Description of a Murine Model of Pneumocystis Pneumonia.

Pneumocystis pneumonia is a serious lung infection caused by an original ubiquitous fungus with opportunistic behavior, referred to as Pneumocystis jirovecii. P. jirovecii is the second most common fungal agent among invasive fungal infections after Candida spp. Unfortunately, there is still an inability to culture P. jirovecii in vitro, and so a great impairment to improve knowledge on the pathogenesis of Pneumocystis pneumonia. In this context, animal models have a high value to address complex interplay between Pneumocystis and the components of the host immune system. Here, we propose a protocol for a murine model of Pneumocystis pneumonia. Animals become susceptible to Pneumocystis by acquiring an immunocompromised status induced by iterative administration of steroids within drinking water. Thereafter, the experimental infection is completed by an intranasal challenge with homogenates of mouse lungs containing Pneumocystis murina. The onset of clinical signs occurs within 5 weeks following the infectious challenge and immunosuppression can then be withdrawn. At termination, lungs and bronchoalveolar lavage (BAL) fluids from infected mice are analyzed for fungal load (qPCR) and immune response (flow cytometry and biochemical assays). The model is a useful tool in studies focusing on immune responses initiated after the establishment of Pneumocystis pneumonia.

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.

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.

Aggregates Associated with Instability of Antibodies during Aerosolization Induce Adverse Immunological Effects.

BACKGROUND: Immunogenicity refers to the inherent ability of a molecule to stimulate an immune response. Aggregates are one of the major risk factors for the undesired immunogenicity of therapeutic antibodies (Ab) and may ultimately result in immune-mediated adverse effects. For Ab delivered by inhalation, it is necessary to consider the interaction between aggregates resulting from the instability of the Ab during aerosolization and the lung mucosa. The aim of this study was to determine the impact of aggregates produced during aerosolization of therapeutic Ab on the immune system. METHODS: Human and murine immunoglobulin G (IgG) were aerosolized using a clinically-relevant nebulizer and their immunogenic potency was assessed, both in vitro using a standard human monocyte-derived dendritic cell (MoDC) reporter assay and in vivo in immune cells in the airway compartment, lung parenchyma and spleen of healthy C57BL/6 mice after pulmonary administration. RESULTS: IgG aggregates, produced during nebulization, induced a dose-dependent activation of MoDC characterized by the enhanced production of cytokines and expression of co-stimulatory markers. Interestingly, in vivo administration of high amounts of nebulization-mediated IgG aggregates resulted in a profound and sustained local and systemic depletion of immune cells, which was attributable to cell death. This cytotoxic effect was observed when nebulized IgG was administered locally in the airways as compared to a systemic administration but was mitigated by improving IgG stability during nebulization, through the addition of polysorbates to the formulation. CONCLUSION: Although inhalation delivery represents an attractive alternative route for delivering Ab to treat respiratory infections, our findings indicate that it is critical to prevent IgG aggregation during the nebulization process to avoid pro-inflammatory and cytotoxic effects. The optimization of Ab formulation can mitigate adverse effects induced by nebulization.

Inhibition of the Arp2/3 complex represses human lung myofibroblast differentiation and attenuates bleomycin-induced pulmonary fibrosis.

