Work

249 A genome-scale, in vivo transcription factor screen identifies a previously unrecognized factor that enhances CAR-T persistence and tumor control under high-dose rechallenge

Year: 2026

Type: conference-abstract

Abstract: Background Chimeric antigen receptor (CAR)-T cell therapy holds promise for cancer and a growing range of immune-mediated diseases. However, responses are not always durable, because CAR-T cells become exhausted and lose persistence under chronic antigen stimulation. Although several transcription factors enhance CAR-T persistence, they were identified individually rather than by unbiased search. We therefore screened all human transcription factors (TFs) in parallel, in vitro and in vivo, for factors that enhance CAR-T persistence and antitumor efficacy.Methods We used the Multiplexed Overexpression of Regulatory Factors (MORF) library, 1 a barcoded lentiviral library encoding 1,836 human TFs (3,548 isoform-resolved barcodes), quantifiable by next-generation sequencing. The library was transduced into CD19/BCMA dual-targeting (dual-CAR) T cells at MOI 0.3 with puromycin selection, then cryopreserved as the Day 0 input. In vitro, cells were repeatedly stimulated with Nalm6 over multiple rounds without exogenous cytokines. In vivo, the library was transferred into immunodeficient mice bearing established Nalm6 leukemia, and CAR-T cells were later recovered from bone marrow, spleen, and blood. Hits were defined by significant enrichment over the Day 0 input. We validated a single TF scoring as a hit in both arms by overexpressing it individually in CAR-T cells, comparing it with established persistence-enhancing factors as positive controls, and assessing efficacy, persistence, and safety across multiple donors in vitro and in vivo.Results The in vitro and in vivo screens yielded 11 and 12 enriched TFs (adjusted p < 0.01), respectively, with three shared. We focused on one — a TF not normally expressed in peripheral T cells and previously unstudied in them — and expressed it individually in CAR-T cells. TF-expressing cells gave better tumor control and higher peak expansion in vivo. After rechallenge at four times the initial tumor burden, positive-control cells relapsed within about two weeks, whereas TF-expressing cells maintained control through Day 105, with all mice tumor-free; one mouse relapsed transiently and cleared again. Under repeated stimulation in vitro, TF-expressing cells outperformed BFP controls across rounds. They also showed a stronger memory phenotype and greater metabolic fitness than BFP controls. Finally, without cytokine support, the TF did not drive antigen-independent proliferation.Conclusions An unbiased screen of all human TFs identified a previously unstudied factor that improves CAR-T persistence and antitumor efficacy under high-dose rechallenge, without antigen-independent proliferation — a broadly applicable strategy for more durable CAR-T cells across hematologic malignancies, autoimmune disease, and solid tumors.Reference Joung J, Ma S, Tay T, et al. A transcription factor atlas of directed differentiation. Cell 2023;186:209–229.e26. doi:10.1016/j.cell.2022.11.026.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0249