CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer

Abstract

The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy toward EBVaGC. These studies reveal possibility of generating PD-1-disrupted CTL by CRISPR-Cas9 system and demonstrate enhanced immune response of these PD-1-disrupted CTLs to the EBV-LMP2A antigen and superior cytotoxicity to the EBV-positive gastric cancer cell. In addition, when combined with low-dose radiotherapy, these PD-1-disrupted CTLs mediated an impressive antitumor effect in a xenograft mouse model of EBVaGC. Taken together, these studies illustrate PD-1/PD-L1-mediated immune tolerance of EBVaGC and provide a new strategy for targeting immune checkpoints to break the tolerance for the T cell-based adoptive therapy.

Engineered cells for costimulatory enhancement combined with IL-21 enhance the generation of PD-1-disrupted CTLs for adoptive immunotherapy

“…We have previously achieved efficient gene disruption of primary human T cells by CRISPR-Cas9 system [7,8]. In this study, PBMCs from healthy donors were isolated and activated for nucleofection for 3 d. To determine the transfection efficiency, we used the reporter plasmids pST1374-Cas9-GFP, encoding the green fluorescent protein (GFP) tagged Cas9, to co-transfect with pGL3-U6-hPD-1-sgRNA, by co-electroporation as previously described [7].…”

“…The Cas9 expression construct pST1374-Cas9-N-NLS-Flag-linker (Addgene 44758) and pGL3-U6-sgRNA-hPD-1 (Addgene 51133) vectors were constructed as previously described [7,8]. The expression of Epstein-Barr virus in the gastric cancer cell line AGS-EBV was verified by the surface expression of the target antigen LMP2A.…”

“…The hPD-1-sgRNA-disrupted T cells were prepared using PBMCs from gastric cancer patients as described previously [7,8]. Cells were transfected with the appropriate plasmids with Nucleo-fector II (Lonza, Germany) using the Amaxa Human T cells Nucleofector Kit, VPA-1002 (Lonza, Germany).…”

“…Our laboratory pioneered a gene editing approach using clustered regularly interspaced short palindromic repeatsassociated protein 9 (CRISPR-Cas9) to disrupt PD-1 on primary T cells [7]. We demonstrated that by reducing immune tolerance, CRISPR-Cas9-modified cytotoxic T lymphocytes (CTLs) ranked above other reagents in terms of immune responses and cytotoxicity [8].…”

“…To generate sufficient number of PD-1-disrupted CTLs for adoptive cell transfer, extensive ex vivo expansion of T cells is required. We have previously demonstrated that PD-1-disrupted CTLs can be activated and ex vivo expanded when cultured with IL-2, IL-7, and IL-15 [7,8]. To facilitate further PD-1disrupted CTLs activation and obtain sufficient number of them with increased antitumor activity, we investigated and compared the effects of 4-1BBL-expressing K562 cells and IL-21 on the expansion behavior, phenotype and function of PD-1-disrupted CTLs.…”