Epigenetic and signaling-based engineering enhances CAR-T cell function in oculo-cerebral lymphoma

Patients with PCNSL, an aggressive B-cell malignancy confined to the central nervous system (brain, cerebrospinal fluid, spinal cord and/or eye), face poor outcomes, particularly upon relapse. Although CD19-directed CAR-T cell therapies have transformed the treatment of systemic B-cell malignancies, their application in PCNSL has been constrained by neurotoxicity concerns in pivotal studies. Recent data demonstrate feasibility and safety in this setting; however, more than half of patients eventually relapse, highlighting the need for more persistent CAR-T cells capable of functioning within the immunosuppressive CNS microenvironment. To address this, we first evaluated a panel of second-generation CD19 CARs, including ITAM-tuned “1XX” variants, using a 3D spheroid model of PCNSL. T cells expressing the (SJ25C1)1XX-CAR format demonstrated the highest antitumor activity. To further improve their durability, we knocked out SUV39H1, a histone methyltransferase that limits memory T cell differentiation through histone H3 lysine 9 trimethylation (H3K9me3), an epigenetic mark associated with chromatin compaction. The resulting (SJ25C1)1XX-CAR SUV39H1 KO T cells showed enhanced cytotoxicity in vitro, and superior tumor control and survival in an orthotopic PCNSL xenograft model compared to conventional second-generation CAR-T cells. To further test persistence, we established an ocular lymphoma rechallenge model. Following initial tumor clearance by both edited and non-edited 1XX CAR-T cells, only SUV39H1 knockout CAR-T cells maintained long-term protection upon rechallenge in the contralateral eye. Ex vivo spleen analysis confirmed their accumulation and enrichment in memory precursors (CD27⁺KLRG1⁻) with elevated Ki67 expression, indicating enhanced proliferative potential. Altogether, our study supports the clinical translation of a next-generation cell therapy for PCNSL based on epigenetically reprogrammed, signaling-optimized CAR-T cells. We are currently developing a GMP-compatible manufacturing process incorporating the RQR8 marker/safety switch to enable rituximab-mediated elimination in the event of unexpected toxicity, in preparation for a future phase I/II clinical trial.