615 Reactivating antitumor immunity in gliomas with osteopontin/integrin blocking peptide

BackgroundGlioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Despite improvements in imaging, surgical techniques, radiotherapy and chemotherapy, the prognosis of patients with GBM remains poor with a median overall survival of 15 months [1,2]. GBM is immunologically a ”cold” tumor with low infiltration of functional T and NK cells, which imposes poor responsiveness of GBM patients to immunotherapies. The immunosuppressive microenvironment in GBM is created by the malignant cells and tumor-associated macrophages (TAMs), such as resident brain microglia and recruited peripheral myeloid cells [3]. Osteopontin/Spp1 is one of glioma-derived factors that is responsible for the protumorigenic reprogramming of TAMs [4]. SPP1 expression is highly elevated in tumor tissues and sera from GBM patients, and inversely correlates with patient survival [5]. Cross-talk between malignant cells and TAMs relays on osteopontin binding to integrin receptors (mainly αvβ3 and αvβ5) via its RGD motif [6]. Thus, with the use of a RGD peptide (our in-house designed competitor of binding to integrins) we interfered with glioma-microglia interaction in vitro and evaluated the in vivo antitumor efficacy of integrin blockade as a monotherapy and in combination with an immune check-point inhibitor.MethodsThe efficacy of the RGD peptide to block microglia-dependent glioma invasion was determined in a Matrigel invasion assay. Antitumor activity of the peptide was assessed in a murine syngeneic orthotopic GL261 glioma model. RGD peptide was administrated intratumorally via osmotic pomps. For combination therapy, the animals received anti-PD-1 or isotype IgG antibody (4 inj. x 10 mg/kg i.p.). Tumor volume was measured using MRI. Heterogeneity of the immune cells compartment of glioma microenvironment was analysed by flow cytometry. The transcriptomes of CD11b+ cells immunosorted from tumor-bearing mouse brains were evaluated using RNAseq. Cytokine levels in the blood and the brain homogenates were measured using Luminex bead-based assays.ResultsThe microglia-stimulated invasion of GL261 glioma cells was reduced significantly in the presence of the RGD peptide in the in vitro co-culture system. The RGD peptide administrated to tumor-bearing mice induced proinflammatory reprogramming of TAMs. Combination of the RGD peptide with anti-PD-1 therapy increased the production of proinflammatory cytokines and the percentage of effector CD8+(CD44+CD62L-) cells in the tumors.ConclusionsThese results demonstrate that blockade of osteopontin/integrin signaling using the RGD peptide can mitigate the immunosuppressive microenvironment, reactivate the antitumor immunity and lay ground for improved response to immunotherapy in GBM.ReferencesJemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ: Cancer statistics, 2005. CA Cancer J Clin 2005, 55(1):10–30.Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K et al: Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC- NCIC trial. Lancet Oncol 2009, 10(5):459–466.Woroniecka KI, Rhodin KE, Chongsathidkiet P, Keith KA, Fecci PE: T-cell Dysfunction in Glioblastoma: Applying a New Framework. Clin Cancer Res 2018, 24(16):3792–3802Denhardt, D.T., M. Noda, A.W. O’Regan, D. Pavlin, and J.S. Berman. 2001. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 107:1055–1061.Grassinger, J., D.N. Haylock, M.J. Storan, G.O. Haines, B. Williams, G.A. Whitty, et al. 2009. Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and alpha4beta1 integrins. Blood 114:49–59.Anborgh, P.H., J.C. Mutrie, A.B. Tuck, and A.F. Chambers. 2010. Role of the metastasis-promoting protein osteopontin in the tumour microenvironment. Journal of cellular and molecular medicine 14:2037–2044Ethics ApprovalAll research protocols conformed to the Guidelines for the Care and Use of Laboratory Animals (European and national regulations 2010/63/UE September 22, 2010 and Dz. Urz. UE L276/20.10.2010, respectively). Animals were decapitated by a qualified researcher. The First Warsaw Local Ethics Committee for Animal Experimentation approved the study (approval no. 812/2019).

