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Background The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM. Methods We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models. Findings B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines. Interpretation We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. Fund Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

Background The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT). Methods In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response. Results Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8+ T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4+ T-cell activation (CD38+/HLA-DR+) was paralleled by an increase in TT-induced CD4+/CD38low/CD127high memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4+ T cells. Conclusions TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.

Dendritic cells (DC) are the most potent antigen-presenting cells, strongly inducers of T cell-mediated immune responses and, as such, broadly used as vaccine adjuvant in experimental clinical settings. DC are widely generated from human monocytes following in vitro protocols which require 5&ndash, 7 days of differentiation with GM-CSF and IL-4 followed by 2&ndash, 3 days of activation/maturation. In attempts to shorten the vaccine&rsquo, s production, Fast-DC protocols have been developed. Here we reported a Fast-DC method in compliance with good manufacturing practices for the production of autologous mature dendritic cells loaded with antigens derived from whole tumor lysate, suitable for the immunotherapy in glioblastoma patients. The feasibility of generating Fast-DC pulsed with whole tumor lysate was assessed using a series of small-scale cultures performed in parallel with clinical grade large scale standard method preparations. Our results demonstrate that this Fast protocol is effective only in the presence of PGE2 in the maturation cocktail to guarantee that Fast-DC cells exhibit a mature phenotype and fulfill all requirements for in vivo use in immunotherapy approaches. Fast-DC generated following this protocol were equally potent to standard DC in inducing Ag-specific T cell proliferation in vitro. Generation of Fast-DC not only reduces labor, cost, and time required for in vitro clinical grade DC development, but can also minimizes inter-preparations variability and the risk of contamination.

Dendritic cells (DC) are the most potent antigen-presenting cells, strongly inducers of T cell-mediated immune responses and, as such, broadly used as vaccine adjuvant in experimental clinical settings. DC are widely generated from human monocytes following in vitro protocols which require 5–7 days of differentiation with GM-CSF and IL-4 followed by 2–3 days of activation/maturation. In attempts to shorten the vaccine’s production, Fast-DC protocols have been developed. Here we reported a Fast-DC method in compliance with good manufacturing practices for the production of autologous mature dendritic cells loaded with antigens derived from whole tumor lysate, suitable for the immunotherapy in glioblastoma patients. The feasibility of generating Fast-DC pulsed with whole tumor lysate was assessed using a series of small-scale cultures performed in parallel with clinical grade large scale standard method preparations. Our results demonstrate that this Fast protocol is effective only in the presence of PGE2 in the maturation cocktail to guarantee that Fast-DC cells exhibit a mature phenotype and fulfill all requirements for in vivo use in immunotherapy approaches. Fast-DC generated following this protocol were equally potent to standard DC in inducing Ag-specific T cell proliferation in vitro. Generation of Fast-DC not only reduces labor, cost, and time required for in vitro clinical grade DC development, but can also minimizes inter-preparations variability and the risk of contamination.

Simple Summary Active communication between GBM cells and tumor-infiltrating immune components contributes to establishing an immunosuppressive environment where T cells are scarce and exhausted. This condition is particularly exacerbated upon recurrence, which is almost inevitable for GBM patients. Immunotherapeutic approaches, including checkpoint inhibitors, have demonstrated limited efficacy and failed to prolong survival or recurrent GBM patients. Nevertheless, many studies have shown that T cell priming is possible in the GBM microenvironment, and that T cell exhaustion or dysfunction can be reprogrammed. We will revisit data from the literature and report original results obtained from recurrent GBM patients treated with dendritic cells to demonstrate the role of the microenvironment in predicting immunotherapy response and influencing decisions for personalized therapies. Abstract The interpretation of the presence and function of immune infiltration in glioblastoma (GBM) is still debated. Over the years, GBM has been considered a cold tumor that is less infiltrated by effector cells and characterized by a high proportion of immunosuppressive innate immune cells, including GBM-associated microglia/macrophages (GAMs). In this context, the failure of checkpoint inhibitors, particularly in recurrent GBM (rGBM), caused us to look beyond the clinical results and consider the point of view of immune cells. The tumor microenvironment in rGBM can be particularly hostile, even when exposed to standard immunomodulatory therapies, and tumor-infiltrating lymphocytes (TILs), when present, are either dysfunctional or terminally exhausted. However, after checkpoint blockade therapy, it was possible to observe specific recruitment of adaptive immune cells and an efficient systemic immune response. In this review article, we attempt to address current knowledge regarding the tumor and immune microenvironment in rGBM. Furthermore, immunosuppression induced by GAMs and TIL dysfunction was revisited to account for genetic defects that can determine resistance to therapies and manipulate the immune microenvironment upon recurrence. Accordingly, we reevaluated the microenvironment of some of our rGBM patients treated with dendritic cell immunotherapy, with the goal of identifying predictive immune indicators of better treatment response.

