Abstract 582: Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy
Authors: Blumenthal Daniel , Gabitova Linara , Menchel Brett , Reyes-Uribe Patricia , DeLong Sabrina Ceeraz , Abramson Sascha , Klichinsky Michael 
Published: June 2022

Introduction: Engineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but responses have been rare in solid tumors. Our previously developed human chimeric antigen receptor macrophage (CAR-M) platform has shown potent anti-tumor activity in pre-clinical solid tumor models1, and the anti-HER2 CAR-M CT-0508 is currently being evaluated in a Phase I trial. The use of myeloid cells as a platform for cell therapy provides the tools to overcome critical solid tumor challenges such as infiltration, immunosuppression within the tumor microenvironment, 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 production feasibility, phenotype, pharmacokinetics, cellular fate, specificity, and anti-tumor activity of human CD14+ CAR monocytes.

Experimental: Using the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to express a CAR (CAR-mono) targeted against HER2. 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 (&gt;90%), and efficiently differentiated into CAR-expressing macrophages. Adenoviral transduction led to pre-conditioning of CAR-mono, resulting in a strong M1 phenotype upon differentiation into CAR-M. CAR-mono derived macrophages 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, CAR-mono induced anti-tumor activity in various HER2+ solid tumor xenograft models. Following IV administration, CAR-mono demonstrated the ability to traffic to both GM-CSFhigh and GM-CSFlow expressing tumors. Notably, CAR-mono showed long-term CAR expression and persistence (&gt;180 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence irrespective of human cytokine support.

Conclusions: The CAR-mono platform enables an automated, same-day manufacturing process while maintaining the key characteristics of CAR-M therapy. The use of Ad5f35 for human monocyte transduction primes the cells toward M1 macrophage differentiation and produces a cell population phenotypically and functionally similar to our established CAR-M platform. These data provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.1Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology. March 2020.

Citation Format: Daniel Blumenthal, Linara Gabitova, Brett Menchel, Patricia Reyes-Uribe, Sabrina Ceeraz DeLong, Sascha Abramson, Michael Klichinsky. Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 582.