Anthracycline-induced cytotoxicity in the GL261 glioma model system

Glioblastoma (GBM) is a lethal astrocyte-derived tumor that is currently treated with a multi-modal approach of surgical resection, radiotherapy, and temozolomide-based chemotherapy. Alternatives to current therapies are urgently needed as its prognosis remains poor. Anthracyclines are a class of compounds that show great potential as GBM chemotherapeutic agents and are widely used to treat solid tumors outside the central nervous system. Here we investigate the cytotoxic effects of doxorubicin and other anthracyclines on GL261 glioma tumor cells in anticipation of novel anthracycline-based CNS therapies. Three methods were used to quantify dose-dependent effects of anthracyclines on adherent GL261 tumor cells, a murine cell-based model of GBM. MTT assays quantified anthracycline effects on cell viability, comet assays examined doxorubicin genotoxicity, and flow cytometry with Annexin V/PI staining characterized doxorubicin-induced apoptosis and necrosis. Dose-dependent reductions in GL261 cell viability were found in cells treated with doxorubicin (EC50 = 4.9 μM), epirubicin (EC50 = 5.9 μM), and idarubicin (EC50 = 4.4 μM). Comet assays showed DNA damage following doxorubicin treatments, peaking at concentrations of 1.0 μM and declining after 25 μM. Lastly, flow cytometric analysis of doxorubicin-treated cells showed dose-dependent induction of apoptosis (EC50 = 5.2 μM). Together, these results characterized the cytotoxic effects of anthracyclines on GL261 glioma cells. We found dose-dependent apoptotic induction; however at high concentrations we find that cell death is likely necrotic. Our results support the continued exploration of anthracyclines as compounds with significant potential for improved GBM treatments.

IMMU-05. B7-H3-SPECIFIC CAR T CELLS HAVE POTENT ANTI-TUMOR ACTIVITY IN THE GL261 IMMUNE-COMPETENT MURINE BRAIN TUMOR MODEL

BACKGROUND We and others have identified B7-H3 (CD276) as a promising target for CAR-based immunotherapies for pediatric brain tumors. So far, B7-H3-CAR T cells have only been studied in xenograft models for brain tumors, which do not recapitulate the immunosuppressive tumor microenvironment (TME). To overcome this obstacle, we decided to adapt the immune-competent GL261 murine glioma model which mimics human disease and host immune barriers. METHODS To evaluate the safety and efficacy of antigen-specific CAR T cells, murine B7-H3-CAR T cells were generated using retroviral particles encoding 2nd generation B7-H3-specific CD28.z CAR. Expansion, persistence, and anti-tumor activity were evaluated in vitro and in vivo. Components of the brain TME were then evaluated using flow cytometry and immunostaining. RESULTS B7-H3-CAR T cells only killed B7-H3+ tumor cells, secreted significant levels of IFNγ and IL-2 in an antigen-dependent manner and expanded an average of 33-fold in repeat stimulation assay with B7-H3+ tumor cells in contrast to control CAR T cells. In vivo, intratumoral injection of B7-H3-CAR T cells into orthotopic GL261 glioma induced complete regression in 60% of treated mice. Preliminary studies show numerous infiltration of suppressive tumor-associated macrophages within the tumor and its periphery. CONCLUSIONS In summary, we successfully generated murine B7-H3-CAR T cells and have demonstrated that these cells have potent anti-tumor activity in the immune-competent GL261 glioma model. However, it is likely that the tumor-associated macrophages are mediating immunosuppressive effects on B7-H3-CAR T cells. Therefore, studies focusing on TME/CAR T cell interactions are in progress.