Evaluating changes induced by immunotherapies (IT) on conventional magnetic resonance imaging (MRI) is difficult because those treatments may produce inflammatory responses. To explore the potential contribution of advanced MRI to distinguish pseudoprogression (PsP) and true tumor progression (TTP), and to identify patients obtaining therapeutic benefit from IT, we examined aMRI findings in newly diagnosed glioblastoma treated with dendritic cell IT added to standard treatment. We analyzed longitudinal MRIs obtained in 22 patients enrolled in the EUDRACT N&deg, 2008-005035-15 trial. According to RANO criteria, we observed 18 TTP and 8 PsP. Comparing MRI performed at the time of TTP/PsP with the previous exam performed two months before, a difference in cerebral blood volume &Delta, rCBVmax &ge, 0.47 distinguished TTP from PsP with a sensitivity of 67% and specificity of 75% (p = 0.004). A decrease in minimal apparent diffusion coefficient rADCmin (1.15 vs. 1.01, p = 0.003) was observed after four vaccinations only in patients with a persistent increase of natural killer cells (response effectors during IT) in peripheral blood. Basal rADCmin &gt, 1 was independent predictor of longer progression free (16.1 vs. 9 months, p = 0.0001) and overall survival (32.8 vs. 17.5 months, p = 0.0005). In conclusion, rADC predicted response to immunotherapy and survival, Apparent Diffusion Coefficient (ADC) and Cerebral Blood Volume (CBV) modifications over time help differentiating PsP from TTP at onset.

Recently, we found that temozolomide (TMZ) can upregulate the expression of the multidrug-resistance protein ABCC3 in NK cells from both glioma-bearing mice and glioblastoma patients treated with dendritic cell immunotherapy combined with TMZ, allowing NK cells to escape apoptosis and favoring their role as antitumor effector cells. Here, we demonstrate that CD56dim NK cells expressing CD16+ are predominant in patients surviving more than 12 months after surgery without disease progression. CD56dim CD16+ NK cells co-expressed high levels of ABCC3 and IFN-γ. Notably, not only basal but also TMZ-induced ABCC3 expression was related to a strong, long-term NK cell response and a better prognosis of patients. The identification of the single nucleotide polymorphism (SNP) rs35467079 with the deletion of a cytosine (−897DelC) in the promoter region of the ABCC3 gene resulted associated with a better patient outcome. ABCC3 expression in patients carrying DelC compared to patients with reference haplotype was higher and modulated by TMZ. The transcription factor NRF2, involved in ABCC3 induction, was phosphorylated in CD56dim CD16+ NK cells expressing ABCC3 under TMZ treatment. Thus, ABCC3 protein and the SNP −897DelC can play a predictive role in patients affected by GBM, and possibly other cancers, treated with dendritic cell immunotherapy combined with chemotherapy.

Although some progress has been made in understanding GBM biology, treatment remains a challenge. There is increasing evidence that radiotherapy (RT) not only provides immunomodulatory effects on tumor microenvironment but also influences systemic immune response, supporting the advantage of combinatorial strategies with immunotherapy (IT). Using immune-competent mice in which syngeneic glioma cells are grown intracranially and treated with local fractionated radiation, we assessed the effects of RT on both local innate and adaptive immune cells. GL261-glioma bearing mice were locally irradiated with a total dose of 15 Gy in three consecutive fractions of 5 Gy on day 7, 8, and 9 after the tumor implantation. Irradiation was delivered both as exclusive (RT) and concomitant treatment in combination with dendritic cell (DC) immunotherapy (RT-IT). DCs were injected subcutaneously on day 16, 23, and 30 after tumor implantation. The potential role of RT in reprogramming the glioma-associated microglia is still poorly characterized. Microglia were isolated and enriched using CD11b microbeads from both the brain tumor hemisphere and contralateral hemisphere of RT, RT-IT, and control mice. A gene expression signature was analyzed on isolated microglia. Expression of mmp14 and trem2, involved in enhancing glioma proliferation, decreased in microglia isolated from RT mice (2.3 and 2.0-fold lower than in controls, day 16 P=0.01). The frequency of CD45dim/CD11b+/CD172A+ microglia showed an early increase at day 16 in the tumor mass and contralateral hemisphere in RT mice, but not in controls (p Citation Format: Serena Pellegatta, Natalia Di Ianni, Martina Maffezzini, Maria Luisa Fumagalli, Valentina Pinzi, Silvia Musio, Laura Fariselli, Gaetano Finocchiaro. Radiotherapy treatment in combination with Dendritic Cell Immunotherapy promotes a microglia activation and a disruption of the SIRPα-CD47 signaling axis in the GL261 glioma model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO087.

