Fourteenth Meeting of the Network Italiano per la Bioterapia dei Tumori (NIBIT) on Cancer Bio-Immunotherapy, Siena, Italy, October 13–15, 2016

Introduction

The XIV annual meeting of the Italian Network for Tumor Biotherapy (NIBIT) took place in Siena, Tuscany, on October 13–15, 2016. As in the past, more than 40 Italian and Foreigner Leaders representing Academia, biotechnological and pharmaceutical industry contributed with their presentations to give an update on therapeutic developments in the bio-immunotherapy field. The aim of the meeting was also to favor a rapid and constructive translation from preclinical research to clinical applications. Here, we report a brief summary of the main topics discussed during the meeting.

Session 1: cancer genetics and immunotherapy

Giuseppe Palmieri (Unit of Cancer Genetics, ICB-CNR, Sassari, Italy) opened the meeting explaining the influence of several molecular or immunological alterations on cancer pathogenesis, defining cancer not only as a genetic but also as an immunological disorder. Through the concept of cancer immunoediting that is composed of the phases of Elimination followed by Equilibrium and Escape, he described how inability to eliminate the tumor during its progression is due to different acquired resistance mechanisms. One of these mechanisms is PD-L1 expression on the surface of many tumors, which when it ligates its receptor PD-1 on T cells plays an important immunosuppressive role. The PD-1/PD-L1 pathway seems to be reactivated also in melanoma cells resistant to BRAF inhibitor (BRAFi) in which PD-L1 expression is induced by the reactivation of the c-Jun/STAT3 signal pathway. Since microRNAs (miRNA) are powerful post-transcriptional regulators of gene expression and play a key role in cancer, the group led by Gennaro Ciliberto (Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale,” Naples, Italy) investigated their possible involvement in the mechanisms responsible for the establishment of drug resistance. In particular, they focused their attention on a potential regulator of melanoma progression, miR-579-3p. High expression level of this miRNA correlates with a better prognosis of melanoma patients, while low expression levels were found in melanoma cells resistant to the combination of BRAFi and MEK inhibitors (MEKi), supporting the role of miR-579-3p in the establishment of drug resistance. Indeed, forced expression of miR-579-3p impairs the establishment of resistance to BRAFi. This miRNA acts by targeting both BRAF and MDM2 oncogenes. Based on these results, they broadened the study to the whole miRNA profile in two different BRAF-mutated melanoma cell lines; the bioinformatic analysis revealed a network of deregulated miRNAs implicated in different intracellular pathways with pro-inflammatory and pro-angiogenetic pathways most frequently affected. Drug resistance in melanoma can be affected by these deregulated miRNAs in a manner not yet clear. Another aspect that could be useful for the development of new treatment modalities also for non-responder patients is the study of the relationship existing between the genotype and the immunophenotype. To this end, Zlatko Trajanoski (Medical University of Innsbruck, Innsbruck, Austria) stratified 19 solid cancers, from the Cancer Genome Atlas (TCGA) database, into three different groups based on their mutational load. They observed common mutations for the high mutational load group suggesting that genomic data can indicate a shared treatment regardless of the tumor histotype. Studying the immune infiltrate composition in each group, they observed in the high mutational load group a prominent presence of effector cells but a depletion of MDSC and regulatory T (Treg) cells. To better define the tumor immunogenicity, they developed an “immunephenoscore” based on major immune parameters such as MHC molecules or the presence of specific effector cells. The response to therapy was well predicted through the immunephenoscore in two validation cohorts of patients treated with antibodies against CTLA-4 and PD-1, respectively. These findings could facilitate the use of these treatments in tumors such as hepatocellular carcinoma where immunotherapy approaches are still lacking. Luigi Buonaguro (Molecular Biology And Viral Oncogenesis Unit, Istituto Nazionale dei Tumori “Pascale,” Naples, Italy) introduced a first-in-man trial entitled HepaVac-101 that was designed to investigate the effects of a multipeptide-based HCC vaccine (16 peptides restricted to HLA-A*02; HLA-A*24 and HLA class II) plus the CV8102 adjuvant (RNA adjuvant^®) in early and intermediate stage of HCC. The main endpoints of the HepaVac-101 vaccine trail are tolerability, safety and immunogenicity. However, several immunological parameters will be investigated in these patients to understand the immunological characteristics of HCC in order to improve immunotherapeutic efficacy. The session was closed by Alberto Mantovani (Humanitas Clinical and Research Center, Humanitas University, Milan, Italy) who demonstrated how not only genetics plays a crucial role in carcinogenesis but also inflammation and the microenvironment which influence its development. In particular, microenvironmental signals guide the polarization of macrophages toward M1, M2 and tumor-associated macrophages (TAM), which represent prototypic M2 cells. Emerging studies suggest a tight link between the genetic events responsible for tumor development and tumor inflammation. Moreover, preclinical and pivotal clinical studies identify TAM as optimal targets in human cancer.

