Abstract Viral insertions of chimeric antigen receptor (CAR) cassettes into specific genes can lead to clonal expansion of the resulting CAR T cell population and complete remission of cancer. Viral integration events provide a unique opportunity to study regulators of CAR T proliferation and function. We analyzed the clonal dynamics of twelve patients enrolled in our glypican 3 (GPC3) CAR T cell and IL15 GPC3 CAR T cell clinical trials using VDJ transcripts from single cell RNA sequencing of products and peripheral blood samples. One patient with hepatocellular carcinoma (HCC) treated with IL15 CAR T cells had a remarkably expanded clone. At peak expansion 14.7x109 CAR+ T cells were circulating in the patient based on flow cytometry. Expansion was accompanied by grade 3 cytokine release syndrome requiring activation of the inducible caspase 9 safety switch which rapidly eliminated all CAR T cells as measured by flow cytometry and completely resolved related symptoms. Response assessment with 3D imaging decreased the primary liver mass and either eliminated or significantly reduced the size all metastatic tumors. Alpha-feto-protein tumor marker (half-life ∼ 1 week) decreased from 14500 to 760 over 5 weeks. To determine whether viral integration was associated with this clone we performed whole genome integration analysis. Sonic abundance of intronic integrations in CBLB, CCDC6 and an exonic integration in ASB2 closely mirrored abundance of the expanded clone. We validated these integrations were present in the clonally expanded IL15 CAR T cells using targeted single cell DNA sequencing. Viral integrations can disrupt gene function through the creation of chimeric transcripts. We identified CAR CBLB, CAR ASB2, and IL15 CCDC6 chimeric transcripts in our single cell RNA sequencing. Transcriptomic analysis revealed a unique gene regulatory program. Seurat clustering identified three clusters enriched for the expanded clone. Two clusters were associated with rapidly dividing cells expressing significantly higher levels of TYMS, PCNA, and CDK1. While the last cluster expressed transcription factors associated with T cell function ZNF683 and RUNX2. In a model of HCC, simultaneous knock out of these genes, or combinatorial knockout of CBLB and CCDC6 using base editing led to increased CAR T cell expansion and anti-tumor activity. To our knowledge this is the first report of integration driven clonal expansion seen in solid tumor CAR T therapy. As an integration in CBL was previously associated with clonal expansion, our CBLB finding suggests integration in this gene family may lead to clonal growth. We identified CCDC6 a gene with unknown function in T cells, as a regulator of CAR T proliferation. These results demonstrate the impact of multiple genetic disruptions in CAR T cells resulting in robust expansion and antitumor activity and support the careful implementation of these perturbations to enhance patient outcomes. Citation Format: Azlann Arnett, David Steffin, Antonino Montalbono, Nisha Ghatwai, Amy Courtney, Gabriel Barragan, Magda Magda Esparza Cerda, Michael Wood, Marie Pouzolles, Kshitij Rai, Frederic D. Bushman, Dimitrios L. Wagner, Andras Attila Heczey. Triple disruption of CCDC6, CBLB, and ASB2 promotes the therapeutic efficacy of Glypican 3 targeted T cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4012.
Arnett et al. (Fri,) studied this question.