Inhaled Amikacin to Prevent Ventilator-Associated Pneumonia.

BACKGROUND: Whether preventive inhaled antibiotics may reduce the incidence of ventilator-associated pneumonia is unclear. METHODS: In this investigator-initiated, multicenter, double-blind, randomized, controlled, superiority trial, we assigned critically ill adults who had been undergoing invasive mechanical ventilation for at least 72 hours to receive inhaled amikacin at a dose of 20 mg per kilogram of ideal body weight once daily or to receive placebo for 3 days. The primary outcome was a first episode of ventilator-associated pneumonia during 28 days of follow-up. Safety was assessed. RESULTS: A total of 850 patients underwent randomization, and 847 were included in the analyses (417 assigned to the amikacin group and 430 to the placebo group). All three daily nebulizations were received by 337 patients (81%) in the amikacin group and 355 patients (83%) in the placebo group. At 28 days, ventilator-associated pneumonia had developed in 62 patients (15%) in the amikacin group and in 95 patients (22%) in the placebo group (difference in restricted mean survival time to ventilator-associated pneumonia, 1.5 days; 95% confidence interval [CI], 0.6 to 2.5; P = 0.004). An infection-related ventilator-associated complication occurred in 74 patients (18%) in the amikacin group and in 111 patients (26%) in the placebo group (hazard ratio, 0.66; 95% CI, 0.50 to 0.89). Trial-related serious adverse effects were seen in 7 patients (1.7%) in the amikacin group and in 4 patients (0.9%) in the placebo group. CONCLUSIONS: Among patients who had undergone mechanical ventilation for at least 3 days, a subsequent 3-day course of inhaled amikacin reduced the burden of ventilator-associated pneumonia during 28 days of follow-up. (Funded by the French Ministry of Health; AMIKINHAL ClinicalTrials.gov number, NCT03149640; EUDRA Clinical Trials number, 2016-001054-17.).

Inhalable Nanofitin demonstrates high neutralization of SARS-CoV-2 virus via direct application in respiratory tract.

Nanofitins are small and hyperthermostable alternative protein scaffolds that display physicochemical properties making them suitable for the development of topical therapeutics, notably for the treatment of pulmonary infectious diseases. Local administration of biologics to the lungs involves a particularly stressful step of nebulization that is poorly tolerated by most antibodies, which limits their application by this delivery route. During the COVID-19 pandemic, we generated anti-SARS-CoV-2 monomeric Nanofitins of high specificity for the spike protein. Hit Nanofitin candidates were identified based on their binding properties with punctual spike mutants and assembled into a linear multimeric construction constituting of four different Nanofitins, allowing the generation of a highly potent anti-SARS-CoV-2 molecule. The therapeutic efficacy of the multimeric assembly was demonstrated both in in vitro and in vivo models. Interestingly, the neutralization mechanism of the multimeric construction seems to involve a particular conformation switch of the spike trimer. In addition, we reported the stability and the conserved activity of the tetrameric construction after nebulization. This advantageous developability feature for pulmonary administration associated with the ease of assembly, as well as the fast generation process position the Nanofitin technology as a potential therapeutic solution for emerging infectious diseases.

Intra-tracheal amikacin spray delivery in healthy mechanically ventilated piglets.

BACKGROUND: Nebulization during mechanical ventilation is impeded by large extra-pulmonary drug deposition and long administration durations which currently limit implementation of inhaled antibiotic therapy. Direct intra-tracheal delivery using a sprayer represents an appealing alternative investigated in small animal models, but large animal data are lacking. METHODS: Amikacin was administered through intravenous infusion (20 mg/kg), nebulization (60 mg/kg) and direct intra-tracheal spray (30 mg/kg) to 10 intubated piglets, in a randomized cross-over design. Amikacin concentrations were measured in the serum and pulmonary parenchyma. Anatomic deposition was investigated using immuno-histochemistry. RESULTS: Spray delivery resulted in higher amikacin outputs than nebulization and infusion. Pulmonary inhaled delivery techniques yielded much higher lung concentrations and much lower serum concentrations than intravenous infusion. However, unlike nebulization and infusion, intra-tracheal spray delivery was associated with more than 100- and 1000-fold variability in lung concentrations between and within animals. Amikacin specific immuno-histochemistry showed consistent bronchial and alveolar drug deposition with all modalities. CONCLUSION: Nebulization remains the most reliable and simple technique to deliver inhaled amikacin uniformly to the lung during mechanical ventilation. Further development of tracheal sprays is required to take advantage of potential benefits related to high drug output and low extra-pulmonary deposition in large animals.