Session 2: tumor microenvironment

The tumor microenvironment is composed of tissue-infiltrating immune and non-immune stromal cell populations. Although different immune effector cells are recruited to the tumor site, their anti-tumor functions may be down-modulated in response to tumor-derived signals. As shown by Wolf H. Fridman (Cordeliers Research Centre Université Paris Descartes, Paris, France), the microenvironmental signatures and the immune contexture could predict patients’ OS. In particular, Fridman’s group developed a “Microenvironment cell populations-counter” (MCP-counter) measuring simultaneously the abundance of 10 microenvironment cellular populations (8 immune populations, plus endothelial cells and fibroblasts) from transcriptomic profiles of human tissues. This method allows the stratification of tumor samples based on the composition of their immune and stromal microenvironments. In clear-cell renal cell carcinoma (ccRCC), the MCP-counter identified 1 out of 4 subgroups exhibiting a strong T lymphocyte infiltration, which expressed checkpoint inhibitor molecules associated with tumors expressing PD-L1 or PD-L2. The use of the MCP-counter might be relevant in a clinical setting to predict patients’ prognosis or response to therapy. Barbara Seliger (Institute for Medical Immunology, Martin Luther University, Halle-Wittenberg, Germany) identified miRNAs regulating HLA-G and components of MHC class I antigen-processing machinery. In addition, RNA-binding molecules were identified demonstrating that the heterogeneous nuclear ribonucleoprotein (HNRNP) R is an important regulator of both classical and non-classical MHC class I molecules. The Seliger group demonstrated that HNRNPR binds MHC class I molecules, enhancing their stability and expression. The positive regulation of MHC class I molecules by HNRNPR supports its use as therapeutic agent in the treatment of cancer. The tumor microenvironment also influenced inter- and intra-clonal phenotypic changes in chronic lymphocytic leukemia (CLL) as presented by Claudio Tripodo (Tumor Immunology Unit, University of Palermo, Palermo, Italy). Tripodo showed how activation of the immune response in myeloid malignancies was accompanied by a decreased Treg cell frequency and increased T cell activation. In particular, he focused on the prototypical inflammatory axis IL-23/IL-23 receptor (IL-23R) that has an emerging role in inflammation-related cancer. The study, conducted on a proportion of CLL patients, showed that a higher expression of the IL-23R chain significantly correlated with time to disease progression and that its expression, in lymph node infiltrates, is accompanied by up-modulation of IL12rB1. These findings suggested that IL23R expression could be a valuable marker for risk stratification in early-stage CLL patients. Mario Mandalà (Department of Oncology and Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy) showed how β-catenin expression and lack of tumor-infiltrating lymphocytes could predict resistance in melanoma patients treated with MAPK inhibitors (MAPKi) and correlated with prognosis. Mandalà’s group reported that, in MAPKi-treated melanoma patients, cytoplasmic expression of β-catenin was inversely correlated with tumor-infiltrating mononuclear cells and that low expression of this molecule, in tumor cells, was associated with longer OS. Moreover, they found increased progression-free survival (PFS) and OS in patients with high levels of tumor-infiltrating CD8 T cells. Finally, Antonio Sica (Department of Pharmaceutical Sciences, University of Piemonte Orientale “A. Avogadro,” Novara, Italy) focused on the association between cell metabolism and immunosuppression induced by nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. In particular, NAMPT inhibitors could restore anti-tumor activity by suppressing tumor growth in tumor-bearing mice. Moreover, NAMPT altered the activity of myeloid-derived suppressor cells, within peripheral tissues, by inhibiting C-X-C chemokine receptor type 4 (CXCR4) expression and induced tumor growth-promoting M2-polarization in tumor-associated myeloid cells.

