Abstract Despite recent advances in T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are usually the most abundant immune cell in the tumor microenvironment (TME) where, as immunosuppressive tumor-associated macrophages (TAMs), they participate in disease progression. The current goals of macrophage-based immunotherapies are to reduce TAM infiltration or enhance TAM phagocytosis. In contrast, we have developed a new paradigm based on the adoptive transfer of genetically engineered chimeric antigen receptor (CAR) macrophages (CAR-M) for the treatment of human cancer. CAR-M can only be produced using a unique adenoviral vector, since human macrophages are highly resistant to other methods of gene transfer. We have previously shown that the primary mechanism of action of CAR-M is phagocytosis, and that a single dose of primary human anti-HER2 CAR-M led to significantly improved overall survival in multiple xenograft models. We now establish that Ad5f35-transduced anti-HER2 CAR-M (CT-0508) adopt a unique proinflammatory and antitumor M1 phenotype. Functional evaluation and RNA sequencing revealed that CT-0508 maintain a proinflammatory M1 phenotype despite challenge with immunosuppressive environments in vitro, highlighting their resistance to subversion. By engrafting immunodeficient mice with human hematopoietic cells and human cancer cells, we established a novel xenografted human TME model. We demonstrate with single-cell resolution that CT-0508 maintain their phenotype within the human TME. Additionally, CT-0508 activated the human TME and generated an activated human dendritic cell signature. To further investigate the potential of CT-0508 for TME activation, we modeled the interaction of CT-0508 with immunosuppressive macrophages, dendritic cells, and T cells. CT-0508 shifted bystander macrophages toward a proinflammatory phenotype, induced activation and maturation markers on DCs, and recruited resting as well as activated T cells in chemotaxis assays. Lastly, CT-0508 demonstrated enhanced antigen presentation when compared to control human macrophages. These results show that in addition to direct antitumor activity, the anti-HER2 CAR macrophage cell product CT-0508 is capable of activating the solid cancer TME and promoting a proinflammatory phenotype. The safety of CT-0508 will be evaluated in an upcoming first-in-human phase I clinical trial. Citation Format: Konrad Gabrusiewicz, Nicholas Anderson, Xueqing Lu, Xinhe Shan, Olga Shestova, Nicholas Petty, Feng Shen, Maggie Schmierer, Andrew Best, Martha Zeeman, Yumi Ohtani, Katherine Cummins, Saar Gill, Michael Klichinsky. CT-0508, a novel CAR macrophage product directed against HER2, promotes a proinflammatory tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B65.

Abstract Despite recent advances in T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are often the most abundant immune cell in the tumor microenvironment (TME) where, as immunosuppressive tumor associated macrophages (TAMs), they participate in disease progression. Currently, most macrophage based immunotherapeutic approaches are focused on the depletion, repolarization, or phagocytic disinhibition of TAMs. We have developed a new paradigm based on the adoptive transfer of genetically engineered CAR macrophages (CAR-M) for the treatment of human cancer. CAR-M can be efficiently produced using the chimeric adenoviral vector Ad5f35. We have previously shown that the primary mechanism of action of CAR-M is antigen dependent phagocytosis, and that a single dose of primary human anti-HER2 CAR-M leads to significantly improved overall survival in multiple solid tumor xenograft models. Given that Ad5f35-transduced anti-HER2 CAR-M (CT-0508) adopt a unique pro-inflammatory M1-like phenotype, we hypothesized that CT-0508 may have the capacity to reprogram the TME toward an activated state. Functional evaluation and transcriptome-wide characterization revealed that CT-0508 maintain a pro-inflammatory phenotype despite challenge with immunosuppressive environments in vitro. By engrafting immunodeficient mice with human hematopoietic cells and human cancer cells we established a novel xenografted human TME model. We demonstrate with single cell resolution that CT-0508 maintain their M1 phenotype within the human TME. Additionally, CT-0508 augmented the human TME by inducing a pro-inflammatory signature in surrounding immune cells, characterized by induction of MHC-II and TNFα. To further investigate the potential of CT-0508 for TME activation, we modeled the interaction of CT-0508 with primary human M2 macrophages, dendritic cells, and T cells in vitro. CT-0508 repolarized bystander M2 macrophages toward a pro-inflammatory phenotype, induced activation and maturation markers on immature dendritic cells, and recruited resting as well as activated T cells in chemotaxis assays. CT-0508 demonstrated enhanced antigen presentation when compared to control human macrophages and cross-presented tumor derived intracellular antigens to CD8 T cells after tumor phagocytosis. Our results show that in addition to direct anti-tumor activity, the anti-HER2 CAR macrophage cell product CT-0508 is capable of promoting a pro-inflammatory tumor microenvironment and has the potential to induce epitope spreading via T cell recruitment and antigen presentation. Citation Format: Michael Klichinsky, Konrad Gabrusiewicz, Nicholas Anderson, Maggie Schmierer, Andrew Best, Martha Zeeman, Sotheavy Chhum, Yumi Ohtani, Olga Shestova, Xueqing Lu, Nicholas Petty, Xinhe Shan, Feng Shen, Saar Gill. CT-0508 is an anti-HER2 chimeric antigen receptor (CAR) macrophage with targeted anti-tumor activity that promotes a pro-inflammatory solid tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3242.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1–4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity. Primary macrophages engineered to express chimeric antigen receptors have anti-tumor activity in humanized mice.

IPEX is a fatal X-linked autoimmune disorder that results from mutations in the canonical regulatory T cell transcription factor FOXP3. In this issue of Cell Stem Cell, Masiuk et al. (2019) demonstrate an effective and potentially durable approach to eliminate the IPEX phenotype in murine models.