A novel complement-fixing IgM antibody targeting GPC1 as a useful immunotherapeutic strategy for the treatment of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a very low survival rate at 5 years. The use of chemotherapeutic agents results in only modest prolongation of survival and is generally associated with the occurrence of toxicity effects. Antibody-based immunotherapy has been proposed for the treatment of PDAC, but its efficacy has so far proved limited. The proteoglycan glypican-1 (GPC1) may be a useful immunotherapeutic target because it is highly expressed on the surface of PDAC cells, whereas it is not expressed or is expressed at very low levels in benign neoplastic lesions, chronic pancreatitis, and normal adult tissues. Here, we developed and characterized a specific mouse IgM antibody (AT101) targeting GPC1. METHODS: We developed a mouse monoclonal antibody of the IgM class directed against an epitope of GPC1 in close proximity to the cell membrane. For this purpose, a 46 amino acid long peptide of the C-terminal region was used to immunize mice by an in-vivo electroporation protocol followed by serum titer and hybridoma formation. RESULTS: The ability of AT101 to bind the GPC1 protein was demonstrated by ELISA, and by flow cytometry and immunofluorescence analysis in the GPC1-expressing "PDAC-like" BXPC3 cell line. In-vivo experiments in the BXPC3 xenograft model showed that AT101 was able to bind GPC1 on the cell surface and accumulate in the BXPC3 tumor masses. Ex-vivo analyses of BXPC3 tumor masses showed that AT101 was able to recruit immunological effectors (complement system components, NK cells, macrophages) to the tumor site and damage PDAC tumor tissue. In-vivo treatment with AT101 reduced tumor growth and prolonged survival of mice with BXPC3 tumor (p < 0.0001). CONCLUSIONS: These results indicate that AT101, an IgM specific for an epitope of GPC1 close to PDAC cell surface, is a promising immunotherapeutic agent for GPC1-expressing PDAC, being able to selectively activate the complement system and recruit effector cells in the tumor microenvironment, thus allowing to reduce tumor mass growth and improve survival in treated mice.

Novel Nanotechnology Approaches to Overcome Drug Resistance in the Treatment of Hepatocellular Carcinoma: Glypican 3 as a Useful Target for Innovative Therapies.

Hepatocellular carcinoma (HCC) is the second most lethal tumor, with a 5-year survival rate of 18%. Early stage HCC is potentially treatable by therapies with curative intent, whereas chemoembolization/radioembolization and systemic therapies are the only therapeutic options for intermediate or advanced HCC. Drug resistance is a critical obstacle in the treatment of HCC that could be overcome by the use of targeted nanoparticle-based therapies directed towards specific tumor-associated antigens (TAAs) to improve drug delivery. Glypican 3 (GPC3) is a member of the glypican family, heparan sulfate proteoglycans bound to the cell surface via a glycosylphosphatidylinositol anchor. The high levels of GPC3 detected in HCC and the absence or very low levels in normal and non-malignant liver make GPC3 a promising TAA candidate for targeted nanoparticle-based therapies. The use of nanoparticles conjugated with anti-GPC3 agents may improve drug delivery, leading to a reduction in severe side effects caused by chemotherapy and increased drug release at the tumor site. In this review, we describe the main clinical features of HCC and the common treatment approaches. We propose the proteoglycan GPC3 as a useful TAA for targeted therapies. Finally, we describe nanotechnology approaches for anti-GPC3 drug delivery systems based on NPs for HCC treatment.

