pA-regulator controlled, JMJD6 reprogrammed GPC3-CAR T cells for patients with hepatocellular carcinoma

CAR T cells mediate remarkable clinical responses in patients with hematologic malignancies but have shown only modest benefit for patients with solid cancers. CAR T cell exhaustion is a critical barrier to therapeutic efficacy in solid neoplasms and is marked by decreased proliferative capacity, cytokine production, and cytolytic function driven by a fundamentally altered transcriptional and epigenetic landscape. Specific transcription and epigenetic factors (TF/EFs) can modulate T cell exhaustion, but it is not known which TF/EF can prevent CAR T cell exhaustion most effectively to safely maximize antitumor activity. We developed the CAR “Model to Optimize Temporal ORFeome Responses” (MOTOR) platform consisting of a barcoded lentiviral library of all human TFs and EFs that can be transduced into CAR T cells at 1 ORF / CAR T cell ratio and assessed in the pooled setting. Our preliminary results demonstrate that specific ORF expressing CAR T cell populations enrich after repeated tumor cell encounters and Jumonji Domain Containing 6 (JMJD6) is identified as top candidate. Precise, on- demand control of TF/EF expression is necessary to maximize therapeutic efficacy and safety. Our recently developed ‘pA-regulator’ platform controls gene expression on the RNA level, post-transcriptionally without using foreign regulatory proteins. The system is based on a small ~400bp, non-protein coding sequence added 5’ of the controlled transgene. It can provide a wide regulatable, dynamic range and exhibits an EC50 within FDA- approved concentrations of tetracycline in humans helping implementation in patients. Thus, we hypothesize that pA-regulator controlled overexpression of JMJD6 will safely prevent exhaustion and improve GPC3-CAR T antitumor activity. In aim 1, we will examine all human TF/EFs (including JMJD6) to prevent exhaustion and promote antitumor activity of GPC3-CAR T cells using repeat tumor challenge assessments in vitro in unbiased forward genomics assessments followed by parallel evaluation of the top 5 candidates in additional functional tests ex vivo and in tumor bearing mice. In aim 2, we will determine the tetracycline concentration for pA- regulator controlled TF/EF expression to prevent T cell exhaustion leading durable antitumor responses of GPC3-CAR T cells in vivo. We will examine the effect of turning “on” and “off” the pA-regulator and whether the corresponding induction of JMJD6 or other top TF/EFs can reinvigorate GPC3-CAR T cells. To understand the specific induced programs of exhaustion resistant CAR T cells, gene expression profiles, epigenetic alterations and T cell subset composition changes will be evaluated in CAR T cells from peripheral blood and tumors. Summary: The proposed research will provide an unbiased evaluation of all human TF/EFs and their role in preventing exhaustion and promoting the antitumor properties of CAR T cells. The findings will provide in-depth insight into regulation of exhaustion and a foundation for further clinical testing of pA-regulator controlled CAR T cells in patients with cancer. Project Number: 1R21CA299361-01 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Andras Heczey (+1 co-PI) | Institution: SEATTLE CHILDREN'S HOSPITAL, SEATTLE, WA | Award Amount: $470,759 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZCA1 SRB-8 (J1)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11111542