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

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

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 5

CAR T cells become exhausted upon repeated antigen stimulation in vitro, although MBBz CAR T cells preferentially retain effector cytokine secretion and cytotoxicity in vitro and upon tumor rechallenge in vivo.

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CAR T cells become exhausted upon repeated antigen stimulation in vitro,...
(A) Both M28z and MBBz CAR T cells retain proliferative capacity in vitro upon repeated antigen stimulation. T cells were also tested for cytotoxicity by chromium-release assay and for cytokine secretion by Luminex assay (B–D). (B) (Left) CAR T cells demonstrate equal killing at the first stimulation and loss of cytolytic function upon repeated antigen stimulation, although MBBz CAR T cells are better able to retain cytolytic function as measured by chromium-release assay. (C) Cytotoxic granule release as measured by CD107a expression correlates with chromium-release assay (B). Data represent the mean ± SD (triplicates) of the fold-change relative to the CD107a mean fluorescence intensity (MFI) of unstimulated CD8+ CAR T cells. (D) Cytokine secretion measurements similarly demonstrate loss of CAR T cell effector function upon repeated antigen encounter; again, MBBz CAR T cells are better able to preserve their function. (E) Persisting MBBz CAR T cells demonstrate superior efficacy in vivo and eradicate MSLN+ tumor cells following tumor rechallenge. Twenty-eight days after pleural tumor eradication (following a single dose of 1 × 105 CAR T cells), 1 × 106 MSLN+ tumor cells were injected into the pleural cavity (tumor rechallenge). Control (white circle) represents mice without any previous injections of tumor or T cells. MBBz CAR T cells prevented tumor growth in all mice, whereas tumor growth and death were observed in 2 of 4 mice initially treated with M28z CAR T cells. Student’s t tests were performed, and statistical significance was determined using the Sidak-Bonferonni correction. *P < 0.05; ***P < 0.001. Data represent the mean ± SEM of 3 replicates or are plotted as individual points and are representative of at least 3 independent experiments.