Transcription factor modulation in T-cells to improve cancer immunotherapy

Thesis (Ph.D.)--University of Washington, 2022Adoptive transfer of T-cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients, but can be limited by the inability of ex vivo culture systems to expand T-cells sufficiently for effective dosing while producing highly active T-cell products. Less-differentiated T-cell states correlate with CAR-T capacity to proliferate and mediate anti-tumor responses in vivo, but signals induced by antigen stimulation and IL-2 supplementation during tumor-specific T-cell culture couple proliferation to differentiation, creating an inverse relationship between ex vivo expansion and anti-tumor fitness. Accordingly, interventions during culture that enhance T-cell potency and/or uncouple T-cell proliferation from loss of activity are of high clinical value. We investigated whether modulation of the activity of two transcription factors, NOTCH and the aryl hydrocarbon receptor (AhR), during CAR-T production could enhance immunotherapy efficacy. NOTCH signaling controls both stem cell self-renewal and cellular differentiation across diverse tissues. NOTCH1 agonism restricted the differentiation of CAR-T derived from naïve CD4+ T-cells, and upregulated expression of AhR and c-MAF, driving heightened production of IL-22, IL-10 and Granzyme B upon tumor encounter. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to co-transferred CD8+ CAR-T, driving improved expansion and curative anti-tumor responses in vivo at low CAR-T doses. AhR is a ligand-activated transcription factor upregulated in response to TCR ligation, and IL-2 signaling induces AhR-mediated upregulation of coinhibitory receptors and attenuation of cytokine production. AhR also participates in regulatory T-cell function and can limit autocrine IL-2 production. Independently of NOTCH1 agonism, AhR was expressed in T-cells under culture conditions conducive to efficient CAR-T production. AhRi culture induced strong autocrine IL-2 production, driving enhanced T-cell expansion even in absence of supplemental IL-2. However, AhRi-expanded T-cells did not display phenotypic evidence of expansion-associated differentiation, and proliferated robustly and produced elevated levels of TH1/TC1-associated cytokines upon in vitro restimulation with tumor. AhRi CAR-T further mediated superior tumor control in mice bearing human lymphoma xenografts. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior, and demonstrate that inducing NOTCH1 signaling during activation ex vivo is a viable strategy for enhancing the function of T-cells engineered with tumor-targeting receptors. Our findings also indicate that AhR activity constitutes a critical autoregulatory axis in human T-cells, and identify AhR inhibition during tumor-specific T-cell manufacturing as an easily-translatable strategy to improve both cell product yields and anti-tumor function. This work shows that modulation of transcription factor activity during ex vivo T-cell culture constitutes a valuable and efficacious strategy for enhancing cancer immunotherapy potency