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104 Development and characterization of human chimeric antigen receptor monocytes (CAR-Mono), a novel cell therapy platform
  1. Daniel Blumenthal,
  2. Linara Gabitova,
  3. Brett Menchel,
  4. Patricia Reyes-Uribe,
  5. Andrew Best,
  6. Michael Lynch,
  7. Sotheavy Chhum,
  8. Maggie Schmierer,
  9. Sascha Abramson and
  10. Michael Klichinsky
  1. Carisma Theraputics, Philadelphia, PA, USA


Background Engineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but solid tumors remain an intractable challenge. We have previously developed a human chimeric antigen receptor macrophage (CAR-M) platform for adoptive cell therapy and shown potent anti-tumor activity in pre-clinical solid tumor models.1 CAR-M overcome critical solid tumor challenges such as tumor 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.

Methods Using the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to express a CAR targeted against human epidermal growth factor receptor 2 (HER2) (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 expression and cell viability exceeded 90%, and cells efficiently differentiated into CAR-expressing macrophages. The adenoviral based gene modification method led to pre-conditioning of CAR-mono cells resulting in a strong M1 phenotype upon differentiation, and potent anti-tumor activity regardless of exposure to GM-CSF, M-CSF, or immunosuppressive factors. Treating CAR-mono cells with GM-CSF and IL-4 resulted in their differentiation to monocyte-derived CAR-DCs, indicating that these cells retain their myeloid differentiation potential. In vivo, CAR-mono treatment induced anti-tumor activity in various HER2+ solid tumor xenograft models. Following intravenous administration, CAR-mono demonstrated the ability to traffic to both GM-CSF < sup >high</sup > and GM-CSF< sup >low</sup >expressing tumors. Notably, CAR-mono showed long-term CAR expression and persistence (>100 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence irrespective of human cytokine support.

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 monocytes are primed toward M1 macrophage differentiation and produce a cell population highly similar to our established CAR-M platform. Collectively, these findings provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.


  1. Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology March 2020.

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