Abstract 1507: Chimeric antigen receptor T cells carrying IDH1 neomorph increase CD8 positive central memory proportion without interfering with cell growth
Authors: Xu Jie , Kaminskiy Yaroslav , Melenhorst Jan Joseph 
Published: July 2021

Background: The efficacy of anti-CD19 Chimeric Antigen Receptor (CAR)-T cells in treating relapsed/refractory chronic lymphoid leukemia has proven effective, but only in a small group of cases. Our group previously reported that TET2 disruption promoted the therapeutic efficacy of CART19 cells via the enrichment of the CD8+ central-memory (Tcm) subsets. TET2 is a tumor suppressor; hence, the permanent ablation via genome engineering could result in oncogenic transformation. Since TET2 utilizes alpha-ketoglutarate as a cofactor in the removal of methylgroups from cytosine residues in DNA, the competitive inhibition of TET2 could prove safer. We here explore this strategy by expressing an R132H variant of isocitrate dehydrogenase-I (IDH1), which generates millimolar quantities of D-2-hydroxyglutarate, a competitive inhibitor of TET2.

Methods: We generated a lentiviral construct carrying the full-length wildtype or mutant IDH1 open reading frame aminoterminal of a fully human, 4-1BB/CD3z-signaling CAR19, separated by a 2A element. A cell-surface expressed marker carrying the epitopes for CD20 and CD34 was added for CAR-T cells detection and purification. The above construct was transduced into healthy donors' T lymphocytes by a lentiviral transfer system. Sorted engineered cells were subsequently stimulated with artificial antigen presenting cells (aAPCs) expressing the target antigen. Cell growth was monitored and flow cytometry immunophenotyping was performed at the given time points upon several rounds of restimulation.

Results: Western blot assay showed IDH1 R132H neomorph only expressed in the CAR-T cells carrying mutant IDH1. The proliferation of IDH1 neomorph-expressing CAR-T cells was similar to the IDH1 wildtype group in this repeat stimulation assay. Similar results were obtained with the anti-CD3 expressing APCs. However, CAR-T cells harboring IDH1 R132H contained a remarkable enrichment of CD8+ anti-CD19 CAR T cells after four rounds of tumor stimulations compared with the control groups. To correct for donor-to-donor variability in the proportion of CD4+ and CD8+ T cells at the start of the experiment, we seeded engineered T cells at a 2:1 ratio. The selective elevation of CD8+ proportion was only observed in CAR-T cells with IDH1 R132H. We further found CAR-T cells having mutant IDH1 displayed an increasing enrichment of CD8+ Tcm (CD45RO+CD27+) subsets after repetitive tumor challenges. In contrast, the other two groups showed a stable or reducing percentage of CD8+ Tcm.

Conclusion: The above findings suggested that the introduction of IDH1[R132H] in CAR-T cells could lead to an increase of CD8+ frequency, as well as CD8+ Tcm proportion upon CAR signaling activation. Meanwhile, this introduction didn't make CAR-T cells grow wildly, indicating it is a safe strategy with the potential for further preclinical and clinical studies.

Citation Format: Jie Xu, Yaroslav Kaminskiy, Jan Joseph Melenhorst. Chimeric antigen receptor T cells carrying IDH1 neomorph increase CD8 positive central memory proportion without interfering with cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1507.