Nathalie Heuzé-Vourc’h

Aerodynamical, Immunological and Pharmacological Properties of the Anticancer Antibody Cetuximab Following Nebulization

PurposeDespite an increasing interest in the use of inhalation for local delivery of molecules for respiratory diseases and systemic disorders, methods to deliver therapy through airways has received little attention for lung cancer treatment. However, inhalation of anticancer drugs is an attractive alternative route to systemic administration which results in limited concentration of the medication in the lungs, and triggers whole-body toxicity. In this study, we investigated the feasibility of nebulization for therapeutic antibodies, a new class of fully-approved anticancer drugs in oncology medicine.Materials and methodsCetuximab, a chimeric IgG1 targeting the epidermal growth factor receptor (EGFR), was nebulized using three types of delivery devices: a jet nebulizer PARI LC+®, a mesh nebulizer AeronebPro® and an ultrasonic nebulizer SYST’AM® LS290. Aerosol size distribution was measured using a cascade impactor and aerosol droplets were observed under optical microscopy. The immunological and pharmacological properties of cetuximab were evaluated following nebulization using A431 cells.ResultsThe aerosol particle clouds generated with the three nebulizers displayed similar aerodynamical characteristics, but the IgG formed aggregates in liquid phase following nebulization with both the jet and ultrasonic devices. Flow cytometry analyses and assays of EGFR-phosphorylation and cell growth inhibitions on A431 demonstrated that both the mesh and the jet nebulizers preserved the binding affinity to EGFR and the inhibitory activities of cetuximab.ConclusionsAltogether, our results indicate that cetuximab resists the physical constraints of nebulization. Thus, airway delivery represents a promising alternative to systemic administration for local delivery of therapeutic antibodies in lung cancer treatment.

Nebulization as a delivery method for mAbs in respiratory diseases

Introduction: The pulmonary route is not invasive and can be used to target drugs directly to the lungs, limiting the exposure of secondary organs. It is, thus, an attractive alternative to the intravenous route, for the delivery of mAbs, which display limited transfer from blood into the lungs. Areas covered: This review provides an overview of the pharmacological properties of antibody-based treatments, describes those for respiratory diseases and discusses preclinical/clinical results of aerosolized antibody-based therapeutics. The advantages and limitations of aerosol devices and the formulation for the administration of aerosolized mAbs are also detailed. Expert opinion: Overall, the inhalation of mAbs for therapeutic purposes is both appropriate and feasible. The size and structure of the biotherapeutic molecule are important properties to be taken into account when trying to achieve long-term retention. Mesh nebulizers currently appear to be the most appropriate devices for the safe delivery of large amounts of active mAb into the lungs. mAbs should be formulated so as to prevent their degradation and possible immunogenicity. General guidelines can be given for mAb aerosolization, but the formulation and device combination should be adapted for each therapeutic and clinical application.

Tenascin-W is a better cancer biomarker than tenascin-C for most human solid tumors

BackgroundTenascins are large glycoproteins found in the extracellular matrix of many embryonic and adult tissues. Tenascin-C is a well-studied biomarker known for its high overexpression in the stroma of most solid cancers. Tenascin-W, the least studied member of the family, is highly expressed in the stroma of colon and breast tumors and in gliomas, but not in the corresponding normal tissues. Other solid tumors have not been analyzed. The present study was undertaken to determine whether tenascin-W could serve as a cancer-specific extracellular matrix protein in a broad range of solid tumors.MethodsWe analyzed the expression of tenascin-W and tenascin-C by immunoblotting and by immunohistochemistry on multiple frozen tissue microarrays of carcinomas of the pancreas, kidney and lung as well as melanomas and compared them to healthy tissues.ResultsFrom all healthy adult organs tested, only liver and spleen showed detectable levels of tenascin-W, suggesting that tenascin-W is absent from most human adult organs under normal, non-pathological conditions. In contrast, tenascin-W was detectable in the majority of melanomas and their metastases, as well as in pancreas, kidney, and lung carcinomas. Comparing lung tumor samples and matching control tissues for each patient revealed a clear overexpression of tenascin-W in tumor tissues. Although the number of samples examined is too small to draw statistically significant conclusions, there seems to be a tendency for increased tenascin-W expression in higher grade tumors. Interestingly, in most tumor types, tenascin-W is also expressed in close proximity to blood vessels, as shown by CD31 co-staining of the samples.ConclusionsThe present study extends the tumor biomarker potential of tenascin-W to a broad range of solid tumors and shows its accessibility from the blood stream for potential therapeutic strategies.

TFPI-2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour-stromal cell interactions.

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α1 integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.

Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor.

Abstract The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in non-small cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic non-small cell lung cancer (NSCLC) cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of β-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.

Monitoring of tumour progression using bioluminescence imaging and computed tomography scanning in a nude mouse orthotopic model of human small cell lung cancer

Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.

Pro-angiogenic effect of human kallikrein-related peptidase 12 (KLK12) in lung endothelial cells does not depend on kinin-mediated activation of B2 receptor

Abstract Kallikrein-12 (KLK12) may play an important role in angiogenesis modulating proangiogenic factor bioavailability and activating the kinin receptor B2 pathway. We studied whether KLK12 had an impact on angiogenesis and the activation of kinin receptor B2 results from the KLK12-dependent generation of kinins. KLK12 efficiently hydrolyzed high molecular weight kininogen, liberating a fragment containing the carboxy-terminal end of kinins. The kininogenase activity of KLK12 was poor, however, due to the cleavage resistance of the N-terminal side of the kinin sequence. A very low amount of kinins was accordingly released after in vitro incubation of high molecular weight kininogen with KLK12 and thus the proangiogenic activity of KLK12 in lung endothelial cells was not related to a kinin release.

VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations

K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-rasLA1 model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations.

Beneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI‐2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI‐2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI‐2 in tumour progression, TFPI‐2 was overexpressed in NCI‐H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI‐2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI‐H209 cells expressing TFPI‐2. We also demonstrated that TFPI‐2 upregulation in NCI‐H209 cells decreased MMP expression, particularly by downregulating MMP‐1 and MMP‐3. Moreover, TFPI‐2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP‐1 was predominant in SCLC tissues and was inversely expressed with TFPI‐2 in 35% of cases. These results suggest that downregulation of TFPI‐2 expression could favour the development of SCLC.

Downregulation of the neonatal Fc receptor expression in non-small cell lung cancer tissue is associated with a poor prognosis.

Lung cancer is the leading cause of cancer-related death worldwide. Although the recommended tumor, node and metastasis (TNM) classification and stage determination are important to select therapeutic options for patients with non-small cell lung carcinoma (NSCLC), additional molecular markers are required to indicate the prognosis, in particular within a specific stage, and help with the management of patients. Because neonatal Fc receptor (FcRn) has recently been involved in colon cancer immunosurveillance, we measured its expression in non-cancerous and NSCLC lung tissues and evaluated its prognostic value in overall survival for patient with NSCLC. FcRn expression was determined at both mRNA and protein levels on cancerous and adjacent non-cancerous tissues from 80 NSCLC patients. In NSCLC, FcRn was mainly found in resident and tumor infiltrating immune cells. The corresponding mRNA and protein were significantly less abundant in lung tumor than non-cancerous tissue. Moreover, analysis of our cohort and datasets from the public data bases show that FCGRT mRNA down-regulation is a robust and independent, unfavorable predictive factor of NSCLC patient survival. We conclude that FCGRT mRNA expression may be a useful additional marker for immunoscoring, reflecting tumor immune system, and help in the decision-making process for NSCLC patients.