Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma.

Glioblastoma (GBM) remains a deadly tumor. Treatment with chemo-radiotherapy and corticosteroids is known to impair the functionality of lymphocytes, potentially compromising the development of autologous CAR T cell therapies. We here generated pre-clinical investigations of autologous anti-GD2 CAR T cells tested against 2D and 3D models of GBM primary cells. We detected a robust antitumor effect, highlighting the feasibility of developing an autologous anti-GD2 CAR T cell-based therapy for GBM patients.

Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells.

BACKGROUND AIMS: The proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy. METHODS: Monocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay. RESULTS: Monocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors' presence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and interleukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures. CONCLUSIONS: In summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.

Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma.

BACKGROUND: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. METHODS: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. FINDINGS: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. INTERPRETATION: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.

GD2 CAR T cells against human glioblastoma.

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF ß1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.

Developing cell therapies as drug products.

In the last 20 years, the global regulatory frameworks for drug assessment have been managing the challenges posed by using cellular products as new therapeutic tools. Currently, they are defined as "Advanced Therapy Medicinal Products", comprising a large group of cellular types that either alone or in combination with gene and tissue engineering technology. They have the potential to change the natural course of still lethal or highly debilitating diseases, including cancers, opportunistic infections and chronic inflammatory conditions. Globally, more than 50 cell-based products have obtained market authorization. This overview describes the advantages and unsolved challenges on developing cells as innovative therapeutic vehicles. The main cell therapy players and the legal framework are discussed, starting from chimeric antigen receptor T-cells for leukaemia and solid tumours, dealing then with lymphocytes as potent anti-microbiological tools and then focusing on mesenchymal stem/stromal cells whose role covers regenerative medicine, immunology and anti-tumour therapy.

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors.

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.

Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

GD2 expression in breast cancer.

Breast cancer (BC) is a heterogeneous disease, including different subtypes having diverse incidence, drug-sensitivity and survival rates. In particular, claudin-low and basal-like BC have mesenchymal features with a dismal prognosis. Disialoganglioside GD2 is a typical neuroectodermal antigen expressed in a variety of cancers. Despite its potential relevance in cancer diagnostics and therapeutics, the presence and role of GD2 require further investigation, especially in BC. Therefore, we evaluated GD2 expression in a cohort of BC patients and its correlation with clinical-pathological features.Sixty-three patients with BC who underwent surgery without prior chemo- and/or radiotherapy between 2001 and 2014 were considered. Cancer specimens were analyzed by immunohistochemistry and GD2-staining was expressed according to the percentage of positive cells and by a semi-quantitative scoring system.Patient characteristics were heterogeneous by age at diagnosis, histotype, grading, tumor size, Ki-67 and receptor-status. GD2 staining revealed positive cancer cells in 59% of patients. Among them, 26 cases (41%) were labeled with score 1+ and 11 (18%) with score 2+. Notably, the majority of metaplastic carcinoma specimens stained positive for GD2. The univariate regression logistic analysis revealed a significant association of GD2 with triple-receptor negative phenotype and older age (> 78) at diagnosis.We demonstrate for the first time that GD2 is highly prevalent in a cohort of BC patients clustering on very aggressive BC subtypes, such as triple-negative and metaplastic variants.

A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing.

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

Mesenchymal progenitors expressing TRAIL induce apoptosis in sarcomas.

Sarcomas are frequent tumors in children and young adults that, despite a relative chemo-sensitivity, show high relapse rates with up to 80% of metastatic patients dying in 5 years from diagnosis. The real ontogeny of sarcomas is still debated and evidences suggest they may derive from precursors identified within mesenchymal stromal/stem cells (MSC) fractions. Recent studies on sarcoma microenvironment additionally indicated that MSC could take active part in generation of a supportive stroma. Based on this knowledge, we conceived to use modified MSC to deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) targeting different sarcoma histotypes. Gene modified MSC expressing TRAIL were cocultured with different osteosarcoma, rhabdomyosarcoma, and Ewing's Sarcoma (ES) cell lines assessing viability and caspase-8 activation. An in vivo model focused on ES was then implemented considering the impact of MSC-TRAIL on tumor size, apoptosis, and angiogenesis. MSC expressing TRAIL induced significantly high apoptosis in all tested lines. Sarcoma death was specifically associated with caspase-8 activation starting from 8 hours of coculture with MSC-TRAIL. When injected into pre-established ES xenotransplants, MSC-TRAIL persisted within its stroma, causing significant tumor apoptosis versus control groups. Additional histological and in vitro studies reveal that MSC-TRAIL could also exert potent antiangiogenic functions. Our results suggest that MSC as TRAIL vehicles could open novel therapeutic opportunities for sarcoma by multiple mechanisms.

Detection of microparticles from human red blood cells by multiparametric flow cytometry.

BACKGROUND: During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as "storage lesions". These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC. MATERIAL AND METHODS: RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody. RESULTS: We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected. DISCUSSION: Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies.