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BackgroundEngineered 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.MethodsUsing 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).ResultsCAR 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 and GM-CSF< sup >lowexpressing 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.ConclusionsThe 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.ReferenceKlichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology March 2020.

BackgroundDespite the remarkable efficacy achieved by CAR-T therapy in hematologic malignancies, application in solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival.1 Given that CAR-M are M1-polarized macrophages with the potential to remodel the tumor microenvironment (TME) and act as professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the interaction of CAR-M with the TME and the adaptive immune system.MethodsMurine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. To evaluate the safety and efficacy of CAR-M therapy, immunocompetent mice were engrafted with HER2+ tumors and treated with syngeneic CAR-M monotherapy or in combination with a PD1 blocking antibody. Tumors were collected at various time points and dynamic changes in the TME were assessed using flow cytometry, immunohistochemistry, and gene expression analysis.ResultsIn addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. CAR-M, but not control macrophages, phagocytosed and killed HER2-overexpressing tumor cell lines. CAR-M induced MHC-I expression on tumor cells and enhanced the cytotoxicity of CD8+ T cells. In vivo, CAR-M led to significant tumor regression and improved overall survival in multiple syngeneic models. Analysis of the TME showed that CAR-M led to increased infiltration of intratumoral CD4+ and CD8+ T, NK, and dendritic cells – as well as an increase in T cell responsiveness to tumor-associated antigens, indicating enhanced epitope spreading. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with anti-PD1 in the CT26-HER2 model, which is resistant to anti-PD1 monotherapy, and found that the combination further reprogrammed the TME, enhanced tumor control, and improved overall survival compared to monotherapy with either agent. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse, indicating long-term anti-tumor immunity. Finally, the combination of CAR-M with anti-PD1 did not trigger sustained elevations of any serum analyte associated with cytokine release syndrome (CRS) and was well tolerated across numerous safety assessmentsConclusionsThese results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors for the treatment of solid tumors.ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953