BACKGROUND AND PURPOSE: The Arp2/3 multiprotein complex regulates branched polymerisation of the actin cytoskeleton and may contribute to collagen synthesis and fibrogenesis in the lung. EXPERIMENTAL APPROACH: Expression of Arp2/3 components was assessed in human lung fibroblasts and in the bleomycin-induced pulmonary fibrosis model in mice. The Arp2/3 complex was repressed with the allosteric inhibitor CK666 and with interfering RNAs targeting the ARP2, ARP3 and ARPC2 subunits (siARP2, siARP3 and siARPC2) in CCD-16Lu human lung fibroblasts in vitro. Mice received daily intraperitoneal injections of CK666 from the 7th to the 14th day after tracheal bleomycin instillation. KEY RESULTS: Expression of Arp2/3 complex subunits mRNAs was increased in fibroblasts treated with TGF-ß1 and in the lungs of bleomycin-treated mice compared with controls. In vitro, CK666 and siARPC2 inhibited cell growth and TGF-ß1-induced α-smooth muscle actin (ACTA2) and collagen-1 (COL1) expression. CK666 also decreased ACTA2 and COL1 expression in unstimulated cells. CK666 reduced Akt phosphorylation and repressed phospho-GSK3ß, ß-catenin and MRTF-A levels in unstimulated fibroblasts. In vivo, CK666 reduced levels of both procollagen-1 and insoluble collagen in bleomycin-treated mice. CONCLUSION AND IMPLICATIONS: Expression of the Arp2/3 complex was increased in profibrotic environments in vitro and in vivo. Inhibition of the Arp2/3 complex repressed ACTA2 and COL1 expression and repressed an Akt/phospho-GSK3ß/ß-catenin/MRTF-A pathway in lung fibroblasts. CK666 exerted antifibrotic properties in the lung in vivo. Inhibition of the Arp2/3 complex could represent an interesting new therapy for idiopathic pulmonary fibrosis and other fibrotic interstitial lung diseases.

Cleavage of Occludin by Cigarette Smoke-Elicited Cathepsin S Increases Permeability of Lung Epithelial Cells.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an irreversible disease mainly caused by smoking. COPD is characterized by emphysema and chronic bronchitis associated with enhanced epithelial permeability. HYPOTHESIS: Lung biopsies from smokers revealed a decreased expression level of occludin, which is a protein involved in the cohesion of epithelial tight junctions. Moreover, the occludin level correlated negatively with smoking history (pack-years), COPD grades, and cathepsin S (CatS) activity. Thus, we examined whether CatS could participate in the modulation of the integrity of human lung epithelial barriers. METHODS AND RESULTS: Cigarette smoke extract (CSE) triggered the upregulation of CatS by THP-1 macrophages through the mTOR/TFEB signaling pathway. In a co-culture model, following the exposure of macrophages to CSE, an enhanced level of permeability of lung epithelial (16HBE and NHBE) cells towards FITC-Dextran was observed, which was associated with a decrease in occludin level. Similar results were obtained using 16HBE and NHBE cells cultured at the air-liquid interface. The treatment of THP-1 macrophages by CatS siRNAs or by a pharmacological inhibitor restored the barrier function of epithelial cells, suggesting that cigarette smoke-elicited CatS induced an alteration of epithelial integrity via the proteolytic injury of occludin. CONCLUSIONS: Alongside its noteworthy resistance to oxidative stress induced by cigarette smoke oxidants and its deleterious elastin-degrading potency, CatS may also have a detrimental effect on the barrier function of epithelial cells through the cleavage of occludin. The obtained data emphasize the emerging role of CatS in smoking-related lung diseases and strengthen the relevance of targeting CatS in the treatment of emphysema and COPD.

Targeting Aspergillus fumigatus Crf Transglycosylases With Neutralizing Antibody Is Relevant but Not Sufficient to Erase Fungal Burden in a Neutropenic Rat Model.

Aspergillus fumigatus is an airborne opportunistic fungal pathogen responsible for severe infections. Among them, invasive pulmonary aspergillosis has become a major concern as mortality rates exceed 50% in immunocompromised hosts. In parallel, allergic bronchopulmonary aspergillosis frequently encountered in cystic fibrosis patients, is also a comorbidity factor. Current treatments suffer from high toxicity which prevents their use in weakened subjects, resulting in impaired prognostic. Because of their low toxicity and high specificity, anti-infectious therapeutic antibodies could be a new alternative to conventional therapeutics. In this study, we investigated the potential of Chitin Ring Formation cell wall transglycosylases of A. fumigatus to be therapeutic targets for therapeutic antibodies. We demonstrated that the Crf target was highly conserved, regardless of the pathophysiological context; whereas the CRF1 gene was found to be 100% conserved in 92% of the isolates studied, Crf proteins were expressed in 98% of the strains. In addition, we highlighted the role of Crf proteins in fungal growth, using a deletion mutant for CRF1 gene, for which a growth decrease of 23.6% was observed after 48 h. It was demonstrated that anti-Crf antibodies neutralized the enzymatic activity of recombinant Crf protein, and delayed fungal growth by 12.3% in vitro when added to spores. In a neutropenic rat model of invasive pulmonary aspergillosis, anti-Crf antibodies elicited a significant recruitment of neutrophils, macrophages and T CD4 lymphocytes but it was not correlated with a decrease of fungal burden in lungs and improvement in survival. Overall, our study highlighted the potential relevance of targeting Crf cell wall protein (CWP) with therapeutic antibodies.

