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

Bryan D Choi,Amanda A Bouffard,William T Curry,Rebecca C Larson,Ana P Castano,Genevieve M Gerhard,Stefanie R Bailey,Daniel B Henderson,Xiaoling Yu,Matthew J Frigault,Mark B Leick,Andrea Schmidts,Irene Scarfò,Henia Darr,Marcela V Maus,Jason G Sagert,Bob S Carter

doi:10.1186/s40425-019-0806-7

Bryan D Choi, Amanda A Bouffard + Show 15 more

Open Access

https://doi.org/10.1186/s40425-019-0806-7

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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.

Highlights

Despite remarkable success in the treatment of hematological malignancies, chimeric antigen receptor (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
Multiplexed gene-editing of EGFRvIII CAR T cells In the current study, we employed the EGFRvIII CAR Tcell construct based on a second-generation backbone containing 4-1BB and CD3ζ intracellular signaling domains, but this time cloned into an AAV6 vector backbone instead of a lentiviral vector (Fig. 1a), the former allowing for integration of the CAR sequence into a specific locus rather than relying on random genomic integration
This was followed by AAV6 transduction, which resulted in CAR T cells with either endogenous or deleted PD-1 (i.e., CART-EGFRvIII and CARTEGFRvIIIΔPD-1) (Fig. 1e)

Summary

Introduction

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. In our clinical study of intravenous CAR T cells targeting a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII) in patients with GBM, we observed that EGFRvIII CAR T cells localized to intracerebral tumors and led to successful reduction of EGFRvIIIexpressing cancer cells [6] This was associated with concomitant upregulation of programmed cell death ligand 1 (PD-L1) expression within treated gliomas, contributing to immune suppression, CAR T-cell dysfunction and subsequent disease progression. CRIPSR-Cas technology has emerged as a simple and efficient method of gene-editing CARs with the potential to address these barriers to therapy This includes the design of universal CAR T cells with reduced potential for both initiating graft-versus-host disease (GVHD) and eliciting donor T-cell rejection, through targeted disruption of the endogenous T-cell receptor (TRAC) and beta-2 microglobulin (B2M), respectively [7, 8]. The use of CRISPR-Cas affords the opportunity to modify the expression of other relevant genes involved in suppressing T-cell function in the microenvironment of GBM tumors

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