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243 NR4A3 gene editing and c-Jun overexpression synergize to limit exhaustion and enhance functional activity of ROR1 CAR T cells in vitro and in vivo
  1. Viola Lam,
  2. Jessica Barragan,
  3. Christina Cheung,
  4. Jia Lu,
  5. David Chian,
  6. Rowena Martinez,
  7. Candace Sims,
  8. Purnima Sundar,
  9. Hajime Hiraragi,
  10. Shobha Potluri and
  11. Rachel Lynn
  1. Lyell Immunopharma, Inc., South San Francisco, CA, USA


Background Next-generation strategies to improve T-cell functional activity, persistence, and durability are needed for effective cellular immunotherapy against solid tumors. Overexpression of the activator protein 1 (AP-1) family transcription factor c-Jun reduces chimeric antigen receptor (CAR) T-cell exhaustion thereby improving functional activity in multiple preclinical models.1 Nuclear receptor subfamily 4A (NR4A) transcription factors may contribute to exhaustion and limit T-cell function by restraining expression of AP-1 regulated genes.2,3 Thus, we hypothesize that NR4A knockout (KO) and c-Jun overexpression may synergize to further limit exhaustion and enhance CAR T-cell function.

Methods Healthy donor T cells were transduced with a ROR1 CAR lentiviral vector with (+) or without (-) c-Jun overexpression. NR4A family genes (NR4A1, NR4A2, or NR4A3) were disrupted using CRISPR/Cas9 ribonucleoprotein delivery via electroporation (EP). CAR T-cell cytotoxicity and cytokine production were evaluated in vitro after primary and repeated antigen-stimulation assays designed to promote exhaustion. Cell phenotypes (flow cytometry) and transcriptional profiling (bulk and single-cell RNA-Seq) were also assessed. Finally, CAR T cells were evaluated in vivo using a ROR1-expressing H1975 lung cancer xenograft model in mice.

Results NR4A3 KO ROR1 CAR T cells consistently demonstrated superior cytotoxic activity and prolonged cytokine production upon repeated antigen stimulation compared to NR4A1 KO, NR4A2 KO, and EP control ROR1 CAR T cells. NR4A3 KO showed significant synergy in ROR1 CAR T cells overexpressing c-Jun (figure 1).

NR4A3 KO + c-Jun ROR1 CAR T cells were phenotypically and functionally indistinguishable at primary antigen stimulation. However, this combination resulted in the highest levels of cytokine production (IFN-γ, IL-2, and TNF-α), increased CAR T-cell persistence, and reduced surface expression of inhibitory receptors after repetitive antigen stimulation, suggesting a mechanism of resistance to exhaustion-induced dysfunction. Transcriptomic analysis indicated that NR4A3 KO + c-Jun increased effector and interferon response-associated T-cell subsets, yet reduced terminal exhaustion compared to control + c-Jun ROR1 CAR T cells following antigen restimulation.

NR4A3 KO + c-Jun ROR1 CAR T cells showed the most robust anti-tumor efficacy in vivo with activity at a 7-fold reduced CAR T-cell dose and demonstrated more than 20-fold greater CAR T-cell expansion in blood compared to control + c-Jun ROR1 CAR T cells (figure 2).

Conclusions These data suggest that reducing NR4A3 expression in combination with c-Jun overexpression has the potential to further limit exhaustion and provide durable ROR1 CAR T-cell functional activity compared to either strategy alone, which may improve cellular immunotherapy against ROR1-expressing solid tumors.

Acknowledgements We thank members of Lyell Immunopharma’s In Vivo Pharmacology team (Abira Bandyopadhyay, Shawn Anderson, Jacob Corpuz, and Quinn Walker), NGS/Flow team (Amir Figueroa, Elizabeth Hernandez, Andrew Jimena, Amanda Sims, Emily Fu-Sum, Yi-Dong Lin, Sheila Lou, Stefan Siebert, Ken Xiong, and Lora Zhao), Research team (Rachel Fukuda, Helle Jensen, Megan Murt , Spencer Park, Blythe Sather, Courtney Simianer, Sydney Spadinger, and Nicole Zhang), and Vector Sciences team (David Anderson, Martin Wohlfahrt, and Chang-Chi Wu) for their experimental contributions.


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  2. Chen J, Lopez-Moyado IF, Seo H, Lio, CJ, Hempleman LJ, Sekiya T, Yoshimura A, Scott-Browne JP, Rao A. NR4A transcription factors limit CAR T cell function in solid tumors. Nature. 2019;567:530-534.

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Ethics Approval Experiments presented in this abstract relied on human donor material that was obtained from commercial vendors. These vendors use their own IRB-approved protocol and consent process. In vivo experiments presented in this abstract were approved by Lyell Immunopharma’s IACUC (EB17-010-117).

Abstract 243 Figure 1

Successive lysis of ROR1-expressing H1975-NucLight Red (NLR) target cells by NR4A1 KO, NR4A2 KO, NR4A3 KO, or control unedited ROR1 CAR T-cells with (+) or without (-) c-Jun overexpression, and mock untransduced T cells in one representative donor of four donors tested. Lysis of H1975-NLR target cells was quantified by measuring total NLR intensity. NLR intensity was normalized relative to the starting intensity after replating for each round of stimulation. NR4A3 KO + c-Jun showed significant synergy at the last timepoint of the fifth stimulation compared to other T-cells tested. Asterisks represent p-value significance of each condition compared to NR4A3 KO + c-Jun (unpaired t-test, ** p < 0.005, *** p < 0.001, **** p < 0.0001).

Abstract 243 Figure 2

Anti-tumor efficacy at three CAR T-cell doses (A) and CAR T-cell expansion (B) of NR4A3 KO or control edited ROR1 CAR T cells with (+) or without (-) c-Jun overexpression, and mock untransduced T cells in one representative experiment (n=3 healthy donors) tested in an in vivo H1975 xenograft model. NR4A3 KO + c-Jun CAR T cells significantly impaired tumor growth at all three CAR T cell doses tested compared to control + c-Jun (* p < 0.05, ** p < 0.005, Tukey one-way ANOVA). At the 0.6x106 CAR T-cell dose, NR4A3 KO + c-Jun CAR T cells demonstrated a significantly higher day 21-fold expansion compared to control + c-Jun (mean of 40.1 vs 1.7, ****p < 0.0001, unpaired t-test, n=10 mice per group). Day 21-fold expansion was normalized relative to day 1.

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