Crucial Role for Immune Complexes but Not FcRn in Immunization against Anti-TNF-α Antibodies after a Single Injection in Mice.

The immunogenicity of infliximab and adalimumab is a major concern because patients may develop Abs also called antidrug Abs (ADA), directed against these anti-TNF-α Abs after just a few weeks of treatment. These ADAs can lead to a decrease in biologic concentration, which is associated with lower treatment efficacy. Our aim was to study the involvement of immune complexes and neonatal Fc receptor (FcRn) in the emergence of ADAs in the case of anti-TNF-α Abs. Wild type and FcRn knockout mice were injected once with either infliximab or adalimumab, alone or preincubated with TNF-α. Adalimumab cross-reacts with murine TNF-α whereas infliximab is species specific. When injected alone, only adalimumab elicited a humoral response. By preforming immune complexes with TNF-α, an anti-infliximab response was elicited. Surprisingly, both wild type and FcRn knockout mice were able to mount an immune response against anti-TNF-α Abs, suggesting that immune complexes are a major determinant of this immunization.

Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases.

The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.

Increment in Drug Loading on an Antibody-Drug Conjugate Increases Its Binding to the Human Neonatal Fc Receptor in Vitro.

Antibody-drug conjugates, such as brentuximab vedotin (BTXv), are an innovative category of monoclonal antibodies. BTXv is bioconjugated via the chemical reduction of cysteine residues involved in disulfide bonds. Species of BTXv containing zero, two, four, six, or eight vedotin molecules per antibody coexist in the stock solution. We investigated the influence of drug loading on the binding of the antibody to FcRn, a major determinant of antibody pharmacokinetics in humans. We developed a hydrophobic interaction chromatography (HIC) method for separating the different species present in the stock solution of BTXv, and we purified and characterized the collected species before use. We assessed the binding of these different species to FcRn in a cellular assay based on flow cytometry and surface plasmon resonance. HIC separated the different species of BTXv and allowed their collection at adequate levels of purity. Physicochemical characterization showed that species with higher levels of drug loading tended to form more aggregates. FcRn binding assays showed that the most conjugated species, particularly those with saturated loading, interacted more strongly than unconjugated BTXv with the FcRn.

Angiogenesis stimulated by human kallikrein-related peptidase 12 acting via a platelet-derived growth factor B-dependent paracrine pathway.

KLK12, a kallikrein peptidase, is thought to take part in the control of angiogenesis. Our analysis of the secretome of endothelial cells (ECs) that had been treated with KLK12 showed that KLK12 converts the extracellular matrix- or membrane-bound precursor of platelet-derived growth factor B (PDGF-B) into a soluble form. Both PDGF-B and vascular endothelial growth factor A (VEGF-A) take part in the induction of angiogenesis by KLK12 in a coculture model of angiogenesis that mimics endothelial tubule formation. We used a cellular approach to analyze the interplay between KLK12, PDGF-B, and VEGF-A and showed that release of PDGF-B by KLK12 leads to the fibroblast-mediated secretion of VEGF-A. This then stimulates EC differentiation and the formation of capillary tube-like structures. Thus, KLK12 favors the interaction of ECs and stromal cells. The released PDGF-B acts as a paracrine factor that modulates VEGF-A secretion by stromal cells, which ultimately leads to angiogenesis. Moreover, the genes encoding KLK12 and PDGFB are both expressed in ECs and up-regulated in tumor cells kept under hypoxic conditions, which is consistent with the physiological involvement of KLK12 in PDGF-B maturation.

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.

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 ß-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.

Development of monoclonal antibodies to human kallikrein-related peptidase 6 (KLK6) and their use in an immunofluorometric assay for free KLK6.

We have raised monoclonal antibodies against KLK6 and constructed a 'sandwich' type immunoassay using 8A8G3 as capture and 3H3E9 as tracer antibodies. 8A8G3 bound to one side of KLK6, whereas 3H3E9 probably bound near its catalytic site. The assay did not detect complexed forms of KLK6, indicating that it quantifies only free KLK6 (fKLK6). fKLK6 was higher in serum of patients with ovarian cancer (11.34 µg/l±1.37) than in controls (3.39 µg/l±0.42). The cerebrospinal fluid contained high concentrations of fKLK6 (257-965 µg/l). This assay could facilitate the evaluation of fKLK6 in human diseases.

The proteolytic airway environment associated with pneumonia acts as a barrier for treatment with anti-infective antibodies.

