Background Interleukin-12 (IL-12) is a powerful immunostimulatory cytokine that has been expressed ectopically in genetically engineered T cells to enhance their antitumor activity. However, the constitutive production of IL-12 by engineered T cells caused severe adverse effects in patients. Thus, to fully harness the immunostimulatory potential of IL-12 while avoiding systemic toxicity, we inserted IL-12 gene into the PDCD1 locus in T cell receptor (TCR)-engineered T cells using the CRISPR/Cas9-based genome editing tool, which allows for IL-12 secretion to be induced strictly in a T cell activation-dependent manner.
Methods As a model TCR, we used a monoclonal TCR that is specific to the NY-ESO-1 (SLLMWITQV) peptide. The PD1-edited NY-ESO-1-specific TCR-T cells were generated by sequential lentiviral transduction and Cas9 RNP/AAV6-based knock-in into human primary T cells. The resulting TCR-T cells were co-cultured with an A375 cell line expressing NY-ESO-1 antigen to evaluate cytokine production, cytotoxicity, and proliferation in vitro. In vivo antitumor activity of the TCR-T cells was investigated in A375 xenograft models using NSG mice.
Results The PDCD1 locus was successfully edited in NY-ESO-1 TCR-T cells by replacing the endogenous PD-1 gene with a single-chain IL-12 transgene, without affecting the viability or expansion of the engineered T cells. Upon recognition of the target cells, the IL-12 transgene was expressed successfully, resulting in the strong phosphorylation of STAT-4 in the TCR-T cells. As compared to control TCR-T cells, these ΔPD-1-IL-12 NY-ESO-1 T cells displayed enhanced in vitro effector function, including increased secretion of IFNγ, TNF, and IL-10 as well as faster tumor cell lysis. In addition, ΔPD-1-IL-12 NY-ESO-1 T cells expanded more robustly after repeated challenges with PD-L1 overexpressing target cells. In xenograft models, ΔPD-1-IL12 NY-ESO-1 T cells potently eliminated established tumors and demonstrated increased tumor infiltration compared to control TCR-T cells.
Conclusions Using the CRISPR/Cas9 system, we demonstrated that the upregulation of PD-1, an immune-suppressive event in T cells, could be reprogrammed to secrete immunostimulatory IL-12 in TCR-T cells. In both in vitro assays and in vivo mouse xenograft studies, these PD-1-edited TCR-T cells demonstrated enhanced cytotoxic activity. Our approach may offer a novel engineering option for adoptive T cell therapy against solid tumors.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.