

“…Degranulation and intracellular cytokine assays were performed as previously described. (50,53–56) UTD or TSHR-CART cells were incubated with various target cells at an E:T ratio of 1:5. Antibodies against CD107a (clone H4A3) FITC (BD Pharmingen, San Diego, CA, USA, Cat# 555800), CD28 (BD Biosciences, San Diego, CA, USA, Cat# 348040), CD49d (BD Biosciences, San Diego, CA, USA, Cat# 340976) and monensin (Biolegend, San Diego, CA, USA, Cat# 420701) were added prior to the incubation.…”

“…Samples were prepared for flow cytometry as previously described. (50,(53)(54)(55)(56) In all analyses, the population of interest was gated based on forward vs side scatter characteristics, then by singlet gating, and then by live cell gating using LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA). Flow cytometry was performed on a three-laser CytoFLEX (Beckman Coulter, Chaska, MN, USA).…”

“…Proliferation assays were performed as previously described. (50,(53)(54)(55)(56) UTD or TSHR-CART cells were resuspended in T cell medium at 1×10 6 /mL, and 100 µL per well were seeded in 96-well plates. Each assay included T cells with media only as a negative control and T cells with PMA and ionomycin as a positive control.…”