CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma

Abstract

Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.

Keywords: CRISPR-Cas systems; EGFRvIII; Glioblastoma; Receptors, chimeric antigen.

Fig. 1

Multiplexed CRISPR-Cas9 gene-editing is efficient in primary human T cells. a Schematic representation of the EGFRvIII targeted CAR construct. b Primary human T cells were stimulated, RNP electroporated and transduced to produce CAR T cells. c Following expansion, cells were subjected to flow cytometry for TCR and B2M expression. d Bivariate plot displays frequency of cells with both TCR and B2M deletion. e EGFRvIII CAR T cells that have been gene-edited for PD-1 (CART-EGFRvIIIΔPD-1) do not have the ability to interact with PD-L1 expressed on target cells. f Effector cells were incubated with irradiated U87vIII for 1 week and subjected to flow cytometric analysis for surface PD-1 expression. The control group contains cells gene-edited for both TCR and B2M, and mock transduced with AAV

Fig. 2

PD-1 disruption promotes favorable differentiation of CAR T cells targeting PD-L1 expressing glioma. Effector cells were cocultured with irradiated target U87vIII at and E:T of 1:1. The phenotype of T cells were assessed at Day 1 (prior to stimulation) and at Day 21 by flow cytometry. Cells were grouped by flow cytometry according to T-cell phenotype as follows: naïve (T_N) CCR7⁺CD45RO⁻, central memory (T_CM) CCR7⁺CD45RO⁺, effector memory (T_EM) CCR7⁻CD45RO⁺, and effector (T_E) CCR7⁻CD45RO⁻

Fig. 3

PD-1 disruption enhances EGFRvIII CAR T cells. a Cytokine production by CAR-transduced primary human T cells when cocultured for 18 h at an E:T of 1:1. b Proliferation assessment of effector cells stimulated weekly with irradiated U87vIII. c Impedance-based cytotoxicity assay measuring activity of effector cells against U87vIII at an E:T of 1:3, with cell index serving as an inverse measure of target cell viability. Assays were performed in triplicate (mean ± SEM is depicted; unpaired, two-tailed t-test, * = P < 0.05, *** = p < 0.001)

Fig. 4

Intravenous delivery of CAR T cells does not significantly prolong survival in mice. a U87vIII cells (5 × 10³) were implanted orthotopically into NSG mice and treated post-implantation with intravenous (IV) effector cells. b Antitumor responses produced by CART-EGFRvIIIΔPD-1 in vivo. Survival curves were estimated for each group using Kaplan–Meier product-limit estimation. Primary comparative analyses of the curves for each group were performed using the log-rank test. c Bioluminescence imaging of U87vIII tumor growth over time, n = 5 mice

Fig. 5

Intraventricular infusion with gene-edited CAR T cells is efficacious against GBM. a U87vIII cells (5 × 10³) were implanted orthotopically into NSG mice and treated post-implantation with intraventricular (IVT) effector cells. b Antitumor responses produced by CART-EGFRvIIIΔPD-1 in vivo. Survival curves were estimated for each group using Kaplan–Meier product-limit estimation. Primary comparative analyses of the curves for each group were performed using the log-rank test (*** = p < 0.001). c Bioluminescence imaging of U87vIII tumor growth over time, n = 5 mice