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Objective There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Methods Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 hours and 10 days after transplantation and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using anti-human CD63 antibody quantum dot on the nanoparticle detector, and via GFP trigger on the nanoparticle flow cytometer (FACS). Results In syngeneic controls, steady state levels of circulating donor exosomes were detected at all post-transplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. Conclusions Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.

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.

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.

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.

Despite rapid advances in cancer immunotherapy, clinical responses in metastatic solid tumors have been limited. Macrophages are the most abundant immune cell in the solid tumor microenvironment (TME) and are primarily recruited as monocytes by TME-derived chemokines. When not under the control of the TME, macrophages are potent immune effector cells capable of phagocytosis, T cell recruitment, and antigen presentation. We have previously demonstrated that CAR macrophages (CAR-M) have potent anti-tumor activity and overcome several of the barriers to success in solid tumor immunotherapy - trafficking, immunosuppression, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. To more closely recapitulate normal biologic behavior, we attempted to create CAR monocytes that could traffic and differentiate into CAR macrophages upon tumor penetration. Toward that goal, we genetically engineered CD14+ human monocytes without ex vivo differentiation and with minimal cell culture. Using the chimeric adenoviral vector Ad5f35, we engineered human CAR-monocytes targeted against HER2. CAR expression and viability both exceeded 90%. Ad5f35 transduced CAR monocytes survived and maintained CAR expression ex vivo for at least 21 days. CAR monocytes efficiently differentiated into CAR-expressing macrophages when treated with GM-CSF as determined by FACS-based phenotypic characterization and Wright-Giemsa staining. Anti-HER2 CAR monocytes eradicated HER2 expressing tumor cells in a time and dose-dependent manner, and had comparable potency to anti-HER2 CAR-M. Additionally, the CAR monocyte manufacturing process offered the logistical advantage of a short manufacturing process (approximately two days). We have previously demonstrated that CAR-M are polarized toward a pro-inflammatory M1 phenotype after transduction with Ad5f35. Similarly, CAR monocytes demonstrated elevated expression of M1 markers, and intriguingly after differentiation into CAR-expressing macrophages, HLA-DR, CD80, CD86, and other M1 markers remained elevated - suggesting that transduction prior to differentiation does not impact the pro-inflammatory impact of adenoviral vectors on myeloid cells. Taken together, this abstract describes the successful development of CAR-monocytes with the potential for a rapid manufacturing process. In addition to direct anti-tumor activity while in the monocyte phase, CAR monocytes have the capacity to differentiate into CAR macrophages in situ, which are in turn capable of phagocytosis, T cell recruitment, TME activation, and antigen presentation. Given the previously demonstrated pre-clinical efficacy of CT-0508 (an anti-HER2 CAR macrophage), the CAR monocyte platform described herein offers a shortened manufacturing process and a potential advantage in tumor penetration, which will be directly evaluated in upcoming studies. Citation Format: Konrad Gabrusiewicz, Maggie Schmierer, Andrew Best, Martha Zeeman, Yumi Ohtani, Linara Gabitova, Daniel Cushing, Saar Gill, Michael Klichinsky. Genetically engineered chimeric antigen receptor (CAR) monocytes demonstrate targeted anti-tumor activity and differentiate into M1-polarized CAR macrophages [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 2180.

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.