Session 3: mesothelioma immune-biotherapy

In the complex therapeutic scenario of malignant mesothelioma (MM), an asbestos-related cancer that develops in the membrane lining of the lungs and abdomen, a complete overview on the efficacy of the current approved therapies for its treatment was given by Giovanni Luca Ceresoli (Medical Oncology, Humanitas Gavazzeni, Bergamo, Italy). At present, the combination of Pemetrexed with platinum compounds is the only therapy approved as first-line treatment. There are no standard treatments for the second-line setting and this underlines the need for looking at more effective therapies for the treatment of MM patients. Dr. Ceresoli hypothesized that the main drivers for clinical research could be: (i) inhibition of angiogenesis, considering that MM patients present high circulating levels of vascular endothelial growth factor (VEGF), which is a negative prognostic factor; (ii) targeting mesothelin, that is a cell surface tumor differentiation antigen highly expressed in solid tumors; (iii) immunotherapy with the use of immune checkpoint inhibitors alone or in combination with other agents. Treatment of MM patients with immunotherapy was also the focus of other speakers. In particular, Luana Calabrò (Medical Oncology and Immunotherapy, University Hospital of Siena, Siena, Italy), stressed the idea that the use of mAbs targeting immune checkpoints in MM patients could be a very attractive strategy to increase anti-tumor immune response due to the very strong immunosuppressive MM microenvironment. Confirming this hypothesis Dr. Calabrò presented the results of two studies (NCT01649024 and NCT01655888) in which MM patients were treated with different schedules of the anti-CTLA-4 mAb Tremelimumab. Results of these studies demonstrated that the increase in the absolute number of circulating CD4 + ICOS + T lymphocytes in the very early phases of treatment is predictive of an improved survival of enrolled MM patients. A correlation of the soluble ligands of the natural killer group 2 member D (NKG2D) receptor (i.e., MIC-B, ULBP-2 and CD25) with the OS of these patients, as described by Carla Chiarucci (Medical Oncology and Immunotherapy, University Hospital of Siena, Siena, Italy), was also demonstrated. Moreover, a trend toward longer survival was observed in MM patients with T cell reactivity to allogeneic HLA-matched MM-cell lines: 29 versus 10.3 months in 3 MM patients at baseline, (p = 0.07) and 15.4 versus 8.8 months in 5 MM patients post-treatment (p = 0.10). TAA-specific T cell responses were constitutively detectable, up-regulated and induced in 11, 8 and 11 MM patients, respectively, by the treatment. These intriguing clinical results and the emerging efficacy of immunomodulatory mAbs targeting other immune checkpoints such as PD-1/PD-L1 in different tumor types, prompted the design of the NIBIT-MESO-1 study (NCT02588131) aimed at investigating the efficacy of Tremelimumab combined with the anti-PD-L1 Durvalumab in MM patients. Results of this study, presented by Dr. Calabrò, although preliminary, are extremely encouraging demonstrating an 80% immune-related disease control rate (DCR) at week 12 for the first 10 patients enrolled. Despite intriguing results obtained with mAbs targeting immune checkpoints, only a small proportion of patients benefit from such therapeutic approaches; therefore, several strategies can be explored to improve the effectiveness of cancer immunotherapy. One of these could be the transformation of cold tumors (without T cell infiltration) into hot tumors (with T cell infiltration). Joachim Aerts (Department of Pulmonary Medicine, Erasmus MC Cancer Institute, Rotterdam, The Netherlands), proposed three different approaches to perform this transformation: decreasing immune suppression, increasing immune cell delivery as well as immune activation by combining different treatments. Demonstrating the role of TAMs as key players in MM pathogenesis, Dr. Aerts utilized PLX3397, a macrophage-CSF receptor tyrosine kinase inhibitor, to deplete TAMs in a murine model. However, no increase in survival or in the numbers of proliferating CD8⁺ T cells was observed after treatment. Noteworthy, when DC pulsed with autologous tumor lysate were added to PLX3397 monotherapy, 100% survival of the mice was achieved. Based on these results and on data generated in a phase I study with allogeneic lysate-based DC therapy against MM, they have already planned a phase III registration trial in which patients after chemotherapy will be treated with modified DC to hopefully increase the response rate of MM patients after the first line of treatment.