EXTH-20. SYNGENEIC B7-H3-SPECIFIC CAR T-CELLS HAVE POTENT ANTI-BRAIN TUMOR ACTIVITY VIA LOCAL OR SYSTEMIC DELIVERY

BACKGROUND We and others have identified B7-H3 (CD276) as a promising target for CAR T-cell-based immunotherapies for pediatric brain tumors. So far, B7-H3-CAR T cells have only been studied in xenograft models for brain tumors, which do not recapitulate the immunosuppressive tumor microenvironment (TME). To overcome this obstacle, we decided to adapt the immune competent GL261 murine glioma model which mimics human disease and host immune barriers. METHODS To evaluate their safety and efficacy, murine B7-H3-CAR T-cells were generated using retroviral particles encoding a 2nd generation B7-H3-CAR with a CD28.z signaling domain. Expansion, persistence, and anti-tumor activity were evaluated in vitro and in vivo. Components of the brain TME were then evaluated using flow cytometry and immunostaining. RESULTS B7-H3-CAR T cells only killed B7-H3+ tumor cells, secreted significant levels of IFNγ and IL-2 in an antigen-dependent manner and expanded an average of 85-fold in repeat stimulation assay with B7-H3+ tumor cells in contrast to control CAR T-cells. In vivo, intratumoral (2x106) or systemic (3x106) injection of syngeneic B7-H3-CAR T-cells into mice with orthotopic GL261 glioma induced complete regression in 60% of treated mice resulting in a significant survival advantage. Mice showed no evidence of acute or long-term toxicities related to CAR T-cell infusions. We confirmed this encouraging safety profile by systemic administration of a high dose (1x107) B7-H3-CAR T-cells and performing histological analyses of all major organs on day 14 post T-cell injection, which showed no notable signs of injury or on-target/off-tumor toxicities. CONCLUSIONS We successfully generated syngeneic B7-H3-CAR T-cells and have demonstrated that these cells have potent anti-tumor activity in the immune competent GL261 glioma model via local or systemic delivery without apparent toxicities. Our study paves the way for future testing of B7-H3-CAR T-cells in early phase clinical studies.

Temporal and spatial modulation of the immune response of the murine Gl261 glioma tumor microenvironment

Glioblastoma, the most aggressive form of glioma, has a 5-year survival rate of <5%. While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. This study quantifies the temporal and spatial localization of immune cell populations and mediators during glioma development.Eight-week old male C57Bl/6 mice were orthotopically inoculated with 1×106 Gl261 cells and tumor morphology, local and systemic immune cell populations, and plasma cytokines/chemokines assessed at Day-0, 1, 3, 7, 14, and 21 post-inoculation by magnetic resonance imaging, chromogenic immunohistochemistry, multiplex immunofluorescent immunohistochemistry, flow cytometry and multiplex immunoassay respectively.From Day-3 tumors were distinguishable with >30% Ki67 and increased tissue vascularization (p<0.05). Increasing tumor proliferation/malignancy and vascularization were associated with significant temporal changes in immune cell populations within the tumor (p<0.05) and systemic compartments (p=0.02 to p<0.0001). Of note, at Day-14 16/24 plasma cytokine/chemokines levels decreased coinciding with an increase in tumor cytotoxic T cells, natural killer and natural killer/T cells. Data derived provide baseline characterization of the local and systemic immune response during glioma development. They reveal that type II macrophages and myeloid-derived suppressor cells are more prevalent in tumors than regulatory T cells, highlighting these cell types for further therapeutic exploration.

