Evaluating Novel Therapeutic Strategies to Enhance CAR-T Cell Efficacy Using Patient-Derived Organotypic Tumor Spheroids

Novel therapeutic strategies are needed to improve the efficacy of chimeric antigen receptor (CAR)-T cells in the treatment of solid tumors. Multiple tumor microenvironmental factors are thought to contribute to CAR-T cell therapy resistance in solid tumors, and appropriate model systems to identify and examine these factors using clinically relevant biospecimens are limited. Here, we examined the activity of B7-H3 (CD276) directed CAR-T (B7-H3.CAR-T) using 3D microfluidic cultures of patient-derived organotypic tumor spheroids (PDOTS) and confirmed activity of B7-H3.CAR-T in PDOTS. While B7-H3 expression in PDOTS was associated with B7-H3.CAR-T sensitivity, mechanistic studies revealed dynamic upregulation of co-inhibitory receptors on CAR-T cells following target cell encounter that led to CAR-T cell dysfunction and limited efficacy against B7-H3 expressing tumors. PD-1 blockade restored CAR-T activity in monotypic and organotypic tumor spheroids with improved tumor control and upregulation of effector cytokines. Given the emerging role of TANK-binding kinase 1 (TBK1) as an immune evasion gene, we examined the effect of TBK1 inhibition on CAR-T efficacy. Similar to PD-1 blockade, TBK1 inhibition restored CAR-T activity in monotypic and organotypic tumor spheroids, prevented CAR-T cell dysfunction, and enhanced T cell proliferation. Inhibition or deletion of TBK1 also enhanced sensitivity of cancer cells to immune-mediated killing. Taken together, our results demonstrate the feasibility and utility of ex vivo profiling of CAR-T cells using PDOTS and suggest that targeting TBK1 is a novel strategy to enhance CAR-T efficacy by overcoming tumor-intrinsic and -extrinsic resistance mechanisms. For scRNAseq analysis of PDOTS +/- CAR-T cell challenge, B7-H3.CAR-T cells and cryopreserved PDOTS (melanoma, 10214) were thawed and rested media to serve as baseline (0 hour) controls. Remaining PDOTS were prepared in collagen for 3D culture. B7-H3.CAR-T cells (E:T 1:1) were added with or without TBK1i (1 μM), 250 μg/mL pembrolizumab (Meck Sharp&Dobine, NDC 000^-3026-01), or the combination, compared to untreated control for 24 hours before PDOTS +/- CAR-T were collected for processing. Droplet-based isolation of single cells and subsequent libraries were generated with the Chromium Controller (10X Genomics) according to the manufacturer’s specifications using the 10x Chromium Next GEM single cell 5’ kit v2 (1000263). Characterization of the sequencing library was performed with the Bioanalyzer High Sensitivity DNA kit (Agilent; 5067-4626) and Qubit (ThermoFisher) instruments. Each pair of sample libraries were were sequenced using Illumina NextSeq 500 with two paired-end reads (read1= 26; read2= 46). *************************************************************** We are submitting raw data to dbGaP ***************************************************************