In a two-stage phase II study, 24 patients with first diagnosis of glioblastoma (GBM) were treated with dendritic cell (DC) immunotherapy associated to standard radiochemotherapy with temozolomide (TMZ) followed by adjuvant TMZ. Three intradermal injections of mature DC loaded with autologous GBM lysate were administered before adjuvant TMZ, while 4 injections were performed during adjuvant TMZ. According to a two-stage Simon design, to proceed to the second stage progression-free survival (PFS) 12 months after surgery was expected in at least 8 cases enrolled in the first stage. Evidence of immune response and interaction with chemotherapy were investigated. After a median follow up of 17.4 months, 9 patients reached PFS12. In these patients (responders, 37.5%), DC vaccination induced a significant, persistent activation of NK cells, whose increased response was significantly associated with prolonged survival. CD8+ T cells underwent rapid expansion and priming but, after the first administration of adjuvant TMZ, failed to generate a memory status. Resistance to TMZ was associated with robust expression of the multidrug resistance protein ABCC3 in NK but not CD8+ T cells. The negative effect of TMZ on the formation of T cell-associated antitumor memory deserves consideration in future clinical trials including immunotherapy.

Different approaches have been explored to raise effective antitumor responses against glioblastoma (GBM), the deadliest of primary brain tumors. In many clinical studies, cancer vaccines have been based on dendritic cells (DCs) loaded with peptides, representing one or more specific tumor antigens or whole lysates as a source of multiple antigens. Randomized clinical trials using DCs are ongoing, and results of efficacy are not yet available. Such strategies are feasible and safe; however, immune-suppressive microenvironment, absence of appropriate specific epitopes to target, and cancer immunoediting can limit their efficacy. The aim of this review is to describe how the definition of novel and more specific targets may increase considerably the possibility of successful DC immunotherapy. By proposing to target glioblastoma stem-like cells (GSCs), the immune response will be pointed to eradicating factors and pathways highly relevant to GBM biology. Preclinical observations on efficacy, and preliminary results of immunotherapy trials, encourage exploring the clinical efficacy of DC immunotherapy in GBM patients using high-purity, GSC-loaded DC vaccines.

In a two-stage phase II study, 24 patients with first diagnosis of glioblastoma (GBM) were treated with dendritic cell (DC) immunotherapy associated to standard radiochemotherapy with temozolomide (TMZ) followed by adjuvant TMZ. Three intradermal injections of mature DC loaded with autologous GBM lysate were administered before adjuvant TMZ, while 4 injections were performed during adjuvant TMZ. According to a two-stage Simon design, to proceed to the second stage progression-free survival (PFS) 12 months after surgery was expected in at least 8 cases enrolled in the first stage. Evidence of immune response and interaction with chemotherapy were investigated. After a median follow up of 17.4 months, 9 patients reached PFS12. In these patients (responders, 37.5%), DC vaccination induced a significant, persistent activation of NK cells, whose increased response was significantly associated with prolonged survival. CD8+ T cells underwent rapid expansion and priming but, after the first administration of adjuvant TMZ, failed to generate a memory status. Resistance to TMZ was associated with robust expression of the multidrug resistance protein ABCC3 in NK but not CD8+ T cells. The negative effect of TMZ on the formation of T cell-associated antitumor memory deserves consideration in future clinical trials including immunotherapy.

Checkpoint inhibitors, like ipilimumab, nivolumab, and pembrolizumab, have provided a breakthrough in cancer immunotherapy, such as in the treatment of melanoma and colorectal and lung cancer. The close relationship between the number of mutations (mutational load) and the response to checkpoint immunotherapy has been convincingly demonstrated in these cancers. Hypermutations in tumors are caused by environmental factors, like UV radiations or cigarette smoking, or by germinal mutations affecting genes of the Mismatch Repair (MMR) machinery, as in the Lynch syndrome. In the context of a high mutational load, a number of neoantigens become visible to the immune system, creating the basis for effective T cell responses. In low- and high-grade gliomas, the most frequent brain tumors, germinal MMR defects are rare; however, hypermutations associated with mutations or decreased expression of MMR genes are rather frequent, occurring in 20-60% of the tumors at recurrence after alkylating chemotherapy with temozolomide. Ongoing clinical trials and genomic investigations will clarify if temozolomide-induced hypermutations, which usually occur in the presence of methylation of the methylguanine methyltransferase gene (MGMT), will be effectively targeted by immunotherapy with checkpoint inhibitors or dendritic cell immunotherapy, thus improving the survival expectations for patients affected by these tumors.

