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.

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The ability of immune cells to expand numerically after infusion distinguishes adoptive immunotherapies from traditional drugs, providing unique therapeutic advantages as well as the potential for unmanageable toxicities. Here, we describe a case of lethal hyperleukocytosis in a patient with neuroblastoma treated on phase 1 clinical trial (NCT03294954) with autologous natural killer T cells (NKTs) expressing a GD2-specific chimeric antigen receptor and cytokine interleukin 15 (GD2-CAR.15). This patient was the first to be treated on dose level (DL) 5 and the first patient whose product was restimulated with K562-derived artificial antigen-presenting cells (aAPCs) instead of autologous peripheral blood mononuclear cells (PBMCs). 12 previously treated patients on DLs 1 through 4 did not experience significant toxicity. Our root-cause analysis revealed no genetic alterations of known clinical significance and excluded the possibility of clonal expansion due to insertional retroviral mutagenesis. We report that the use of aAPCs instead of PBMCs for CAR-NKT restimulation contributed to a hyperproliferative state associated with distinct gene expression that possibly led to explosive lymphocyte expansion and uncontrolled toxicity in the patient. These findings warrant the implementation of measures to control immune cell activation during manufacture of cell therapy products, especially those armed with transgenic cytokines.

Abstract Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L– cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/β-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/β-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/β-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory–like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell–based immunotherapy. See related Spotlight by Van Kaer, p. 144

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

Supplementary Figures 1-2. Supplementary Figure 1. Expression of CD80, CD83, CD11c and HLA-DR by monocytes 72 hours after co-culture with irradiated K/pp65 (in blue), K/OX40L/pp65 (in violet), K/CD40L/pp65 (in orange) and K/OX40L/CD40L/pp65 (in green); Supplementary Figure 2. Cytotoxic activity (51Cr-release assay at 40:1, 20:1, 10:1 and 5:1 effector:target ratios) of CAR-CMV-CTLs stimulated in vitro by K/pp65 or K/CD40L/pp65 or K/CD40L/OX40L/pp65.

Abstract Purpose: Vα24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs. Experimental Design: Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma. Results: Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activation-induced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2.CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround in vitro tumor cell killing. Following transfer into mice bearing neuroblastoma xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared with GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathologic analysis. Conclusions: Our results informed selection of the CD28-based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).

Poor antigen delivery is one of the major limitations of modern cancer vaccine vectors. To overcome this challenge, we exploited Salmonella Pathogenicity Island 2 and type III secretion system to deliver tumor-associated antigen (TAAs) into the cytosol of antigen-presenting cells in situ. In a recent report, we demonstrated that an attenuated strain of S. typhimurium, which was engineered to express SPI2-regulated oncoprotein survivin (SVN), induced potent CD8 T-cell-mediated antitumor response that was curative in a murine model of highly aggressive lymphoma. In further development of this technology for clinical use, the vaccine has been transferred from an experimental strain to a clinically validated strain of S. typhi, CVD908. To adapt CVD908 to stably express recombinant antigens without antibiotic-dependent selection, we used a recently reported plasmid stabilization system that encodes the single-stranded binding protein (SSB), an essential protein in DNA metabolism, which was deleted from the bacterial chromosome. The SPI2-regulated expression cassette was then cloned into the SSB plasmid, so that the resultant construct maintained bacterial vector stability while expressing and translocating TAAs. We found that CVD908Δssb vector effectively infects human dendritic cells in vitro and translocates recombinant SVN and MYCN oncoproteins into their cytosol. Furthermore, CVD908Δssb remains stable in mice and induces generation of antigen-specific CD8 T cells. Therapeutic vaccination of tumor-bearing mice with SVN or MYCN vaccine produced potent antitumor activity in murine models of lymphoma or neuroblastoma. The results justify clinical testing of CVD908Δssb-based SVN and MYCN vaccines in cancer patients.

Background aims. The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4-chimeric antigen receptor (CAR) that was transduced intoT cells to mediate anti-leukemia activity. Methods. Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. Results. Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. Conclusions. Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.

