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 (2022)

<h3>Background</h3> 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.<sup>1</sup> Nuclear receptor subfamily 4A (NR4A) transcription factors may contribute to exhaustion and limit T-cell function by restraining expression of AP-1 regulated genes.<sup>2,3</sup> Thus, we hypothesize that NR4A knockout (KO) and c-Jun overexpression may synergize to further limit exhaustion and enhance CAR T-cell function. <h3>Methods</h3> 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 <i>in vitro</i> 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 <i>in vivo</i> using a ROR1-expressing H1975 lung cancer xenograft model in mice. <h3>Results</h3> 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 <i>in vivo</i> 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). <h3>Conclusions</h3> 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. <h3>Acknowledgements</h3> 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. <h3>References</h3> Lynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, Anbunathan H, Lattin J, Jones R, Tieu V, Nagaraja S, Granja J, de Bourcy CFA, Majzner R, Satpathy AT, Quake SR, Monje M, Chang HY, and Mackall CL. c-Jun overexpressing CAR-T cells are exhaustion-resistant and mediate enhanced antitumor activity. <i>Nature</i>. 2019;<b>576</b>:293–300. 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. <i>Nature</i>. 2019;<b>567</b>:530-534. Liu X, Wang Y, Lu H, Li J, Yan X, Xiao M, Hao J, Alekseev A, Khong H, Chen T, Huang R, Wu J, Zhao Q, Wu Q, Xu S, Wang X, Jin W, Yu S, Wang Y, Wei L, Wang A, Zhong B, Ni L, Liu X, Nurieva R, Ye L, Tian Q, Bian X, Dong C. Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction. <i>Nature</i>. 2019;<b>567</b>:525-529. <h3>Ethics Approval</h3> 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).