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma.

A number of studies in murine models have suggested that the immune system may edit different tumors by forcing their expression profiles so that they escape immune reactions and proliferate. Glioblastoma (GB), the most frequent and aggressive primary brain tumor, provides a good example of this, thanks to the production of numerous immunosuppressive molecules (with TGF-β being of paramount importance), downregulation of the MHC complex and deregulation of the potential for antigen presentation by the surrounding microglia. Given that surgery, radiotherapy and chemotherapy with available protocols have limited effects on the survival of GB patients, different immunotherapy strategies have been developed, based on the use of dendritic cells, antibodies and peptide vaccination. Presently, bevacizumab, a humanized anti-VEGF antibody, provides the most successful example for immune-based treatment of GB, however, its action is limited in time, as the often tumor relapses due to still undefined immunoediting mechanisms. Altered function of EGF receptor-driven pathways is common in GB and is most frequently due to the presence of a deleted form named EGFRvIII, providing a unique cancer epitope that has been targeted by immunotherapy. A recent trial of GB immunotherapy based on vaccination with the EGFRvIII peptide has shown clinical benefit: interestingly most GBs at relapse were negative for EGFRvIII expression, a relevant, direct example of cancer immunoediting. Investigations on the mechanisms of GB immunoediting will lead to an increased understanding of the biology of this malignancy and hopefully provide novel therapeutic targets.

e14053Background: Recurrent glioblastoma (GBM) are highly aggressive tumors allowing 6-8 month survival. Recent data suggested that tetanus/ diphtheria toxoid (Td) preconditioning could increase de...

Cell therapy based on dendritic cells (DCs) pulsed with tumor lysate is a promising approach in addition to conventional therapy for the treatment of patients with glioblastoma (GB). The success of this approach strongly depends on the ability to generate high-quality, functionally mature DCs (mDCs), with a high level of standardization and in compliance with Good Manufacturing Practices. In the cell factory of the Carlo Besta Foundation, two phase I clinical trials on immunotherapy with tumor lysate-loaded DCs as treatment for GB are ongoing. From 2010 to 2014, 54 patients were enrolled in the studies and 54 batches of DCs were prepared. We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating yield of mDCs and their quality in terms of microbiological safety and immunological efficacy. The number of mDCs obtained allowed the treatment of all the enrolled patients. All 54 batches were sterile, conformed to acceptable endotoxin levels, and were free of Mycoplasma species and adventitious viruses. During culture, cells maintained a high percentage of viability (87%–98%), and all batches showed high viability after thawing (mean ± SD: 94.6% ± 2.9%). Phenotype evaluation of mDCs showed an evident upregulation of markers typical of DC maturation; mixed lymphocyte reaction tests for the functional evaluation of DCs demonstrated that all batches were able to induce lymphocyte responses. These results demonstrated that our protocol for DC preparation is highly reproducible and permits generation of large numbers of safe and functional DCs for in vivo use in immunotherapy approaches. Significance Cell therapy based on antigen-pulsed dendritic cells (DCs) is a promising approach for the treatment of glioblastoma patients. The success of this approach strongly depends on the ability to generate high-quality, functional DCs with a high level of standardization, ensuring reproducibility, efficacy, and safety of the final product. This article summarizes the results of the quality controls on 54 batches, to demonstrate the feasibility of producing a therapeutic cell-based vaccine via a well-controlled Good Manufacturing Practices (GMP)-compliant production process. The findings may be of scientific interest to those working in the field of preparation of GMP-compliant products for cell-therapy applications.

The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas. Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261, creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies. Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0180-0) contains supplementary material, which is available to authorized users.

