Background Chimeric antigen receptor (CAR) T-cells have revolutionized the treatment of hematological malignancies. However, the current autologous approach to CAR T-cell production suffers several drawbacks (difficulty to obtain high quality starting material from patients, limited manufacturing capacity, complex logistics, long vein-to-vein time, high costs), to the point that to date only approximately 10% of eligible patients can receive the treatment. Allogeneic ‘off-the-shelf’ CAR T-cells could help circumvent these issues, but their successful clinical application is hampered by the difficulty to manage graft-versus-host disease (GvHD) and host-versus-graft (HvG). A variety of genetic engineering strategies have been proposed to address these issues, all burdened by the complexity of multiple, potentially risky genome edits. Here we describe a non-gene edited one-step approach for the effective generation of allogeneic CAR T-cells. To this aim, we silenced CD3ζ to target T-cell receptor (TCR) expression, and we downregulated β2M and CIITA to target HLA class I and HLA class II expression, respectively. We aimed at preventing apoptosis induced by CD95L, often expressed by cancer cells, by knocking down CD95.
Methods A microRNA (miRNA)-based short hairpin RNA (shRNA) platform was developed, to allow for the tunable modulation of multiple target genes simultaneously. The platform was equipped with shRNA-derived guide sequences (shGuides) targeting CD3ζ, β2M, CIITA and CD95 simultaneously, and combined with an anti-CD19 CAR. Each shRNA was screened and assessed functionally in vitro.
Results The miRNA-derived shRNA platform targeted against CD3ζ, β2M, CIITA and CD95 in the CAR construct led to a substantial downregulation of TCR, HLA class I, HLA class II and CD95. This, in turn, granted the engineered CAR T-cells protection in in vitro assays mimicking GvHD, HvG and CD95L-induced autophagy, ultimately leading to significantly improved CAR T-cell survival. Notably, thanks to the tunability of gene expression allowed by our platform, we were able to achieve functionally relevant downregulation of HLA class I to avoid recognition by donor T-cells, ensuring at the same time sufficient expression to let CAR T-cells escape NK recognition, without the need of further editing (e.g., HLA E overexpression).
Conclusions These data validate our technology for the effective introduction of multiple functionally relevant edits in CAR T-cells. Moreover, we show in vitro proof-of-concept that our approach can improve allogeneic CAR T-cell viability by allowing evasion from GvHD, HvG and CD95L-induced autophagy. Our technology stands out as an easy, safe, and effective strategy for the generation of allogeneic CAR T-cells.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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.