Session 4: immune checkpoints

The immune system continuously interacts with cancer cells, playing a fundamental role in attempting to reject the tumor. However, the induction of protective anti-tumor immunity is often compromised by innate immunosuppressive mechanisms that may dominate the tumor microenvironment. CD4 + Treg cells are overall master regulators of the immune system and have a pivotal role in the establishment of the immunosuppressive tumor microenvironment. In general, tumors have an altered balance with high proportions of Treg cells and low proportions of T effector (Teff) cells. It has been reported, in a mouse melanoma model, that CTLA-4-blockade can alter this balance, improving the number of Teff cells, ultimately promoting the rejection of tumors by depletion of Treg cells via antibody-dependent cell-mediated cytotoxicity (ADCC) in an Fc-Gamma receptor-dependent manner. To this end, Sergio Quezada (Research Department of Haematology, UCL Cancer Institute, London, UK), demonstrated that preferential Treg cell depletion depends mostly on the differential expression of the CTLA-4 molecule on the surface of the target (higher on Treg cells as compared to Teff cells). He also demonstrated the possibility of developing a next generation of immune modulatory antibodies, engineering their Fc receptors, with an enhanced ADCC activity. The final anti-tumor response depends on the presence of neoantigens, particularly clonal neoantigens that induce effective T cell responses. In this context, the presence of subclonal neoantigens is not protective. The association between a more favorable Teff/Treg cells ratio and a better anti-tumor response in melanoma and prostate adenocarcinoma mouse models was also discussed by Angela Rita Elia (Cellular Immunology Unit, San Raffaele Scientific Institute, Milan, Italy). To improve tumor immunity, she modified tumor permeability targeting endothelial cancer cells by NGR-TNF alpha. The co-administration of NGR-TNF alpha and anti-PD-1 or anti-CTLA-4 mAbs, increasing the presence of Granzyme B +/interferon-γ+ , CD8 + and CD4 + effector T cells within the tumor, could be combined with adoptive cell therapy (ACT) to improve clinical responses in cancer patients. Tumor-infiltrating T regulatory (TI-Treg) cells, acquiring specific features of the tissue in which they reside, have a different phenotype from those present in normal tissues, as described by Massimiliano Pagani (National Institute of Molecular Genetics “Romeo and Enrica Invernizzi,” Milan, Italy). The isolation and characterization of tumor-resident Treg cells in colorectal cancer (CRC) and in non-small-cell lung cancer (NSCLC) demonstrated that TI-Treg cells are characterized by specific signature genes, such as Layilin (LAYN), MAGE Family Member H1 (MAGE-H1), or Chemokine C–C motif receptor 8 (CCR8) expression. Dr. Pagani demonstrated that the high expression of the three genes correlated, in all cases analyzed, with significantly reduced survival in both types of cancer, i.e., CRC and NSCLC. Giorgio V. Scagliotti (Department of Oncology, University of Turin, Turin, Italy), presented a series of potential mechanism(s) for immune evasion in lung cancer: defective antigen presentation, upregulation/secretion of immunosuppressive cytokines, infiltrates of MDSC and Treg cells. In addition, he showed results obtained from different clinical studies in NSCLC involving patients treated with anti-CTLA-4 (ipilimumab), anti-PD-1 (Nivolumab and Pembrolizumab) and anti-PD-L1 (Atezolizumab) mAbs focusing his attention on the still unclear role of PD-L1 expression as a predictive biomarker. He suggested the need to create a “cancer immunogram” already proposed by Blank (Blank C.U. et al., Science, 2016) that can take into account several types of information regarding cancer and immune system crosstalk, trying to identify patients who can benefit from immunotherapeutic approaches. An overview of the clinical results obtained with anti-CTLA-4, anti-PD-1 or the combination of these two mAbs in melanoma patients was clearly described by Steven J. O’Day (Immuno-Oncology & Clinical Research, John Wayne Cancer Institute, Santa Monica, USA). He highlighted the different OS rate and the time required to reach a survival plateau comparing single agent therapy (20% for anti-CTLA-4 mAb in 2 years, and 40% with anti-PD1 mAb in 3–4 years) to the combination (70% in 12 months). Finally, Carmelo Carlo-Stella (Hematology and Cancer Experimental Therapeutics at Humanitas Research Hospital, Milan, Italy) presented the results of a phase I study (CA209-039) in which classical Hodgkin lymphoma (HL) patients were treated with Nivolumab. The overall response rate (ORR) was 87% in patients with a median duration of response and a median PFS after two years not yet reached. These results are likely related to the genetic dependence of HL on the PD-1/PD-L1 pathway through genetic amplification of chromosome 9p24. Analyses performed combining molecular and protein data confirmed that the majority of HL patients presented this genetic amplification, which is also associated with PD-L1/PD-L2 overexpression. Dr Carlo-Stella also highlighted the importance of TAMs in the pathogenesis of HL and suggested the use of RP6530, a phosphatidylinositol 3-kinase delta/gamma inhibitor, reducing M2 macrophages polarization and inhibiting tumor angiogenesis, as a new strategy for the clinical treatment of HL patients.

