O’Connor, Roddy S.

Abstract Despite recent landmark 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 they may convert into immunosuppressive (M2) tumor-associated macrophages (TAMs) and participate in disease progression. Currently, macrophage-orientated immunotherapeutic approaches under clinical development in oncology seek to reduce TAM infiltration (CSF-1 antagonists) or enhance TAM phagocytosis (CD47 antagonists). Transfer of autologous, activated, but nontargeted macrophages failed to demonstrate antitumor efficacy in past clinical trials. 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 antitumor T-cell response. We first demonstrate that CD3-zeta-based CARs are capable of inducing phagocytosis in human THP-1 macrophages, while truncated intracellular-domain deficient CARs were not. 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. We demonstrate that primary human macrophages, which are resistant to most viral vectors, are efficiently transduced by the chimeric fiber adenoviral vector Ad5f35 (~70% in 10 normal donors). Using Ad5f35, we engineered primary human macrophages with a CD3-zeta-based CAR against HER2. Anti-HER2 primary human CAR macrophages demonstrated targeted phagocytosis against HER2+ but not HER2- cell lines, with phagocytic activity dependent on both the CAR and antigen densities. Furthermore, CAR, but not UTD, macrophages led to potent dose-dependent killing of three distinct HER2-high cell lines in vitro. We sought to test the efficacy of anti-HER2 primary human macrophages in xenograft models of human HER2+ ovarian cancer. A single dose of CAR, but not UTD macrophages, led to tumor regression and improved overall survival in both intraperitoneal and disseminated models of disease. We show that macrophage transduction with Ad5f35, a double-stranded DNA virus, leads to a broad gene expression change, an interferon signaling signature, and phenotypic clustering toward classically activated M1 macrophages. 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, we show that primary human CAR macrophages are capable of targeted tumor phagocytosis, lead to improved overall survival in xenograft models, and demonstrate enhanced T-cell stimulation. CAR macrophages are a novel cell therapy platform for the treatment of human cancer. This abstract is also being presented as Poster B29. Citation Format: Michael Klichinsky, Marco Ruella, Olga Shestova, Andrew Best, Kristin Blouch, Xueqing M. Lu, Saad S. Kenderian, Miriam Y. Kim, Roddy O'Connor, Stephen Wallace, Miroslaw Kozlowski, Dylan M. Marchione, Maksim Shestov, Benjamin A. Garcia, Carl June, Saar Gill. Human chimeric antigen receptor (CAR) macrophages for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR07.

Abstract Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated profound results in hematologic malignancies but clinical efficacy in the solid tumor setting has not been observed. Barriers to T cell entry and function may partially explain this observation. As solid tumors actively recruit myeloid cells, we hypothesized that macrophages have the potential to be a powerful cellular immunotherapeutic agent in this setting if properly activated and redirected. We here describe the development of CAR macrophages (CARMA), demonstrating the feasibility, mechanism, and efficacy of this platform. To examine the function of CARs in macrophages, first generation anti-CD19, anti-mesothelin, or anti-HER2 CARs with a CD3ζ intracellular domain were introduced into the THP1 macrophage model. In vitro function was assessed via quantitative phagocytosis and luciferase-based specific killing assays. CARMA selectively phagocytosed and cleared cognate antigen-bearing tumor cells. To demonstrate the requirement for CAR-mediated intracellular signaling for activity, a CD3ζ-null CAR construct was tested in vitro. The deletion of CD3ζ significantly reduced the phagocytic and killing capacity (p<0.01) of CARMA. We identified Ad5f35, a chimeric adenovirus, as a novel and highly efficient viral vector for the transduction of normal donor and cancer patient macrophages (>70% CAR expression). Ad5f35 transduction polarized human macrophages toward a durable immunostimulatory M1 phenotype and rendered CARMA resistant to subversion toward the immunosuppressive M2 phenotype, as defined by surface markers and metabolomics. CARMA enhanced the proliferative capacity of CD8+ T cells in phytohemagglutinin activation assays and secreted factors that activated by-stander macrophages. Primary human anti-HER2 CARMA demonstrated targeted phagocytosis and killing of HER2 expressing ovarian and breast cancer cell lines, and exhibited a six-fold higher luciferase-based killing capacity of SKOV3 cells compared to trastuzumab in vitro (p=0.002). Anti-HER2 CARMA was evaluated in vivo in an intraperitoneal (IP) SKOV3 ovarian cancer xenograft model. Mice that received IP CARMA had a decrease in tumor burden of approximately two orders of magnitude and had a 30-day survival benefit relative to untreated or control macrophage treated mice (p=0.018). In a systemically disseminated SKOV3 model, a single dose of IV CARMA led to a durable anti-tumor response (38-fold reduction relative to control on day 31 post-treatment; p=0.016) Lastly, we demonstrated that the blockade of the anti-phagocytic CD47/SIRPα axis enhanced the phagocytic capacity of CARMA. In summary, we here demonstrate that human macrophages engineered with a CAR exhibit targeted anti-tumor function in both in vitro and in vivo preclinical models. This novel cellular immunotherapeutic approach has a clear translational potential for the treatment of solid tumors. Citation Format: Michael Klichinsky, Marco Ruella, Olga Shestova, Saad S. Kenderian, Miriam Y. Kim, Roddy O'Connor, John Scholler, Carl June, Saar Gill. Chimeric antigen receptor macrophages (CARMA) for adoptive cellular immunotherapy of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2017-4575