2065 Background: The MRI follow –up of patients affected by glioblastoma (GBM) and treated with immunotherapy may be difficult, as immune responses may mimic tumor progression. To explore the potential contribution of quantitative MRI to the identification of patients with clinical benefit benefit, we used advanced MRI to study GBM patients treated with dendritic cell (DC) immunotherapy added to standard treatment (surgery, radiotherapy with concomitant temozolomide (TMZ) followed by adjuvant TMZ; DENDR1 trial, EUDRACT N° 2008-005035-15). Methods: A retrospective analysis was performed on longitudinal MRIs obtained soon after radiotherapy, within two days before the first vaccination (basal MRI) and every two months, in 22 patients enrolled in DENDR1. The following parameters were collected: tumor volume of contrast–enhanced lesions, mean rCBV, maximal rCBV, mean ADC, minimal ADC, ADC skewness. Receiver Operating Characteristic (ROC) curves were used to determine optimal sensitivity and specificity in differentiating patients as responder or not responder. Association with PFS (as per RANO criteria) and OS was analyzed using log-rank test and Cox regression. Results: Ten patients with PFS > 12 months were defined as responders Their basal mean ADC was significantly higher than in non responders (1.34 ± 0.17 vs 1.14 ± 0.34, p = 0.03). After four DC vaccinations mean ADC significantly decreased in responders only from 1.34 ± 0.17 to 1.23 ±0.23 (p = 0.028); the decrease persisted during immunotherapy. A basal mean ADC value ≥ 1.07 and a decrease in mean ADC value ≥ 0.13 were significant predictors of longer PFS (15.4 vs 9 m p = 0.0006; 17.2 vs 10.2 p = 0.04) and OS (29 vs 12.5 mo p = 0.002; 33 vs 19.9 p = 0.04 No significant correlations between the other parameters and the outcome were observed. Conclusions: Association with prolonged survival may suggest that in DENDR1responders decreased ADC is partly contributed by immune cells infiltrating the tumor.

BACKGROUND: Chemotherapy can influence the immune response by inducing immunogenic death of tumor cells or modulating tumor microenvironment (Galluzzi et al. 2012). The limitations of chemotherapy and immunotherapy as single therapeutic modalities have generated considerable interest in combinatorial strategies (Zitvogel L et al 2008). Here we focused on understanding the molecular mechanisms directly induced by temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma, that can activate immune cells. METHODS: We studied the in vivo effects of TMZ on immune cells by treating mice 9 days after intracranial implantation of GL261 gliomas with intraperitoneal injections of TMZ (5 mg/kg) or vehicle (control mice) for 5 days. Mice were sacrificed 20 hours after treatment on days 1 to 5: tumor and peripheral blood were harvested and analyzed by flow cytometry. RESULTS: Trafficking of NK1.1+ CD3- NK cells in blood, but not of CD8+ T cells, and their homing ability into the brain significantly increased in TMZ-treated mice on day 12 after the second administration of TMZ. At this early time point TMZ led to a significant enrichment of CD11b(low) CD27(high) and CD11b(high) CD27(high) subsets. Microarray analysis revealed differentially expressed genes represented in different clusters of which chemotaxis, cell cycle, multidrug resistance and anti-apoptosis were predominantly up-regulated in blood-derived NK cells isolated from TMZ-treated compared to control mice. We focused validation experiments on genes related to multidrug resistance and chemotaxis. Chemotaxis was assayed using the in vitro transwell system confirming that the migration ability of NK cells from treated mice significantly increased by 3-fold compared with controls. Increased expression of three ATP transporter genes was confirmed by real time PCR (2.6-fold higher than controls, P = 0.006). Five days after the end of the treatment we found an enrichment of CD11(high) CD27(low), the most potent effector subset of NK cells (Chiossone et al 2009). In agreement, IFN-gamma production evaluated by flow cytometry and the in vitro cytotoxic activity of NK cells from TMZ-treated mice were higher than that of NK cells from control mice. CONCLUSIONS: Our results support the idea that NK cells can be resistant to chemotherapy (Lowdell 2003; MacDonald HR et al 1995). In particular at early time points TMZ leads to an enrichment of NK cells with migratory function, in the later phase TMZ leads to an increase of NK cells promoting cytotoxic ability. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

The stem cell–determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor–induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells. Cancer Res; 74(6); 1833–44. ©2014 AACR.

2039Background: In the two stage phase I-II study DENDR-I, 24 patients with first diagnosis of glioblastoma (GBM) were treated with DC immunotherapy associated to standard radio-chemotherapy. To go...