Like pneumonia, coronavirus disease 2019 (COVID-19) is characterized by a massive infiltration of innate immune cells (such as polymorphonuclear leukocytes) into the airways and alveolar spaces. These cells release proteases that may degrade therapeutic antibodies and thus limit their effectiveness. Here, we investigated the in vitro and ex vivo impact on anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) IgG1s and other IgG subclasses (IgG2 and IgG4) of the neutrophil elastase, proteinase 3 and cathepsin G (the three main neutrophil serine proteases) found in endotracheal aspirates from patients with severe COVID-19. Although the IgGs were sensitive to neutrophil serine proteases, IgG2 was most resistant to proteolytic degradation. The two anti-SARS CoV2 antibodies (casirivimab and imdevimab) were sensitive to the lung's proteolytic environment, although neutrophil serine protease inhibitors only partly limited the degradation. Overall, our results show that the pneumonia-associated imbalance between proteases and their inhibitors in the airways contributes to degradation of antiviral antibodies.

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

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.

Barriers for orally inhaled therapeutic antibodies.

INTRODUCTION: Respiratory diseases represent a worldwide health issue. The recent Sars-CoV-2 pandemic, the burden of lung cancer, and inflammatory respiratory diseases urged the development of innovative therapeutic solutions. In this context, therapeutic antibodies (Abs) offer a tremendous opportunity to benefit patients with respiratory diseases. Delivering Ab through the airways has been demonstrated to be relevant to improve their therapeutic index. However, few inhaled Abs are on the market. AREAS COVERED: This review describes the different barriers that may alter the fate of inhaled therapeutic Abs in the lungs at steady state. It addresses both physical and biological barriers and discusses the importance of taking into consideration the pathological changes occurring during respiratory disease, which may reinforce these barriers. EXPERT OPINION: The pulmonary route remains rare for delivering therapeutic Abs, with few approved inhaled molecules, despite promising evidence. Efforts must focus on the intertwined barriers associated with lung diseases to develop appropriate Ab-formulation-device combo, ensuring optimal Ab deposition in the respiratory tract. Finally, randomized controlled clinical trials should be carried out to establish inhaled Ab therapy as prominent against respiratory diseases.

Influence of mesh nebulizer characteristics on aerosol delivery in non-human primates.

Non-Human Primates (NHPs) are particularly relevant for preclinical studies during the development of inhaled biologics. However, aerosol inhalation in NHPs is difficult to evaluate due to a low lung deposition fraction and high variability. The objective of this study was to evaluate the influence of mesh nebulizer parameters to improve lung deposition in macaques. We developed a humidified heated and ventilated anatomical 3D printed macaque model of the upper respiratory tract to reduce experiments with animals. The model was compared to in vivo deposition using 2D planar scintigraphy imaging in NHPs and demonstrated good predictivity. Next, the anatomical model was used to evaluate the position of the nebulizer on the mask, the aerosol particle size and the aerosol flow rate on the lung deposition. We showed that placing the mesh-nebulizer in the upper part of the mask and in proximal position to the NHP improved lung delivery prediction. The lower the aerosol size and the lower the aerosol flow rate, the better the predicted aerosol deposition. In particular, for 4.3 ± 0.1 µm in terms of volume mean diameter, we obtained 5.6 % ± 0.2 % % vs 19.2 % ± 2.5 % deposition in the lung model for an aerosol flow rate of 0.4 mL/min vs 0.03 mL/min and achieved 16 % of the nebulizer charge deposited in the lungs of macaques. Despite the improvement of lung deposition efficiency in macaques, its variability remained high (6-21 %).

Mucosal administration of anti-bacterial antibodies provide long-term cross-protection against Pseudomonas aeruginosa respiratory infection.

Bacterial respiratory infections, either acute or chronic, are major threats to human health. Direct mucosal administration, through the airways, of therapeutic antibodies (Abs) offers a tremendous opportunity to benefit patients with respiratory infections. The mode of action of anti-infective Abs relies on pathogen neutralization and crystallizable fragment (Fc)-mediated recruitment of immune effectors to facilitate their elimination. Using a mouse model of acute pneumonia induced by Pseudomonas aeruginosa, we depicted the immunomodulatory mode of action of a neutralizing anti-bacterial Abs. Beyond the rapid and efficient containment of the primary infection, the Abs delivered through the airways harnessed genuine innate and adaptive immune responses to provide long-term protection, preventing secondary bacterial infection. In vitro antigen-presenting cells stimulation assay, as well as in vivo bacterial challenges and serum transfer experiments indicate an essential contribution of immune complexes with the Abs and pathogen in the induction of the sustained and protective anti-bacterial humoral response. Interestingly, the long-lasting response protected partially against secondary infections with heterologous P. aeruginosa strains. Overall, our findings suggest that Abs delivered mucosally promotes bacteria neutralization and provides protection against secondary infection. This opens novel perspectives for the development of anti-infective Abs delivered to the lung mucosa, to treat respiratory infections.