Session 5: how immunotherapy shapes company pipelines

In this session of the meeting, representatives of the most important pharma companies involved in developing immunotherapy compounds presented an update of their pipelines. Starting from data on consolidated mAb immunotherapy, Cosimo Paga (Oncology & Immunology, Bristol-Myers Squibb, Rome, Italy) presented the results from different clinical trials testing checkpoint blocking mAbs (Nivolumab and Ipilimumab) in combination with conventional chemotherapy in different tumor types. Dr. Paga highlighted that tumors use complex, overlapping mechanisms to evade and suppress the immune system and stressed the need to look beyond T cells, by investigating the role of other immune cells (NK cells, macrophages) and of agents targeting the tumor microenvironment, including stromal elements and extracellular matrix to improve the efficacy of cancer therapeutic approaches. Aiman Shalabi (Immuno-Oncology Global Medical Affairs, AstraZeneca/Medimmune, London, UK) presented the four key platforms in their Oncology portfolio: 1. Tumor drivers and resistance; 2. DNA damage response; 3. Immuno-oncology and 4. Antibody conjugates, focusing on the power of combination therapies as the cornerstone of their strategy. The combination therapy they have advanced farthest in the pipeline involves Durvalumab, an anti-PD-L1 mAb and Tremelimumab, an anti-CTLA4 mAb that is under investigation across a broad range of treatment settings in NSCLC. A new and interesting target for tumor therapy in combination with immunotherapy is represented by IDO-1. Michael Lahn (Clinical Development, Incyte, Geneva, Switzerland) presented the latest research on the IDO-1 inhibitor Epacadostat in combination with anti-PD-1/PD-L1 mAbs in multiple tumor types, including melanoma, NSCLC, head & neck, colorectal, ovarian and pancreatic carcinoma. IDO-1 is a tryptophan-catabolizing enzyme that is over-expressed in many cancers and induces immune tolerance by suppressing T cell responses. Results presented by Dr. Lahn showed that IDO-1 inhibition leads to increased numbers of TILs with enhanced secretion of IFNγ and decreased Treg cells in tumors. Responses observed in the ECHO-202 Study combining Epacadostat + anti-PD-1 strongly supported advancing to a broad phase III program. Aiming to move toward precision medicine approaches customizing healthcare on individual patients, Zhen Su (Oncology Medical, Merck KGaA, Darmstadt, Germany) stressed the necessity to identify unique and useful biomarkers as well as the need to activate the right effector cells after immunotherapy with different mAbs. Mechanisms of resistance to therapy and management of non-responder patients were also discussed during the presentation. The analysis of the expression of PD-L1 in the tumor microenvironment has been recently indicated as a critical point in immunotherapy as presented by William Grossmann (GI & Cancer Immunotherapy Combinations, Genentech, South San Francisco, CA, USA). The modulation of PD-L1 expression by IFNγ can occur in tumor cells and immune cells in the tumor microenvironment, leading to the inhibition of anti-tumor T cell responses. Differential PD-L1 expression observed between tumor and immune cells in patients is associated with distinct biological features and clinical outcomes. Preliminary data on IMvigor 210, a pivotal, single-arm study of the anti-PD-L1 mAb, TECENTRIQ, in metastatic urothelial carcinoma (mUC), were presented. The mean OS in all patients was 14.8 months with a median follow-up of 14.4 months. Results from the phase III OAK study for locally advanced or metastatic NSCLC were also presented. Novel technology platforms like cell and gene therapy and recombinant proteins were presented by Claudio Bordignon (Chairman, MolMed, Milan, Italy). The two novel proprietary investigational treatments are represented by Zalmoxis, a cell-based therapy enabling bone marrow transplants from partially compatible donors in the absence of post-transplant immune suppression currently in phase III clinical trial for the treatment of high-risk acute leukemia patients, and CAR-CD44v6, an immune-gene therapy project potentially effective for many hematological malignancies and several epithelial tumors, currently under preclinical development. CAR-CD44v6 is specific for the CD44v6 antigen, which is expressed by hematological tumors and by several solid tumors of different histotypes. An