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Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition
Leonid Cherkassky, … , Michel Sadelain, Prasad S. Adusumilli
Leonid Cherkassky, … , Michel Sadelain, Prasad S. Adusumilli
Published July 25, 2016
Citation Information: J Clin Invest. 2016;126(8):3130-3144. https://doi.org/10.1172/JCI83092.
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Research Article Oncology Therapeutics

Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

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Abstract

Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies.

Authors

Leonid Cherkassky, Aurore Morello, Jonathan Villena-Vargas, Yang Feng, Dimiter S. Dimitrov, David R. Jones, Michel Sadelain, Prasad S. Adusumilli

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Figure 1

CARs with CD28 or 4-1BB costimulation exhibit similar cytolytic functions, effector cytokine secretion, and proliferation in vitro upon initial antigen stimulation.

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CARs with CD28 or 4-1BB costimulation exhibit similar cytolytic function...
(A) First- and second-generation CARs. (B) MSLN–targeted CARs contain the CD3ζ endodomain either alone (Mz, first-generation CAR) or in combination with the CD28 (M28z) or 4-1BB (MBBz) costimulatory domain (second-generation CAR). PSMA-directed CARs with CD28 costimulation (P28z) as well as PSMA-expressing targets (PSMA+) are included in experiments as negative controls. CYT, cytoplasmic domain; LS, leader sequence; LTR, long terminal repeat; SA, splice acceptor; SD, splice donor; TM, transmembrane. (C–E) Antigen-specific effector functions of CAR-transduced T cells. (C) Lysis of MSLN-expressing targets (MSLN+), but not PSMA+ targets, as measured by chromium-release assays. (D) 4-1BB and CD28 costimulations enhance cytokine secretion, as assessed by Luminex assay, after coculture of CAR T cells with MSLN+ cells. (E) M28z and MBBz CARs facilitate robust T cell accumulation after stimulation with MSLN+ cells. (F) 4-1BB and CD28 costimulations decrease the rate of apoptosis as assessed by annexin V/7-AAD+ staining every 7 days after coculture with MSLN+ target. (D and E) ***P < 0.001, comparing costimulated CAR T cells (M28z or MBBz) with the first-generation receptor (Mz), by Student’s t test; significance was determined using the Sidak-Bonferonni correction for multiple comparisons. Data are representative of at least 3 independent experiments and represent the mean ± SEM (C and E) of 3 replicates or are plotted as individual points.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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