A critical requirement of an efficient cancer immunotherapy is the generation of long-lasting, specific memory. In a clinical trial active at Istituto Besta, first diagnosis glioblastoma (GBM) patients, with post-surgery volume ≤10 cc, underwent leukapheresis before radiotherapy and chemotherapy with the alkylating agent temozolomide (TMZ). Three intradermal injections of mature dendritic cells (DCs) loaded by autologous tumor lysates were done before adjuvant chemotherapy. Subsequent 4 injections were performed in association with six cycles of adjuvant TMZ. Peripheral blood lymphocytes (PBLs) were analyzed by flow cytometry to characterize immune response before and after DC vaccines. The ratio of vaccination/baseline frequencies and counts (V/B ratio) of all of the immunological parameters for each patient was calculated, and the median of all of the observations used as the cut off value to separate patients. V/B ratio was correlated with the progression free survival (PFS) of each patient. Preliminary results from the interim analysis on 24 patients showed that an increased NK, but not CD8+ T cell response was significantly associated with prolonged survival (p In a group of responder patients (PFS>12), during the first three vaccines, CD8+ T cells underwent a rapid expansion and produced higher levels of IFN-γ compared to the baseline (p After the third vaccine and TMZ administration, primed CD8+ T cells failed to form an effector memory phenotype (CCR7negCD45RAnegCD62Llow/neg). Our results support a possible interference of adjuvant chemotherapy on effector memory formation. Further investigations are required to understand how memory T cells behave in response to repeated exposure to TMZ. Citation Format: Serena Pellegatta, Marica Eoli, Elena Anghileri, Sara Pessina, Carlo Antozzi, Simona Frigerio, Gabriele Cantini, Maria Grazia Bruzzone, Bianca Pollo, Eugenio A. Parati, Gaetano Finocchiaro. CD8+T cells fail to form an effector memory in glioblastoma patients treated with dendritic cell immunotherapy in combination with chemotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A031.

During recent years different approaches have been explored to raise effective antitumor responses against brain tumors and particularly glioblastomas (GBMs). In most cases, cancer vaccines were based on autologous dendritic cells loaded with GBM peptides or whole tumor lysates. Many phase I-II studies showed that such strategy is feasible and nontoxic but failed to provide convincing evidence of its efficacy. This was due to study design and other biological issues: local immune suppression and insufficient characterization of appropriate epitopes appear as particularly relevant.In neuro-oncology intriguing data have been obtained by vaccinating patients with the epidermal growth factor receptor variant III (EGFRvIII) peptide, reproducing a specific epitope arising because of large deletion of the EGFR gene. In other cancers immunotherapy is obtaining clinically meaningful results: in prostate cancer vaccination with dendritic cells loaded with a cancer peptide, and in metastatic melanoma antibodies against an immune gate-keeper, CTL4, both led to increased survival and have been approved by FDA.On the basis of these and other clinical and preclinical findings it appears that several approaches may have chances of clinical success. They include combinatorial treatments of chemotherapy and immunotherapy, systemic and local immunotherapy, vaccination against specific targets, for example cytomegalovirus protein or stem cell markers re-expressed during brain cancer progression.

Pilot data showed that adding intratumoral (IT) injection of dendritic cells (DCs) prolongs survival of patients affected by glioblastoma multiforme (GBM) treated by subcutaneous (SC) delivery of DCs. Using a murine model resembling GBM, we investigated the immunological mechanisms underlying this effect. C57BL6/N mice received brain injections of GL261 glioma cells. Seven days later, mice were treated by 3 SC injections of DCs with or without 1 IT injection of DCs. DC maturation, induced by pulsing with GL261 lysates, was necessary to develop effective immune responses. IT injection of pulsed (pDC), but not unpulsed DCs (uDC), increased significantly the survival, either per se or in combination with SC-pDC (P.001 vs controls). Mice treated by IT-pDC plus SC-pDC survived longer than mice treated by SC-pDC only (P = .03). Injected pDC were detectable in tumor parenchyma, but not in cervical lymph nodes. In gliomas injected with IT-pDC, CD8+ cells were significantly more abundant and Foxp3+ cells were significantly less abundant than in other groups. Using real-time polymerase chain reaction, we also found enhanced expression of IFN-gamma and TNF-alpha and decreased expression of transforming growth factor-beta (TGF-beta) and Foxp3 in mice treated with SC-pDC and IT-pDC. In vitro, pDC produced more TNF-alpha than uDC: addition of TNF-alpha to the medium decreased the proliferation of glioma cells. Overall, the results suggest that IT-pDC potentiates the anti-tumor immune response elicited by SC-pDC by pro-immune modulation of cytokines in the tumor microenvironment, decrease of Treg cells, and direct inhibition of tumor proliferation by TNF-alpha.

