318 Pre-clinical development of a CAR monocyte platform for cancer immunotherapy

Background

Engineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but responses against solid tumors remain rare. Our previously developed human chimeric antigen receptor macrophage (CAR-M) platform has shown potent anti-tumor activity in pre-clinical solid tumor models,¹ and an anti-HER2 CAR-M product (CT-0508) is currently being evaluated in a Phase I trial. Use of myeloid cells for immunotherapy has the potential to overcome the main challenges presented by solid tumors – tumor infiltration, immunosuppression within the tumor microenvironment (TME), lymphocyte exclusion, and target antigen heterogeneity. Currently, CAR-M are generated in a week-long ex-vivo process in which peripheral blood monocytes are differentiated into macrophages prior to genetic manipulation. Here, we demonstrate the feasibility, phenotype, pharmacokinetics, durable CAR expression, cellular fate, specificity, and anti-tumor activity of human CD14+ CAR monocytes.

Methods

Using the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to durably express an anti-HER2 CAR (CAR-mono). Using a partially automated approach, we established a process that allowed for same day manufacturing (from Leukopak to cryopreserved CAR-mono cell product).

Results

CAR-mono showed high CAR expression and viability (>90%), and efficiently differentiated into CAR-expressing macrophages. The production process was designed to pre-condition CAR-mono to differentiate into M1-like CAR macrophages with strong pro-inflammatory effector functions. CAR-mono derived CAR-M (cmdCAR-M) demonstrated potent anti-tumor activity regardless of exposure to GM-CSF or M-CSF, and were protected against M2 switching by immunosuppressive factors. Treating CAR-mono with GM-CSF and IL-4 resulted in their differentiation to monocyte-derived CAR-DCs with an activated phenotype, indicating that these cells retained their myeloid differentiation potential. In vivo, intravenous administrated CAR-mono demonstrated the ability to traffic to tumors and showed remarkable long-term CAR expression and persistence (>180 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence and CAR expression irrespective of human cytokine support. CAR-mono differentiated into strong pro-inflammatory CAR-M even when injected directly into well-established tumors. Finally, CAR-mono induced anti-tumor activity in various HER2+ solid tumor xenograft models.

Conclusions

The CAR-mono platform allows for a rapid, same-day manufacturing process while maintaining the key characteristics of CAR-M therapy. Ad5f35 engineered human CAR monocytes are primed toward M1 macrophage differentiation, demonstrate durable CAR expression and persistence, and produce a cell population highly similar to our established CT-0508 product. These data provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.

Reference

Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology. 2020; 38: 947-953.