Administration of Bacteriophages via Nebulization during Mechanical Ventilation: In Vitro Study and Lung Deposition in Macaques.

Bacteriophages have been identified as a potential treatment option to treat lung infection in the context of antibiotic resistance. We performed a preclinical study to predict the efficacy of delivery of bacteriophages against Pseudomonas aeruginosa (PA) when administered via nebulization during mechanical ventilation (MV). We selected a mix of four anti-PA phages containing two Podoviridae and two Myoviridae, with a coverage of 87.8% (36/41) on an international PA reference panel. When administered via nebulization, a loss of 0.30-0.65 log of infective phage titers was measured. No difference between jet, ultrasonic and mesh nebulizers was observed in terms of loss of phage viability, but a higher output was measured with the mesh nebulizer. Interestingly, Myoviridae are significantly more sensitive to nebulization than Podoviridae since their long tail is much more prone to damage. Phage nebulization has been measured as compatible with humidified ventilation. Based on in vitro measurement, the lung deposition prediction of viable phage particles ranges from 6% to 26% of the phages loaded in the nebulizer. Further, 8% to 15% of lung deposition was measured by scintigraphy in three macaques. A phage dose of 1 × 109 PFU/mL nebulized by the mesh nebulizer during MV predicts an efficient dose in the lung against PA, comparable with the dose chosen to define the susceptibility of the strain.

Kallikrein-related peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors.

Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF(165) binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-ß1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners.

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

BACKGROUND: Tenascins 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. METHODS: We 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. RESULTS: From 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. CONCLUSIONS: The 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.

Alternative Routes of Administration for Therapeutic Antibodies-State of the Art.

BACKGROUND: For the past two decades, there has been a huge expansion in the development of therapeutic antibodies, with 6 to 10 novel entities approved each year. Around 70% of these Abs are delivered through IV injection, a mode of administration allowing rapid and systemic delivery of the drug. However, according to the evidence presented in the literature, beyond the reduction of invasiveness, a better efficacy can be achieved with local delivery. Consequently, efforts have been made toward the development of innovative methods of administration, and in the formulation and engineering of novel Abs to improve their therapeutic index. OBJECTIVE: This review presents an overview of the routes of administration used to deliver Abs, different from the IV route, whether approved or in the clinical evaluation stage. We provide a description of the physical and biological fundamentals for each route of administration, highlighting their relevance with examples of clinically-relevant Abs, and discussing their strengths and limitations. METHODS: We reviewed and analyzed the current literature, published as of the 1 April 2022 using MEDLINE and EMBASE databases, as well as the FDA and EMA websites. Ongoing trials were identified using clinicaltrials.gov. Publications and data were identified using a list of general keywords. CONCLUSIONS: Apart from the most commonly used IV route, topical delivery of Abs has shown clinical successes, improving drug bioavailability and efficacy while reducing side-effects. However, additional research is necessary to understand the consequences of biological barriers associated with local delivery for Ab partitioning, in order to optimize delivery methods and devices, and to adapt Ab formulation to local delivery. Novel modes of administration for Abs might in fine allow a better support to patients, especially in the context of chronic diseases, as well as a reduction of the treatment cost.

Pneumocystis Pneumonia: Pitfalls and Hindrances to Establishing a Reliable Animal Model.

Pneumocystis pneumonia is a severe lung infection that occurs primarily in largely immunocompromised patients. Few treatment options exist, and the mortality rate remains substantial. To develop new strategies in the fields of diagnosis and treatment, it appears to be critical to improve the scientific knowledge about the biology of the Pneumocystis agent and the course of the disease. In the absence of in vitro continuous culture system, in vivo animal studies represent a crucial cornerstone for addressing Pneumocystis pneumonia in laboratories. Here, we provide an overview of the animal models of Pneumocystis pneumonia that were reported in the literature over the last 60 years. Overall, this review highlights the great heterogeneity of the variables studied: the choice of the host species and its genetics, the different immunosuppressive regimens to render an animal susceptible, the experimental challenge, and the different validation methods of the model. With this work, the investigator will have the keys to choose pivotal experimental parameters and major technical features that are assumed to likely influence the results according to the question asked. As an example, we propose an animal model to explore the immune response during Pneumocystis pneumonia.