Dendritic cell (DC) immunotherapy is gaining momentum in clinical cancer immunotherapy. We set-up a phase I-II study in patients with first diagnosis of glioblastoma (GBM) in which DC immunotherapy was associated to standard radiochemotherapy. The primary goal was to evaluate progression free survival (PFS) 12 months after surgery (PFS-12) as defined by RANO criteria. Safety, feasibility and evidence of immune response were also considered. Twenty-four patients were assessed. After gross-total resection (

Growing evidence suggests that chemotherapy can influence the immune response by inducing lymphopenia or enhancing immunogenicity of dying tumor cells. In a clinical trial currently active in our Institution, patients with first diagnosis of glioblastoma are treated with dendritic cells (DC) loaded with autologous tumor lysate together with standard radio and chemotherapy with temozolomide. Peripheral blood lymphocytes from 22 patients were analyzed by flow cytometry to identify immune cell activation before and after DC vaccines. The ratio of vaccine/baseline frequencies (V/B ratio) was correlated with the survival of each patient. Increased V/B ratio for NK cells was significantly associated with prolonged PFS and OS (median 15.0 vs 8.0 mo, p = 0.003; 22.0 vs 12.0 mo, p = 0.02, respectively). Using the murine malignant glioma GL261, we investigated the molecular mechanisms induced by TMZ on NK and CD8 T cells. We treated mice 9 days after intracranial implantation of GL261 cells with intraperitoneal injections of TMZ (5 mg/kg) or vehicle (control mice) for 5 days. Mice were sacrificed at different time points and tumor and peripheral blood harvested and analyzed by flow cytometry. Gene expression profiling on peripheral blood lymphocytes revealed an up-regulation of multidrug resistance genes in NK cells, but not in CD8 T lymphocytes, in TMZ-treated mice compared to controls. The increased expression of the ATP transporter gene Abcc3 was confirmed by real time PCR (4.2-fold higher than controls, P = 0.006). Using eFluxx-ID multidrug resistance (MDR) assay based on specific inhibitors, we also demonstrated that Abcc3 was functionally active during TMZ treatment. NK cell resistance to chemotherapy was accompanied by an increase in migration and homing ability into the brain at early time point and in cytotoxicity at later phases (beyond the end of TMZ treatment). Our data show that murine NK cells are resistant to and activated by TMZ chemotherapy. Further investigations in patients treated by radio-chemotherapy with or without DC immunotherapy are warranted. Note: This abstract was not presented at the meeting. Citation Format: Serena Pellegatta, Sara Pessina, Gabriele Cantini, Emanuela Cazzato, Dimos Kapetis, Gaetano Finocchiaro. Abcc3 up-regulation confers protection from chemotherapy to NK cells in a murine model of malignant glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1343. doi:10.1158/1538-7445.AM2015-1343

Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

In recent years, the use of dendritic cells (DC), the most powerful antigen presenting cells, has been proposed for the creation of vaccines against gliomas. This approach has been demonstrated to be safe and non-toxic in phase I or I-II trials (2, 3). Immunotherapy plays a central role in the search for new treatments for glioblastoma multiforme (GBM). In particular, several phase I studies have been performed using DC pulsed by GBM proteins as a vaccine for patients with relapsing GBM. The studies demonstrated that DC vaccination is safe and may produce a significant increase in overall survival. As the first step in the preparation of appropriate conditions for a clinical evaluation in Italy, we have performed pre-clinical experiments on immune-competent mice injected intra-cerebrally with syngeneic GL261GBM cells and treated subcutaneously and intra-tumorally with DC loaded with a GL261 homogenate. These results show that vaccination with DC pulsed with a tumor lysate increases considerably survival in mice bearing intracranial glioblastomas and supports the development of DC-based clinical trials for patients with glioblastomas that do not respond to standard therapies.

During their growth, malignant gliomas interact with the immune system and are able to escape immune reactions. Attempts to instruct the immune system to develop anti-glioma reactions have been partly unsuccessful. Recent advances in the molecular and cellular biology of dendritic cells (DC), however, may increase the chances of preparing effective “vaccines” against these tumours. We show that vaccination with DC pulsed with a tumour lysate considerably increases survival in mice bearing intracranial glioblastomas. These results support the development of DC-based clinical trials for patients with glioblastomas that do not respond to standard therapies.

2087 Background: DENDR1 is a phase I-II phase study aimed at evaluating dendritic cell (DC) immunotherapy in patients with first diagnosis of glioblastoma multiforme (GBM). Here we provide results ...

