Shestova, Olga

Abstract Chimeric antigen receptor T (CART) cell therapy results in impressively high remission rates in B cell neoplasms but is limited by the development of cytokine release syndrome (CRS). CRS is characterized by the development of high-grade fevers, hypotension, fluid overload and respiratory compromise, coincides with T cell expansion and is associated with marked elevation of interleukin-6, interferon-ɣ and other inflammatory cytokines. Severe CRS is seen in 25-80% of patients treated with CD19 directed CART cell therapy and mortality has been reported. While the use of the anti-IL6 receptor antibody tocilizumab with or without steroids can usually reverse this syndrome, there is concern that the early introduction of immunosuppressive medications could impair the anti-tumor activity and therefore most investigators currently reserve tocilizumab as therapy for severe (grade 3-4) CRS. Ruxolitinib is a JAK/STAT pathway inhibitor that is FDA approved for myelofibrosis and polycythemia vera and has resulted in a significant reduction of inflammatory cytokines in clinical studies. In preclinical models, treatment with ruxolitinib ameliorates cytokine production and manifestations of hemophagocytic lymphohistiocytosis. Therefore, there is a compelling rationale to investigate and develop ruxolitinib as a modality to prevent CRS after CART cell therapy. To our knowledge, there are no relevant models for CRS after human CART therapy, thus severely limiting the development of CRS prevention modalities that would in turn enhance the clinical feasibility of CART therapy. In this study, we established a novel acute myeloid leukemia (AML) xenograft model to study the development of CRS. Here, NSG-S (Non Obese Diabetic, SCID ɣ -/- mice that are additionally transgenic for human stem cell factor, IL-3 and GM-CSF) are engrafted with blasts from AML patients and treated with 1x106 CD123-directed CART cells (CART123), ten-fold higher than we previously used in our primary AML xenograft models. These animals develop an illness characterized by progressive weight loss, generalized weakness, emaciation, hunched bodies, withdrawal and poor motor response. This illness starts within one week of CART cell injection, correlates with T cell expansion and rapidly evolves and results in the death of the animals in 7-14 days (Figure 1A). Serum from these mice seven days after CART123 shows an extreme elevation of human IL-6, Interferon-γ, tumor necrosis factor-α, and other inflammatory cytokines (Figure 1B), resembling human CRS after CART cell therapy. NSG-S mice bearing primary AML were treated with CART123 and randomized to receive different doses of ruxolitinib (30, 60, or 90 mg/kg) or vehicle by oral gavage twice a day. Treatment started on the day of CART123 injection and continued for a week. Mice treated with ruxolitinib 60 or 90 mg/kg exhibited less severe clinical illness as manifested by attenuated weight loss when compared with mice treated with CART123 alone (Figure 2A). With the exception of ruxolitinib-only treated mice, all other groups exhibited an equivalent anti-leukemic effect (Figure 2B). We therefore established ruxolitinib 60 mg/kg for further experimental use. Most importantly, ruxolitinib treatment attenuated inflammatory cytokines (figure 2C) and led to long-term survival (figure 2D). Here we have described for the first time a clinically relevant animal model of human CRS and demonstrated that the JAK/STAT inhibitor ruxolitinib can prevent the development of severe CRS without impairing the anti-tumor effect of CART cells. These findings provide a useful platform for the future study of CRS prevention and treatment modalities. These experiments indicate that ruxolitinib could also be combined with CART cell therapy for the prevention of CRS in patients identified to be at high risk for the development of CRS. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Pfizer: Honoraria; Johnson & Johnson: Research Funding; University of Pennsylvania: Patents & Royalties; Tmunity: Equity Ownership, Other: Founder, stockholder ; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership. Gill:Novartis: Patents & Royalties, Resear\ch Funding.

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

Abstract Patients with otherwise treatment-resistant Hodgkin lymphoma could benefit from chimeric antigen receptor T-cell (CART) therapy. However, Hodgkin lymphoma lacks CD19 and contains a highly immunosuppressive tumor microenvironment (TME). We hypothesized that in Hodgkin lymphoma, CART should target both malignant cells and the TME. We demonstrated CD123 on both Hodgkin lymphoma cells and TME, including tumor-associated macrophages (TAM). In vitro, Hodgkin lymphoma cells convert macrophages toward immunosuppressive TAMs that inhibit T-cell proliferation. In contrast, anti-CD123 CART recognized and killed TAMs, thus overcoming immunosuppression. Finally, we showed in immunodeficient mouse models that CART123 eradicated Hodgkin lymphoma and established long-term immune memory. A novel platform that targets malignant cells and the microenvironment may be needed to successfully treat malignancies with an immunosuppressive milieu. Significance: Anti-CD123 chimeric antigen receptor T cells target both the malignant cells and TAMs in Hodgkin lymphoma, thereby eliminating an important immunosuppressive component of the tumor microenvironment. Cancer Discov; 7(10); 1154–67. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047

Key Points Depletion of CD123-redirected CAR T cells with monoclonal antibodies preserves leukemia remission in human AML xenograft models. AML CAR T-cell depletion enhances feasibility of subsequent allogeneic stem cell transplantation.

Abstract Purpose: Responses to therapy with chimeric antigen receptor T cells recognizing CD19 (CART19, CTL019) may vary by histology. Mantle cell lymphoma (MCL) represents a B-cell malignancy that remains incurable despite novel therapies such as the BTK inhibitor ibrutinib, and where data from CTL019 therapy are scant. Using MCL as a model, we sought to build upon the outcomes from CTL019 and from ibrutinib therapy by combining these in a rational manner. Experimental Design: MCL cell lines and primary MCL samples were combined with autologous or normal donor-derived anti-CD19 CAR T cells along with ibrutinib. The effect of the combination was studied in vitro and in mouse xenograft models. Results: MCL cells strongly activated multiple CTL019 effector functions, and MCL killing by CTL019 was further enhanced in the presence of ibrutinib. In a xenograft MCL model, we showed superior disease control in the CTL019- as compared with ibrutinib-treated mice (median survival not reached vs. 95 days, P < 0.005) but most mice receiving CTL019 monotherapy eventually relapsed. Therefore, we added ibrutinib to CTL019 and showed that 80% to 100% of mice in the CTL019 + ibrutinib arm and 0% to 20% of mice in the CTL019 arm, respectively, remained in long-term remission (P < 0.05). Conclusions: Combining CTL019 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Clin Cancer Res; 22(11); 2684–96. ©2016 AACR.