Current methods for engineering T cells for cancer immunotherapy use agonistic anti-CD3 and -CD28 monoclonal antibodies (mAb) to activate T cells, which induce their proliferation but do not recapitulate other fate-determining signals delivered by antigen-presenting cells. One such signaling axis, the NOTCH pathway, controls CD4+ T cell effector function acquisition and strongly influences behavior. To study NOTCH signaling during chimeric antigen receptor (CAR) T cell production, we developed a culture system using anti-CD3/CD28 mAb-coated beads and plate-coated agonistic NOTCH1-specific mAb to induce simultaneous T cell activation and NOTCH signaling. When transferred into NSG mice bearing CD19+ Raji lymphoma, CD19-specific NOTCH1-agonized (N1) CD4+ CAR T cells displayed a marked proliferative advantage over control (IgG) cells. Tumor-bearing mice given both IgG CD8+ CAR T cells and N1 CD4+ CAR T cells demonstrated superior expansion of both subsets compared to mice given IgG CD8+ and IgG CD4+ CAR T cells, resulting in rapid tumor clearance and protection from tumor re-challenge. These data demonstrate that NOTCH1 agonism could represent a significant improvement to adoptive T cell therapy, but the mechanisms by which NOTCH signaling improves T cell anti-tumor function are currently not understood. NOTCH is known to induce aryl hydrocarbon receptor (AhR) transcriptional activity. Pharmacologic inhibition of AhR activity during N1 CD4+ CAR T cell culture reduced characteristic differences between N1 and IgG cells in surface phenotype, cytokine production and proliferation upon in vitro restimulation. I hypothesize that NOTCH1 agonism organizes AhR-dependent transcriptomic changes that augment proliferative cytokine production in CD4+ CAR T cells, promoting CD4-dependent proliferation that improves CAR T cell efficacy. To test this hypothesis, I will assess the functional effects of AhR inhibition and activation in N1 and IgG CD4+ CAR T cells in vitro and in vivo, and characterize fate-determining transcriptional events in N1, IgG and AhR-inhibited N1 CD4+ CAR T cells using bulk and single-cell RNA sequencing. Collectively, these experiments will investigate the utility of NOTCH and AhR signaling in the generation of superior CAR T cell products and establish the mechanisms by which NOTCH1 agonism improves CAR T cell therapeutic efficacy, laying the groundwork for translation of this promising biomedical advance into immunotherapy clinics.
This project seeks to characterize the extent to which agonism of the NOTCH1 receptor improves the anti- tumor activity of CD4+ T cells engineered to express synthetic tumor-targeting chimeric antigen receptors (CARs), and investigate the mechanisms by which NOTCH signaling accomplishes this improvement. Preliminary data suggest that the aryl hydrocarbon receptor (AhR) mediates many of the effects NOTCH1 agonism has on CD4+ CAR T cell behavior. Information gained from the proposed studies will improve the scientific community?s understanding of how NOTCH and AhR signaling control T cell fate, and may improve the efficacy of the next generation of adoptive T cell therapies.