exciting challenge in immunotherapy is represented by the combination of epigenetic drugs with immune checkpoints mAbs as presented by Pietro Taverna (Translational Pharmacology, Astex Pharmaceuticals, Pleasanton, CA, USA). Astex is exploring guadecitabine, a second-generation DNA Hypomethylating Agent (DHA) formed by a dinucleotide of decitabine and deoxyguanosine to improve in vivo exposure/efficacy. Dose-dependent long interspersed nuclear elements-1 (LINE-1) demethylation profiles in phase I patients with hematological malignancies allowed the definition of a biologically effective dose (BED) of guadecitabine (60 mg/m² daily × 5). In addition to the immunomodulatory activity observed in vitro in many cancer cell lines, preclinical in vivo data showed synergy when guadecitabine was combined with anti-CTLA4 mAb. Moreover, clinical data demonstrated that in myelodysplastic syndromes (MDS) and AML patients, guadecitabine achieves a dose-dependent demethylation and induces the expression of different cancer testis antigens (CTAs) and up-regulates or induces de novo the expression of PD-1, PD-L1, PD-L2 and CTLA-4 in AML patients. Philippe Legenne (Oncology Medical, Amgen, Zug, Switzerland) presented the company pipeline aimed at expanding immuno-oncology platforms beyond immune checkpoints. They are focusing on the glucocorticoid-induced TNFR-related protein (GITR), a key stimulatory protein involved in the priming and activation of T effector cell responses against cancer-specific antigens. Preclinical evidence has demonstrated that signaling through GITR can enhance activation of T effector cells and can abrogate the activity of Treg cell-mediated suppression during immunotherapy and support effective anti-tumor immune responses. Dr. Legenne also presented data on oncolytic virus therapy based on Herpes simplex type 1 virus (HSV-1) that can be used for immunotherapy. These viruses have the capacity to lyse tumor cells, which can be seen when certain key genes are mutated and also have the ability to amplify anti-tumor immune responses. Data on the T-VEC clinical trial were also presented. T-VEC is an oncolytic HSV-1 strain engineered to replicate in tumor cells and to express human granulocyte–macrophage colony-stimulating factor (hGM-CSF). T-VEC replication results in oncolysis of the infected tumor cells and the release of more T-VEC, which goes on to infect neighboring tumor cells, thereby repeating the cycle. Oncolysis together with the production of GM-CSF by T-VEC, induces the recruitment and maturation of dendritic cells, which then process and present the tumor antigens to T cells in the lymph nodes, inducing the activation and expansion of CD8 + T cells and the initiation of a systemic anti-tumor immune response that results in the death of distant tumor cells through the cytotoxic effects of the activated CD8 + T cells. Finally, Ramy Ibrahim (Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA) presented the Parker Institute for Cancer Immunotherapy as a new model fostering the collaboration between academic centers. He also gave an update on the ongoing clinical studies currently being conducted at different medical centers coordinated by the Parker Institute: (i) the combination therapy of Ipilimumab + Nivolumab versus Ipilimumab in melanoma paying attention to a centralized biomarkers analysis; (ii) the CRISPR study, an exciting first-in-human protocol attempting to maintain adoptively transferred T cell function through simultaneously deleting the endogenous T cell receptor and PD-1 genes using a CRISPR-Cas9 gene editing approach performed in 3 centers on melanoma, myeloma and sarcoma patients; (iii) the Pancreatic cancer project investigating the use of CD40 ± PD-1 in combination with the standard of care performed in 6 centers. A Keynote Lecture by Dr. Soldano Ferrone (Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA) closed this session. Dr. Ferrone presented the defects of the HLA class I antigen-processing machinery in checkpoint inhibitor-based therapy of cancer. HLA class I components play a key role in the response to checkpoint inhibitor-based immunotherapy, since they present immunogenic peptides to cognate T cells. Defects in HLA class I components, caused by structural or functional abnormalities, are likely to cause resistance to this type of immunotherapy, because of defective presentation of immunogenic peptides to cognate T cells.

