Project title: Multipronged approaches to develop immunotherapy targeting NETs

General Description

This 4-year study will develop nanobody-directed CAR T-cell therapy with various combinatory approaches in laboratory models to kill NET cells. The research will be carried out in collaboration with Dr. Carl June at the University of Pennsylvania, whose work was integrally involved with the approval of CAR T-cell therapy, a personalized, breakthrough immunotherapy for blood cancer. The multipronged approaches will significantly increase the opportunity to develop efficacious NET-specific immunotherapy.

Outcomes:

Our research focuses on enhancing the ability of VHH1CDH17 chimeric antigen receptor T cells (CAR T cells) to target and kill cancer cells. We investigated whether adding the immune-enhancing proteins IL-23 and CD226 would strengthen the antitumor response of these T cells. Our findings show that third-generation VHH1CDH17 CAR T cells, engineered to express these proteins, significantly improve their tumor-killing activity both in lab settings and in animal models.

By analyzing single-cell RNA sequencing data from T cells isolated from mouse tumor models, we discovered a greater prevalence of Th17 lineage immune cells in third-generation CAR T cells compared to second-generation CAR T cells. Th17 cells are pivotal in promoting immune system activation and inflammation, which are essential for a strong anti-cancer response. Concurrently, bulk RNA sequencing data further confirmed that genes involved in the Th17 immune response were upregulated in these third-generation CAR T cells, suggesting that they are inherently better equipped for combatting tumors.

IL-23, which plays a central role in promoting Th17 differentiation and maintenance, was found to be highly expressed in third-generation CAR T cells. Components of the IL-23 signaling pathway, including its major downstream effector STAT3, were also significantly upregulated. These findings imply that IL-23 signaling plays a crucial role in augmenting the anti-cancer activity of these third-generation CAR T cells.

Moreover, CD226, a cell surface receptor protein that binds to tumor-associated ligands and enhances T cell activation, was similarly upregulated in third-generation CAR T cells following activation. Our studies demonstrated that overexpressing IL-23 and CD226 in CAR T cells led to increased tumor cell killing and improved cytokine production in vitro. These engineered CAR T cells also demonstrated superior antitumor activity against BON cell-derived tumor xenografts in mice, significantly reducing tumor size and improving overall survival. Further flow cytometry analysis revealed that more CD3-positive CAR T cells persisted in the peripheral blood of treated mice, underscoring their sustained and robust antitumor response.

Together, these findings provide compelling evidence that overexpression of IL-23 and CD226 enhances the efficacy of VHH1CDH17 CAR T cells. The results highlight the promising potential of these enhanced CAR T cells for further evaluation and development in treating neuroendocrine tumors, potentially improving outcomes for patients by offering a more effective and targeted treatment approach.

This work has led to a recently opened (June 2024) clinical trial, “A Phase 1/2 Study to Evaluate CHM-2101, an Autologous Cadherin 17 Chimeric Antigen Receptor (CAR) T Cell Therapy.” Read more about the trial here and here.

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