Role of NOTCH1 Signaling in Engineering CD4+ T Cells for Cancer Immunotherapy

PROJECT SUMMARY 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.