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297 Improvement of CAR T-cell performance by simultaneous downregulation of multiple co-inhibitory receptors
  1. Matteo Rossi,
  2. Fanny Huberty,
  3. Thuy Nguyen,
  4. Jerome Marijsse,
  5. Celine Jacques-Hespel and
  6. Eytan Breman
  1. Celyad Oncology, Mont-Saint-Guibert, Belgium
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Co-inhibitory receptors, such as PD-1 and LAG-3, have a crucial role in regulating T-cell activity, as their expression on the cell surface upon chronic T-cell activation is associated with T-cell exhaustion. Immunotherapies directed against co-inhibitory receptors exhibited unprecedented efficacy in several cancer indications. However, many patients do not respond to these therapies and some tumor types remain largely refractory. A key determinant of CAR T-cell failure against solid tumors is T-cell exhaustion induced by checkpoint inhibitors on cancer cells. The coordinated targeting of different co-inhibitory receptors may help overcome these hurdles. Here we show that the simultaneous downregulation of co-inhibitory receptors PD-1, LAG-3, TIM-3 and TIGIT endows CAR T-cells with superior efficacy.

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 PD-1, LAG-3, TIM-3 and TIGIT, and combined with an anti-CD19 CAR. The shRNA platform-equipped CAR T-cells were challenged with target cancer cells expressing the co-inhibitory receptors’ ligands. The impact of the simultaneous downregulation of the four co-inhibitory receptors was assessed in vitro by monitoring T-cell activation (via surface activation markers and cytokine secretion), killing activity, and persistence (via repeated challenges with the target cells).

Results The miRNA-derived shRNA platform targeting PD-1, LAG-3, TIM-3 and TIGIT led to a significant downregulation of the four co-inhibitory receptors in the CAR T-cells. Upon activation by the target cancer cells, the simultaneous knock-down of the four genes enhanced cytokine secretion compared to CAR T-cells not engineered with the shRNA platform. Moreover, the CAR T-cells carrying the quadruple knock-down showed superior killing ability, increased persistence and prolonged activity when repeatedly challenged in sequential cycles with the target cancer cells.

Conclusions These data validate our technology for the effective introduction of multiple functionally relevant edits in CAR T-cells. Co-inhibitory receptors concomitantly contribute at regulating T-cell responses, making it difficult to inhibit or knock-out multiple receptors together. With our approach we proved the feasibility of the simultaneous knock-down of PD-1, LAG-3, TIM-3 and TIGIT. Moreover, we show increased performance of the engineered CAR T-cells, suggesting that the simultaneous downregulation of the four co-inhibitory receptors may improve the antitumor activity of adoptive cell therapy. This strategy may empower the CAR T-cells to successfully target tumors reliant on high expression of immune checkpoint molecules and therefore often refractory to immunotherapy, such as solid tumors.

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

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