Session 6: cell and gene therapy of cancer

This session of the meeting focused on various applications of cell and gene therapy to the treatment of different cancer types. Antonio Rosato (Department of Surgery Oncology and Gastroenterology, University of Padua, Padua, Italy) showed that cytokine-induced killer (CIK) cells, a population of ex vivo expanded T lymphocytes with functional characteristics similar to NK and T cells, are highly suitable for ACT approaches, capable of recognizing target tumor cells without the need for antigen-specific priming. CIK cells express CD16 and are able to kill tumor cells also through ADCC. Their activity can be enhanced by the co-administration of different mAbs (like trastuzumab or cetuximab). In fact, mice injected intraperitoneally with ovarian tumor cells and treated with the combined therapy (CIK + trastuzumab or + cetuximab) demonstrated a delay in tumor growth and an increase in survival. The adoptive transfer of T cells recognizing leukemia-associated lipid antigen is an interesting therapeutic approach, as discussed by Giulia Casorati (Experimental Immunology, San Raffaele Scientific Institute, Milan, Italy). Data showed that a group of CD1c-restricted T cells play an important role in leukemia immune surveillance. These T cells recognize methyl-lysophosphatidic acid (mLPA), a lipid antigen produced by leukemia cells that represents a new target of the immune response. Dr. Casorati showed that mLPA-specific T cells are able to kill CD1c⁺ leukemia blasts and presented data on their therapeutic efficacy in xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) and AML. In this scenario, oncolytic virotherapy (OV) is perhaps the next major breakthrough in cancer treatment following the success of immunotherapy using immune checkpoint inhibitors. Oncolytic viruses are genetically engineered or naturally occurring viruses that kill cancer cells but not normal tissues. John Hiscott (Immunotherapy of Cancer & Infectious Diseases, Istituto Pasteur-Italia, Rome, Italy) in his talk stated that OV represents a novel approach for cancer treatment, that can be improved by the combination with other therapies. Therefore, the combination of chemotherapeutic compounds and suicide gene strategies represents another important approach to improve the OV efficacy against breast cancer, melanoma and B-lymphoma. Marinos Kallikourdis (Adaptive Immunity Lab, Humanitas University, Milan, Italy) showed that tumors promote the recruitment of pro-tumoral cell populations through the secretion of chemokines. The modification of receptors that match these chemokines improved the migration of ACT T cells into lymph node metastases in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Due to the increased targeting efficacy of the ACT, the modified CD8 + T cells demonstrated a notable in vivo protective effect, as shown by the reduction of tumor growth in the E.G7-OVA model (mouse thymoma EL4 cells stably transfected with the complementary DNA of chicken OVA). Surprisingly, the formation of some of the extracellular matrix structures that impede T cell access to tumor was found to be dependent itself on T cell function. Roberto Bernabei (Department of Aging, Neurosciences, Head-Neck and Orthopaedics, Università Cattolica del Sacro Cuore, Rome, Italy) showed that treatment in elderly cancer patients represents a complex process that needs to be enriched considering a multiparametric/multidimensional evaluation. DCs can be targeted precisely through the intravenous injection/administration of RNA-lipoplexes for an efficient antigen presentation, as discussed by Lena Kranz (TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University gGmbH, Mainz, Germany). These RNA-lipoplexes coding neoantigens or endogenous self-antigens induce strong effector and memory T cell responses and mediate IFNα-dependent rejection of the tumor. De novo CD4 and CD8 T cell responses and enhanced immunity against the major CTAs (such as New York-Esophageal cancer-1 (NY-ESO-1) and Melanoma associated antigen 3 (MAGE-A3) were developed in three advanced melanoma patients from a first-in-human phase I clinical trial. This strategy represents a multivalent method for DC targeting and the induction of type-I-IFN-mediated immune responses for immunotherapy treatment. One of the most confirmed cell therapy strategies for the treatment of patients with hematological malignancies is allogeneic hematopoietic stem cell transplantation. Suicide gene transfer into donor T lymphocytes, as discussed by Fabio Ciceri (Unit of Hematology and Bone Marrow Transplantation, IRCSS San Raffaele Scientific Institute, Milan, Italy) allows to strengthen the graft-versus-tumor effect and immune reconstitution, preserving the anti-tumor effect of allogeneic hematopoietic stem cell transplantation. The Herpes simplex virus thymidine kinase (HSV-TK), which activates the prodrug ganciclovir is the suicide gene most studied in humans, as confirmed by phase I-II clinical trials that tested the safety/efficacy of this strategy. In TK007 phase II clinical trial, only patients with TK-cell engraftment progressed to immune reconstitution (IR) while in absence of TK-cells the IR was very slow. This session was closed by Andrea Velardi (Department of Medicine, Hematopoietic Stem Cell Transplantation Program, University of Perugia, Perugia, Italy) who gave an overview of alloreactive NK cells for haploidentical hematopoietic transplantation in adult AML patients. NK cells alloreactivity mediated by donor NK cells is an important therapeutic tool in HLA haplotype mismatched hematopoietic transplantation, associated with better outcome and protecting the host from graft-versus-host disease. Evaluation of donor NK cell is one of the most important criteria for donor selection determined by Killer cell Ig-like receptor genotyping. Moreover, transplantation from NK alloreactive donors with concomitant activating killer cell Ig-like receptors reduced non-relapse and infectious mortalities and improved survival.