Recurrent glioblastoma (GB) are highly aggressive tumors allowing 6-8 month survival. Here we evaluated the possible benefits of immunotherapy with mature dendritic cells (DC) loaded with autologous tumor lysate in 15 patients with recurrent GB. After a median follow-up of 8 months median progression free survival (PFS) was 4.4 months and median overall survival (OS) 8.0 months. Patients with small tumors at first vaccination (< 20 cc; n = 8) had significantly longer PFS and OS than others: 6.0 vs 3.0 months (p = 0.01) and 16.5 vs 7.0 months (p = 0.003), respectively. Patients were analysed for CD8+ T cells, CD56+ NK cells and other relevant immunological parameters in peripheral blood before and after immunization, defining a vaccination/baseline ratio (V/B ratio). Increased V/B ratio of NK but not CD8+ T cells was significantly associated to longer PFS and OS. Patients showing NK cell responses had higher levels of IFN-γ and E4BP4, the NK cell transcription factor. Furthermore, NK V/B ratio was inversely correlated with TGF-β2 and VEGF V/B ratio. The results suggest that tumor-loaded DC may increase survival of recurrent GB after effective tumor debulking and emphasize the role of NK cell responses in this therapeutic setting. Citation Format: Serena Pellegatta, Marica Eoli, Simona Frigerio, Carlo Antozzi, Maria Grazia Bruzzone, Gabriele Cantini, Sara Nava, Elena Anghileri, Lucia Cuppini, Valeria Cuccarini, Emilio Ciusani, Marta Dossena, Bianca Pollo, Renato Mantegazza, Eugenio A. Parati, Gaetano Finocchiaro. NK cell response and tumor debulking are associated to prolonged survival in recurrent glioblastoma treated by dendritic cells loaded with autologous tumor lysate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2839. doi:10.1158/1538-7445.AM2013-2839 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Recurrent glioblastomas (GBs) are highly aggressive tumors associated with a 6–8 mo survival rate. In this study, we evaluated the possible benefits of an immunotherapeutic strategy based on mature dendritic cells (D cs) loaded with autologous tumor-cell lysates in 15 patients affected by recurrent GB. The median progression-free survival ( pFs) of this patient cohort was 4.4 mo, and the median overall survival (O s) was 8.0 mo. p atients with small tumors at the time of the first vaccination (< 20 cm 3 ; n = 8) had significantly longer p Fs and Os than the other patients (6.0 vs. 3.0 mo, p = 0.01; and 16.5 vs. 7.0 mo, p = 0.003, respectively). c D8 + T cells, cD56 + natural killer (NK) cells and other immune parameters, such as the levels of transforming growth factor β , vascular endothelial growth factor, interleukin-12 and interferon γ (IFN γ), were measured in the peripheral blood and serum of patients before and after immunization, which enabled us to obtain a vaccination/baseline ratio (V/B ratio). an increased V/B ratio for NK cells, but not cD8 + T cells, was significantly associated with prolonged pFs and Os. patients exhibiting NK-cell responses were characterized by high levels of circulating IFN γ and e4Bp4, an NK-cell transcription factor. Furthermore, the NK cell V/B ratio was inversely correlated with the TGF β2 and VeGF V/B ratios. These results suggest that tumor-loaded D cs may increase the survival rate of patients with recurrent GB after effective tumor debulking, and emphasize the role of the NK-cell response in this therapeutic setting.

Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE(2), IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with "new method" lysate compared to DC pulsed with "classical method" lysate. Our results indicate that immunomagnetic isolation of CD14(+) monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.

The glutamate-aspartate transporter GLAST is a radial glia marker that is highly expressed in GL261 stem-like cells (GSCs). To target GLAST, we treated glioma-bearing mice with three subcutaneous injections of four GLAST peptides emulsified with Montanide ISA-51 in association with granulocyte macrophage colony-stimulating factor (GM-CSF) injections. Vaccination with GLAST peptides significantly prolonged survival, effectively enhanced systemic T-cell and NK-cell responses and promoted robust antitumor cytotoxicity. GLAST expression significantly decreased in gliomas from immunized mice, as evaluated by histological analysis and real-time PCR (RT-PCR). Moreover, the immunization protocol led to the upregulation of interferonγ (IFNγ) and tumor necrosis factorα (TNFα) as well as to the downregulation of transforming growth factor (TGF) β1 and β2 in the tumor. Beyond these changes, gliomas from immunized mice exhibited an increased recruitment of NK cells and antigen-specific CD8+ T cells expressing the tumor homing molecule VLA-4, as well as a local chemotactic gradient featuring expression of CXCL10 (which may be responsible for the recruitment of CTLs), CCL3, CCL4 and CCL5 (which are involved in NK-cell migration), and NKG2D ligand on glioma cells. Importantly, although GLAST is expressed in the central nervous system, autoimmune reactions were not observed in immunized mice. Altogether, these results support the contention that GLAST may constitute a glioma antigen against which immune responses can be efficiently induced without major safety concerns.