Abstract Purpose: Adoptive transfer of Epstein–Barr virus (EBV)–specific and cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) genetically modified to express a chimeric antigen receptor (CAR) induces objective tumor responses in clinical trials. In vivo expansion and persistence of these cells are crucial to achieve sustained clinical responses. We aimed to develop an off-the-shelf whole-cell vaccine to boost CAR-redirected virus-specific CTLs in vivo after adoptive transfer. As proof of principle, we validated our vaccine approach by boosting CMV-specific CTLs (CMV-CTLs) engineered with a CAR that targets the GD2 antigen. Experimental Design: We generated the whole-cell vaccine by engineering the K562 cell line to express the CMV-pp65 protein and the immune stimulatory molecules CD40L and OX40L. Single-cell–derived clones were used to stimulate CMV-CTLs in vitro and in vivo in a xenograft model. We also assessed whether the in vivo boosting of CAR-redirected CMV-CTLs with the whole-cell vaccine enhances the antitumor responses. Finally, we addressed potential safety concerns by including the inducible safety switch caspase9 (iC9) gene in the whole-cell vaccine. Results: We found that K562-expressing CMV-pp65, CD40L, and OX40L effectively stimulate CMV-specific responses in vitro by promoting antigen cross-presentation to professional antigen-presenting cells (APCs). Vaccination also enhances antitumor effects of CAR-redirected CMV-CTLs in xenograft tumor models. Activation of the iC9 gene successfully induces growth arrest of engineered K562 implanted in mice. Conclusions: Vaccination with a whole-cell vaccine obtained from K562 engineered to express CMV-pp65, CD40L, OX40L and iC9 can safely enhance the antitumor effects of CAR-redirected CMV-CTLs. Clin Cancer Res; 21(13); 2952–62. ©2015 AACR.

xvii, 417 p. : ill., maps. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.

Rapamycin has been demonstrated to be a potent antiproliferative agent. In vitro, it has been instrumental in further defining intracellular signaling pathways associated with mitogenic stimuli. While the direct intracellular target of rapamycin-FKBP complexes remains elusive, its use in the analysis of the regulation of cell cycle progression and proliferation has led to a greater understanding of the complexity of cell growth control. The potential clinical utility of rapamycin is beginning to be appreciated. Whether it will be efficacious as an antifungal or an antitumor drug remains uncertain; it is likely to be useful as a potent immunosuppressive agent. It has clear efficacy in a number of animal models of allogeneic transplantation, and may be useful in the treatment of autoimmune disease. Its unique mechanism of action complements that of CsA, and it appears to be largely nonoverlapping for toxicity. Several in vivo models have shown that the combination of rapamycin and CsA is synergistic, and this approach may allow treatment with doses of each drug which are individually less toxic. However, human trials with rapamycin have not yet been reported, and the spectrum of toxicity is therefore incompletely defined. Nevertheless, rapamycin is likely to be useful in the treatment of autoimmune disease and graft rejection and to be an effective agent used alone or in combination with other immunosuppressive agents.

The development of the western Newfoundland tree-ring chronologies from cross- sections collected by the Newfoundland Forest Service is the basis for this study. Sampling undertaken by the Newfoundland Forest Service started in 1985 and continued through the summer of 1997 and beyond. The data obtained from the Newfoundland Forest Service were cross-dated, time-series evaluated and standardized to achieve five ring-width indices. -- Abies balsamea is the dominant species used in the Western Newfoundland chronologies. Northern Peninsula ring widths are correlated with mean summer and winter temperatures. These ring widths are also correlated with sea-surface temperature and mean monthly surface-air temperature. The Northern Peninsula chronologies show evidence of 1820-1830s warming and subsequent cooling as well as the 1960-1970s North Atlantic anomalies. -- Comparison between published data and developed tree-ring chronologies depicts much similarity in areas from the North Atlantic sector. Comparison between temperature correlation significance in Scandinavia and the Northern Peninsula chronologies shows a definite relationship between mean monthly surface temperature for winter months and ring-width indices. -- Monthly climate data for western insular Newfoundland do not extend backward beyond the early 1930s. The development of tree-ring chronologies, extending as far back as 1760, will improve the understanding of environmental change in the Northwest Atlantic sector.