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.

Effect of CRISPR/Cas9-Mediated PD-1-Disrupted Primary Human Third-Generation CAR-T Cells Targeting EGFRvIII on In Vitro Human Glioblastoma Cell Growth

“…To the best of our knowledge, this is the first study to report the disruption of the PD-1 gene by two PD1 exon 1-targeting sgRNAs and Cas9 expression vectors in third-generation EvCAR-T cells (under the control of CD3zeta, CD28, and 4-1BB signaling molecules) derived from healthy human T cells. In a similar published study, Choi et al reported that CRISPR-Cas9 disruption of PD-1 enhances the activity of second-generation EvCAR-T cells under the control of CD3zeta and 4-1BB signaling molecules in a preclinical model of human GBM [38]. However, our study is distinguished by the use of PD-1-disrupted third-generation EvCAR-T cells, as opposed to the second-generation EvCAR-T cells used in the Choi et al study [38].…”

“…In a similar published study, Choi et al reported that CRISPR-Cas9 disruption of PD-1 enhances the activity of second-generation EvCAR-T cells under the control of CD3zeta and 4-1BB signaling molecules in a preclinical model of human GBM [38]. However, our study is distinguished by the use of PD-1-disrupted third-generation EvCAR-T cells, as opposed to the second-generation EvCAR-T cells used in the Choi et al study [38].…”

Recent Advances in Allogeneic CAR-T Cells

“…For example, CD19-specific CAR-T cells without endogenous expression of TR was created using designer zinc finger nuclease (ZFN) systems harboring CD19-specific CAR that uses the Sleeping Beauty transposon/transposase system [53]. More efficient CAR-T cells were also generated by disrupting both TR and beta-2 microglobulin that are responsible for GvHD and Host-versus-Graft Effect (HvGE), respectively, as well as by disrupting binding of PDCD1 (PD1) to CD274 (PDL-1), and thereby increased effectiveness of CAR-T cell therapy [54]. Recently, advanced multiplex engineering of T cells using CRISPR/Cas9 without double strand break-induced translocations was developed and showed improved expansion of engineered T cells and reduced safety risks associated with unintended genomic alterations and genotoxicity [55].…”

Overhauling CAR T Cells to Improve Efficacy, Safety and Cost

“…The presence of NK cells during the expansion processes aided in the promotion of T cells bearing the CD62L and CCR7 markers which are important for T cell migration to lymph nodes. This expansion protocol was associated with long term CAR expression and killing of B cell leukemia cells in a mouse model [ 136 ]. They have also shown that this approach can be used to reduce the production time from 15 days to 8 days, which may translate into cost savings.…”

“…As mentioned previously, inhibition of the PD-1/PD-L1 axis is known to improve CAR-T cell therapy. For example, use of gene editing, such as CRISPR/Cas9, to engineer CAR-T cells that lack PD1 expression have been explored [ 136 , 156 ]. Alternatively, the CARs may be engineered to secrete a protein, such as a single chain antibody, that inhibits PD1 [ 157 ].…”