The Proteoglycan Glypican-1 as a Possible Candidate for Innovative Targeted Therapeutic Strategies for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all pancreatic cancers, with a 5-year survival rate of 7% and 80% of patients diagnosed with advanced or metastatic malignancies. Despite recent advances in diagnostic testing, surgical techniques, and systemic therapies, there remain limited options for the effective treatment of PDAC. There is an urgent need to develop targeted therapies that are able to differentiate between cancerous and non-cancerous cells to reduce side effects and better inhibit tumor growth. Antibody-targeted strategies are a potentially effective option for introducing innovative therapies. Antibody-based immunotherapies and antibody-conjugated nanoparticle-based targeted therapies with antibodies targeting specific tumor-associated antigens (TAA) can be proposed. In this context, glypican-1 (GPC1), which is highly expressed in PDAC and not expressed or expressed at very low levels in non-malignant lesions and healthy pancreatic tissues, is a useful TAA that can be achieved by a specific antibody-based immunotherapy and antibody-conjugated nanoparticle-based targeted therapy. In this review, we describe the main clinical features of PDAC. We propose the proteoglycan GPC1 as a useful TAA for PDAC-targeted therapies. We also provide a digression on the main developed approaches of antibody-based immunotherapy and antibody-conjugated nanoparticle-based targeted therapy, which can be used to target GPC1.

A Novel Epigenetic Machine Learning Model to Define Risk of Progression for Hepatocellular Carcinoma Patients.

Although extensive advancements have been made in treatment against hepatocellular carcinoma (HCC), the prognosis of HCC patients remains unsatisfied. It is now clearly established that extensive epigenetic changes act as a driver in human tumors. This study exploits HCC epigenetic deregulation to define a novel prognostic model for monitoring the progression of HCC. We analyzed the genome-wide DNA methylation profile of 374 primary tumor specimens using the Illumina 450 K array data from The Cancer Genome Atlas. We initially used a novel combination of Machine Learning algorithms (Recursive Features Selection, Boruta) to capture early tumor progression features. The subsets of probes obtained were used to train and validate Random Forest models to predict a Progression Free Survival greater or less than 6 months. The model based on 34 epigenetic probes showed the best performance, scoring 0.80 accuracy and 0.51 Matthews Correlation Coefficient on testset. Then, we generated and validated a progression signature based on 4 methylation probes capable of stratifying HCC patients at high and low risk of progression. Survival analysis showed that high risk patients are characterized by a poorer progression free survival compared to low risk patients. Moreover, decision curve analysis confirmed the strength of this predictive tool over conventional clinical parameters. Functional enrichment analysis highlighted that high risk patients differentiated themselves by the upregulation of proliferative pathways. Ultimately, we propose the oncogenic MCM2 gene as a methylation-driven gene of which the representative epigenetic markers could serve both as predictive and prognostic markers. Briefly, our work provides several potential HCC progression epigenetic biomarkers as well as a new signature that may enhance patients surveillance and advances in personalized treatment.

Targeted chitosan nanobubbles as a strategy to down-regulate microRNA-17 into B-cell lymphoma models.

Introduction: MicroRNAs represent interesting targets for new therapies because their altered expression influences tumor development and progression. miR-17 is a prototype of onco-miRNA, known to be overexpressed in B-cell non-Hodgkin lymphoma (B-NHL) with peculiar clinic-biological features. AntagomiR molecules have been largely studied to repress the regulatory functions of up-regulated onco-miRNAs, but their clinical use is mainly limited by their rapid degradation, kidney elimination and poor cellular uptake when injected as naked oligonucleotides. Methods: To overcome these problems, we exploited CD20 targeted chitosan nanobubbles (NBs) for a preferential and safe delivery of antagomiR17 to B-NHL cells. Results: Positively charged 400 nm-sized nanobubbles (NBs) represent a stable and effective nanoplatform for antagomiR encapsulation and specific release into B-NHL cells. NBs rapidly accumulated in tumor microenvironment, but only those conjugated with a targeting system (antiCD20 antibodies) were internalized into B-NHL cells, releasing antagomiR17 in the cytoplasm, both in vitro and in vivo. The result is the down-regulation of miR-17 level and the reduction in tumor burden in a human-mouse B-NHL model, without any documented side effects. Discussion: Anti-CD20 targeted NBs investigated in this study showed physico-chemical and stability properties suitable for antagomiR17 delivery in vivo and represent a useful nanoplatform to address B-cell malignancies or other cancers through the modification of their surface with specific targeting antibodies.