IMMU-04. TEMPORAL AND SPATIAL MODULATION OF THE IMMUNE RESPONSE OF THE MURINE GL261 GLIOMA TUMOUR MICROENVIRONMENT

INTRODUCTION Glioma is a debilitating and early fatal cancer arising in the glial cells of brain. Glioblastoma, the most aggressive form of gliomas, has a 5-year survival rate of 5% with treatment options limited to surgery, radiotherapy or chemotherapy with temozolomide. OBJECTIVE While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. In this study we quantified the temporal and spatial localisation of immune cell populations and mediators during glioma development. METHODS Mice were inoculated with 1x106 Gl261 cells at AP0.1mm, ML1.0mm, DV2.6mm Bregma. Tumour morphology, local and systemic immune cell populations were assessed at Day-0, 1, 3, 7, 14, and 21 post-inoculation by MRI, immunohistochemistry for Ki67+ proliferation and CD31+ vessel density; local immune infiltrate by multiplex immunofluorescence (VECTRA®;PerkinElmer); systemic immunity in spleen, bone marrow and peripheral blood by 16-parameter flow cytometry (LSRFortessa™;BD Biosciences); and multiplex immunoassay (Bio-Plex®;Bio-Rad) for plasma cytokines/chemokines. RESULTS From Day-3 tumours were distinguishable with high Ki67+ (> 30%) and increased tissue vascularisation (26.38±1.45 vessels/field; p< 0.05). Ki67 remained high until Day-21 with visible necrotic regions, increased vessel density and lumen area within tumour region. Increasing tumour proliferation/malignancy and vascularisation were associated with significant temporal changes in immune cell populations within the tumour (p< 0.05) and systemic compartments (p=0.02- p< 0.0001). Of note, at Day-14 NK, M1, PMN-MDSCs and M-MDSCs in the tumour infiltrate declined, coinciding with a decrease in 16/24 plasma cytokine/chemokines levels. Tumour infiltrating immune cell populations did not correlate with peripheral blood populations. However, a decrease in plasma cytokine/chemokine levels may indicate ‘immune exhaustion’. CONCLUSIONS The data derived provide baseline characteristics to study changes associated with multi-modal treatment strategies and to sequence therapies to maintain immune modulation. This information will contribute to the identification of novel combination therapies.

EXTH-27. ACTIVATING THE IMMUNITY WITHIN THE TUMOR USING VIROIMMUNOTHERAPY: DELTA-24-RGD ONCOLYTIC ADENOVIRUS ARMED WITH THE IMMUNOPOSITIVE REGULATOR GITRL

Based on promising results of recent clinical trials using oncolytic viruses, virotherapy is evolving as an alternative to treat patients with malignant glioma. Our group developed the oncolytic adenovirus Delta-24-RGD (DNX-2401) that is being tested, alone or in combination with anti-PD1, in clinical trials for recurrent glioblastoma (NCT00805376; NCT01956734; NCT02798406). The results suggest that, besides the expected oncolytic effect, the injection of the pathogen initiated, in a subset of patients, an anti-tumoral immunity that led to 20% of long-term survivors (3.5–5 years). To further enhance this effect, we have armed Delta-24-RGD to express the co-stimulatory ligand GITRL, and generated Delta-24-GREAT. The intracranial injection of Delta-24-GREAT prolonged the survival of GL261 glioma-bearing immunocompetent mice when compared to Delta-24-RGD treatment (P=0.002, log-rank test). Delta-24-GREAT treatment resulted in enhanced frequency of tumor-infiltrating lymphocytes: T lymphocytes (CD45+/CD3+) and cytotoxic T lymphocytes (CD45+CD3+CD8+). Functional studies performed by culturing splenocytes from Delta-24-GREAT-treated mice with glioma cells and analyzing secretion of Th1 cytokines, such as IL2 and IFN-γ, showed that lymphocytes recognized not only viral antigens but also tumoral antigens, suggesting the triggering of anti-tumoral immunity. Of interest, Delta-24-GREAT treatment resulted in an antigen-restricted anti-tumor memory effect and in the generation of central immune memory. Thus, rechallenging the survivor mice from the first experiment with a second implantation of glioma cells did not lead to tumor growth, and we detected an increased frequency of central memory CD8+ T cells (CD45+CD62L+). However, survivor mice developed lethal tumors when implanted intracranially with B16/F10 melanoma cells, strongly indicating that the developed immune response was specific for GL261 glioma antigens. This is a novel approach using an oncolytic adenovirus expressing GITRL to target cancer and to stimulate the immunity within the tumor. Our data strongly indicate that this type of strategy may be further developed to treat patients with malignant glioblastoma.