Session 7: ongoing and prospective NIBIT activities and collaborations

Given the high number of preclinical and clinical research projects dealing with innovative approaches in immunotherapy of cancer, and considering the increasing number of institutions and professional people involved, in 2004 a collaborative network, the Italian Network for Tumor Biotherapy (NIBIT) was set up with the aims of coordinating and integrating the different Italian research groups interested in the biotherapy of tumors. In 2012, supported by the NIBIT Board of Directors, the NIBIT Foundation was established to best focus the comprehensive activities connected with the conception, planning and implementation of clinical trials. Anna Maria Di Giacomo (University Hospital Siena, Siena, Italy) presented the different clinical trials designed and conducted by NIBIT and the NIBIT Foundation. The first study was NIBIT-M1, an open-label, single-arm phase II trial aimed to investigate the efficacy of the combination of an immune checkpoint inhibitor, the anti-CTLA-4 mAb ipilimumab, with fotemustine in patients with unresectable locally or metastatic melanoma with or without asymptomatic brain metastases (BM). Though limited by the number of patients enrolled, disease control in the brain was long lasting achieving a 3-year survival rate of 28%. Based on these results, the NIBIT-M2 study was designed to further explore the efficacy of immunotherapy with checkpoint blocking mAb in metastatic melanoma patients with BM. Despite demonstrated success, only a minority of cancer patients responds to a single checkpoint inhibitor. This evidence has forced researchers to investigate several strategies combining different checkpoint blocking mAbs together or in combination with other immunotherapeutic agents. In this scenario, two premieres, supported by the NIBIT Foundation, in immuno-oncology international research are the NIBIT-MESO-1 and the NIBIT-M4 studies. The first study is a single arm, phase II clinical trial testing the anti-CTLA-4 mAb Tremelimumab in combination with the anti-PD-L1 mAb Durvalumab in patients with unresectable MM. Moreover, based on the preclinical evidence of the broad immunomodulatory activity of the second-generation DHA guadecitabine, the exploratory phase I/II combination study NIBIT-M4 has been designed to provide proof-of-concept of the immunological and clinical efficacy of CTLA-4 blockade combined with DHA in stage III or IV metastatic melanoma patients, amenable to serial tumor biopsies. In the same session of the meeting, a new clinical trial (A-Brave) coordinated, by Istituto Oncologico Veneto was presented by Maria Vittoria Dieci (Istituto Oncologico Veneto, IRCCS, Padua, Italy). A-Brave is a phase III randomized trial that compares an adjuvant or neoadjuvant treatment with the anti-PD-L1 antibody, Avenumab, in high-risk triple negative breast cancer patients. This study is currently recruiting participants. In spite of the success of innovative clinical studies of cancer immuno-biotherapy, still a lot remains to be understood about the identification of the mechanisms of response and resistance in treated patients as well as on predictive and prognostic biomarkers that will allow the selection of the best candidates for this therapeutic approach. In this regard, translational studies are of considerable importance: in particular the analysis at the tumor site becomes fundamental as suggested by Andrea Anichini (Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy). The meeting ended with the Meet-the-editor session that provided the opportunity to discuss all aspects regarding scientific publications with the Co-Editor in Chief of Cancer Immunology Immunotherapy, Graham Pawelec (University of Tübingen Medical School, Tübingen, Germany).

Conclusions

The NIBIT meeting, in the awareness that the key to further success is fostering and strengthening scientific collaborations and exchanging of ideas, presented a great opportunity for young scientists and leaders in immuno-oncology to meet and discuss emerging clinical and preclinical data arising in the field. The meeting provided the attendees with a broad overview of the most recent achievements in cancer bio-immunotherapy.

Abbreviations

ACT

Adoptive cell therapy

ADCC

Antibody-dependent cell-mediated cytotoxicity

AML

Acute myeloid leukemia

BED

Biologically effective dose

BM

Brain metastases

BRAFi

BRAF inhibitor

ccRCC

Clear-cell renal cell carcinoma

CIK

Cytokine-induced killer

CLL

Chronic lymphocytic leukemia

CRC

Colorectal cancer

CTA

Cancer testis antigen

DCR

Disease control rate

GITR

Glucocorticoid-induced TNFR-related protein

HCC

Hepatocellular carcinoma

HL

Hodgkin lymphoma

HNRNP

Heterogeneous nuclear ribonucleoprotein

HSV-TK

Herpes simplex virus thymidine kinase

HSV-1

Herpes simplex type 1 virus

IL23R

IL-23 receptor

MAPKi

MAPK inhibitor

MCP-counter

Microenvironment cell populations-counter

MDS

Myelodysplastic syndromes

MEKi

MEK inhibitors

miRNA

MicroRNA

mLPA

Methyl-lysophosphatidic acid

MM

Malignant mesothelioma

mUC

Metastatic urothelial carcinoma

NAMPT

Nicotinamide phosphoribosyltransferase

NSCLC

Non-small-cell lung cancer

ORR

Overall response rate

OV

Oncolytic virotherapy

PFS

Progression free survival

T-ALL

T cell acute lymphoblastic leukemia

TAM

Tumor-associated macrophage

Teff

T effector

TI-Treg

Tumor infiltrating regulatory T

TRAMP

Transgenic adenocarcinoma of the mouse prostate

Treg

Regulatory T

VEGF

Vascular endothelial growth factor

Author contributions

Vincenzo Russo, Carla Chiarucci, Maria Fortunata Lofiego, Carolina Fazio, Erica Bertocci, Ornella Cutaia, Gianluca Giacobini, Andrea Lazzeri, Antonello Lamboglia contributed to writing the summary of the different meeting sessions; Maresa Altomonte, Patrizia Tunici, Alessia Covre and Michele Maio supervised and critically contributed to the final revision of the manuscript.

Compliance with ethical standards

Conflict of interest

Michele Maio is a consultant/advisory board member for Bristol-Meyers Squibb, Incyte, MSD Oncology, Roche, Astex Pharmaceuticals, Amgen, AstraZeneca and Merck Serono. No potential conflicts of interest were disclosed by the other authors.