Glucose Metabolism and Oxidative Stress in Hepatocellular Carcinoma: Role and Possible Implications in Novel Therapeutic Strategies.

Hepatocellular carcinoma (HCC) metabolism is redirected to glycolysis to enhance the production of metabolic compounds employed by cancer cells to produce proteins, lipids, and nucleotides in order to maintain a high proliferative rate. This mechanism drives towards uncontrolled growth and causes a further increase in reactive oxygen species (ROS), which could lead to cell death. HCC overcomes the problem generated by ROS increase by increasing the antioxidant machinery, in which key mechanisms involve glutathione, nuclear factor erythroid 2-related factor 2 (Nrf2), and hypoxia-inducible transcription factor (HIF-1α). These mechanisms could represent optimal targets for innovative therapies. The tumor microenvironment (TME) exerts a key role in HCC pathogenesis and progression. Various metabolic machineries modulate the activity of immune cells in the TME. The deregulated metabolic activity of tumor cells could impair antitumor response. Lactic acid-lactate, derived from the anaerobic glycolytic rate of tumor cells, as well as adenosine, derived from the catabolism of ATP, have an immunosuppressive activity. Metabolic reprogramming of the TME via targeted therapies could enhance the treatment efficacy of anti-cancer immunotherapy. This review describes the metabolic pathways mainly involved in the HCC pathogenesis and progression. The potential targets for HCC treatment involved in these pathways are also discussed.

Novel immunotherapeutic approaches for hepatocellular carcinoma treatment.

INTRODUCTION: The introduction of immune checkpoint inhibitors has been lately proposed for the treatment of hepatocellular carcinoma (HCC) with respect to other cancer types. Several immunotherapeutic approaches are now under evaluation for HCC treatment including: i) antibodies acting as immune checkpoint inhibitors; ii) antibodies targeting specific tumor-associated antigens; iii) chimeric antigen receptor redirected T (CAR-T) cells targeting specific tumor-associated antigens; iv) vaccination strategies with tumor-specific epitopes. Areas covered: The review provides a wide description of the clinical trials investigating the efficacy of the main immunotherapeutic approaches proposed for the treatment of patients affected by HCC. Expert opinion: The balancing between immunostimulative and immunosuppressive factors in the context of HCC tumor microenvironment results in heterogeneous response rates to immunotherapeutic approaches such as checkpoint inhibitors, among HCC patients. In this context, it becomes crucial the identification of predictive factors determining the treatment response. A multiple approach using different biomarkers could be useful to identify the subgroup of HCC patients responsive to the treatment with a checkpoint inhibitor (as an example, nivolumab) as single agent, and to identify those patients in which other treatment regimens, such as the combination with sorafenib, or with locoregional therapies, could be more effective.

New Insight into Therapies Targeting Angiogenesis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a malignancy characterized by neoangiogenesis that is determined by an augmented production of proangiogenesis factors by tumor and adjacent cells. This unbalanced angiogenesis process is a key feature of HCC carcinogenesis and progression. Proangiogenic factors also have a relevant role in the generation and maintenance of an immunosuppressive tumor microenvironment. Several therapeutic options for HCC treatment are based on the inhibition of angiogenesis, both in the early/intermediate stages of the disease and in the late stages of the disease. Conventional treatment options employing antiangiogenic approaches provide for the starving of tumors of their blood supply to avoid the refueling of oxygen and nutrients. An emerging alternative point of view is the normalization of vasculature leading to enhance tumor perfusion and oxygenation, potentially capable, when proposed in combination with other treatments, to improve delivery and efficacy of other therapies, including immunotherapy with checkpoint inhibitors. The introduction of novel biomarkers can be useful for the definition of the most appropriate dose and scheduling for these combination treatment approaches. The present review provides a wide description of the pharmaceutical compounds with an antiangiogenic effect proposed for HCC treatment and investigated in clinical trials, including antibodies and small-molecule kinase inhibitors.