Human Chimeric Antigen Receptor Macrophages For Cancer Immunotherapy

Despite recent landmark advances in chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Myeloid cells are actively recruited to the tumor microenvironment (TME), where tumor associated macrophages (TAMs) are often the most abundant infiltrating immune cell. Currently, macrophage orientated immunotherapeutic approaches under clinical development in oncology seek to reduce TAM infiltration or enhance TAM phagocytosis. We hypothesized that genetically engineering human macrophages with CARs against tumor-associated antigens could redirect their phagocytic activity and lead to therapeutic efficacy with the potential for the induction of an anti-tumor T cell response. In this thesis, we demonstrate that CD3-zeta based CARs are capable of inducing phagocytosis by human macrophages. Notably, an active intracellular CAR signaling domain was required for activity. Targeted phagocytosis and clearance of CD19+, mesothelin+, and HER2+ cells by CARs targeted against each respective antigen was significantly superior to that by control untransduced (UTD) macrophages. Importantly, CAR macrophages were capable of polyphagocytosis and serial phagocytosis of tumor cells. We demonstrate that primary human monocyte derived macrophages, which are resistant to most viral vectors, are efficiently transduced by the chimeric-fiber adenoviral vector Ad5f35. Ad5f35 transduced primary human CAR macrophages demonstrated targeted phagocytosis, with phagocytic activity dependent on both the CAR and antigen densities. CAR, but not UTD, macrophages led to potent dose-dependent killing of tumor cells in vitro and led to tumor regression and improved overall survival in murine xenograft models of human cancer. Macrophage transduction with Ad5f35 leads to a broad gene expression change, an interferon signaling signature, and induction of a classically activated M1 phenotype. CAR macrophages upregulated co-stimulatory ligand and antigen processing/presentation genes and led to enhanced T cell stimulation in vitro and in vivo. Lastly, CAR, but not UTD, macrophages showed a broad resistance for M2 conversion in response to immunosuppressive cytokines. In conclusion, human CAR macrophages display targeted tumor phagocytosis, lead to improved overall survival in xenograft models, and demonstrate enhanced T cell stimulation. Taken together, these data show that CAR macrophages are a novel cell therapy platform for the treatment of human cancer

Human Chimeric Antigen Receptor Macrophages for Cancer Immunotherapy

Despite recent landmark advances in chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Myeloid cells are actively recruited to the tumor microenvironment (TME), where tumor associated macrophages (TAMs) are often the most abundant infiltrating immune cell. Currently, macrophage orientated immunotherapeutic approaches under clinical development in oncology seek to reduce TAM infiltration or enhance TAM phagocytosis. We hypothesized that genetically engineering human macrophages with CARs against tumor-associated antigens could redirect their phagocytic activity and lead to therapeutic efficacy with the potential for the induction of an anti-tumor T cell response. In this thesis, we demonstrate that CD3-zeta based CARs are capable of inducing phagocytosis by human macrophages. Notably, an active intracellular CAR signaling domain was required for activity. Targeted phagocytosis and clearance of CD19+, mesothelin+, and HER2+ cells by CARs targeted against each respective antigen was significantly superior to that by control untransduced (UTD) macrophages. Importantly, CAR macrophages were capable of polyphagocytosis and serial phagocytosis of tumor cells. We demonstrate that primary human monocyte derived macrophages, which are resistant to most viral vectors, are efficiently transduced by the chimeric-fiber adenoviral vector Ad5f35. Ad5f35 transduced primary human CAR macrophages demonstrated targeted phagocytosis, with phagocytic activity dependent on both the CAR and antigen densities. CAR, but not UTD, macrophages led to potent dose-dependent killing of tumor cells in vitro and led to tumor regression and improved overall survival in murine xenograft models of human cancer. Macrophage transduction with Ad5f35 leads to a broad gene expression change, an interferon signaling signature, and induction of a classically activated M1 phenotype. CAR macrophages upregulated co-stimulatory ligand and antigen processing/presentation genes and led to enhanced T cell stimulation in vitro and in vivo. Lastly, CAR, but not UTD, macrophages showed a broad resistance for M2 conversion in response to immunosuppressive cytokines. In conclusion, human CAR macrophages display targeted tumor phagocytosis, lead to improved overall survival in xenograft models, and demonstrate enhanced T cell stimulation. Taken together, these data show that CAR macrophages are a novel cell therapy platform for the treatment of human cancer

EBV latency types adopt alternative chromatin conformations.

Epstein-Barr Virus (EBV) can establish latent infections with distinct gene expression patterns referred to as latency types. These different latency types are epigenetically stable and correspond to different promoter utilization. Here we explore the three-dimensional conformations of the EBV genome in different latency types. We employed Chromosome Conformation Capture (3C) assay to investigate chromatin loop formation between the OriP enhancer and the promoters that determine type I (Qp) or type III (Cp) gene expression. We show that OriP is in close physical proximity to Qp in type I latency, and to Cp in type III latency. The cellular chromatin insulator and boundary factor CTCF was implicated in EBV chromatin loop formation. Combining 3C and ChIP assays we found that CTCF is physically associated with OriP-Qp loop formation in type I and OriP-Cp loop formation in type III latency. Mutations in the CTCF binding site located at Qp disrupt loop formation between Qp and OriP, and lead to the activation of Cp transcription. Mutation of the CTCF binding site at Cp, as well as siRNA depletion of CTCF eliminates both OriP-associated loops, indicating that CTCF plays an integral role in loop formation. These data indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19– leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019