TBK1 Targeting Is Identified as a Therapeutic Strategy to Enhance CAR T-Cell Efficacy Using Patient-Derived Organotypic Tumor Spheroids

  • Cancer Immunol Res. 2025 Feb 3;13(2):210-228. doi: 10.1158/2326-6066.CIR-23-1011.
Yi Sun  #  1  2 ,  Luke Maggs  #  3 ,  Apekshya Panda  2 ,  Samuel J Wright  2 ,  Angelina M Cicerchia  1 ,  Anne Jenney  4 ,  Matthew D Perricone  4 ,  Caitlin E Mills  4 ,  Giulia Cattaneo  3 ,  Marco Ventin  3 ,  Feng Chen  3 ,  Martin Q Rasmussen  1  2 ,  Alex Miranda  1  2 ,  Or-Yam Revach  1  2 ,  Jacy Fang  1  2 ,  Amina Fu  1  3 ,  Peter J Bowling  3 ,  Tatyana Sharova  3 ,  Aleigha Lawless  3 ,  Peter K Sorger  4 ,  Nabeel Bardeesy  1  2  5 ,  Xinhui Wang  3 ,  Keith T Flaherty  1 ,  Genevieve M Boland  2  3 ,  Arnav Mehta  1  2 ,  Moshe Sade-Feldman  1  2 ,  Cristina R Ferrone  3  6 ,  Russell W Jenkins  1  2  4
Affiliations
  • 1. Mass General Cancer Center, Krantz Family Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.
  • 2. Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
  • 3. Division of Gastrointestinal and Oncologic Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.
  • 4. Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, Massachusetts.
  • 5. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts.
  • 6. Department of Surgery, Cedars-Sinai Medical Center Los Angeles, Los Angeles, California.
  • # Contributed equally.
PMID: 39785827 DOI: 10.1158/2326-6066.CIR-23-1011
Abstract

Novel therapeutic strategies are needed to improve the efficacy of chimeric antigen receptor (CAR) T cells as a treatment of solid Tumors. Multiple tumor microenvironmental factors are thought to contribute to resistance to CAR T-cell therapy in solid Tumors, and appropriate model systems to identify and examine these factors using clinically relevant biospecimens are limited. In this study, we examined the activity of B7-H3-directed CAR T cells (B7-H3.CAR-T) using 3D microfluidic cultures of patient-derived organotypic tumor spheroids (PDOTS) and then confirmed the activity of B7-H3.CAR T cells in PDOTS. Although 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-cell 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-cell efficacy. Similar to PD-1 blockade, TBK1 inhibition restored CAR T-cell activity in monotypic and organotypic tumor spheroids, prevented CAR T-cell dysfunction, and enhanced CAR T-cell proliferation. Inhibition or deletion of TBK1 also enhanced the 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 could be used to enhance CAR T-cell efficacy by overcoming tumor-intrinsic and -extrinsic resistance mechanisms.

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