In a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration.

Due to growing antibiotic resistance, pneumonia caused by Pseudomonas aeruginosa is a major threat to human health and is driving the development of novel anti-infectious agents. Preventively or curatively administered pathogen-specific therapeutic antibodies (Abs) have several advantages, including a low level of toxicity and a unique pharmacological profile. At present, most Abs against respiratory infections are administered parenterally; this may not be optimal for therapeutics that have to reach the lungs to be effective. Although the airways constitute a logical delivery route for biologics designed to treat respiratory diseases, there are few scientific data on the advantages or disadvantages of this route in the context of pneumonia treatment. The objective of the present study was to evaluate the efficacy and fate of an anti-P. aeruginosa Ab targeting pcrV (mAb166) as a function of the administration route during pneumonia. The airway-administered mAb166 displayed a favorable pharmacokinetic profile during the acute phase of the infection, and was associated with greater protection (relative to other delivery routes) of infected animals. Airway administration was associated with lower levels of lung inflammation, greater bacterial clearance, and recruitment of neutrophils in the airways. In conclusion, the present study is the first to have compared the pharmacokinetics and efficacy of an anti-infectious Ab administered by different routes in an animal model of pneumonia. Our findings suggest that local delivery to the airways is associated with a more potent anti-bacterial response (relative to parenteral administration), and thus open up new perspectives for the prevention and treatment of pneumonia with Abs.

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis.

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.

Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment.

The neonatal Fc receptor (FcRn) is responsible for the recycling and transcytosis of IgG and albumin. FcRn level was found altered in cancer tissues and implicated in tumor immunosurveillance and neoplastic cell growth. However, the consequences of FcRn down-regulation in the anti-tumor immune response are not fully elucidated. By using the B16F10 experimental lung metastasis model in an FcRn-deficient microenvironment (FcRn-/- mice), we found lung metastasis associated with an abnormal natural killer (NK) cell phenotype. In FcRn-/- mice, NK cells were immature, as shown by their surface marker profile and their decreased ability to degranulate and synthesize interferon γ after chemical and IL-2 or IL-12, IL-15 and IL-18 activation. These new findings support the critical role of FcRn downregulation in the tumor microenvironment in anti-tumor immunity, via NK cell maturation and activation.

Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin.

The high toxicity of ricin and its ease of production have made it a major bioterrorism threat worldwide. There is however no efficient and approved treatment for poisoning by ricin inhalation, although there have been major improvements in diagnosis and therapeutic strategies. We describe the development of an anti-ricin neutralizing monoclonal antibody (IgG 43RCA-G1) and a device for its rapid and effective delivery into the lungs for an application in humans. The antibody is a full-length IgG and binds to the ricin A-chain subunit with a high affinity (KD=53pM). Local administration of the antibody into the respiratory tract of mice 6h after pulmonary ricin intoxication allowed the rescue of 100% of intoxicated animals. Specific operational constraints and aerosolization stresses, resulting in protein aggregation and loss of activity, were overcome by formulating the drug as a dry-powder that is solubilized extemporaneously in a stabilizing solution to be nebulized. Inhalation studies in mice showed that this formulation of IgG 43RCA-G1 did not induce pulmonary inflammation. A mesh nebulizer was customized to improve IgG 43RCA-G1 deposition into the alveolar region of human lungs, where ricin aerosol particles mostly accumulate. The drug delivery system also comprises a semi-automatic reconstitution system to facilitate its use and a specific holding chamber to maximize aerosol delivery deep into the lung. In vivo studies in monkeys showed that drug delivery with the device resulted in a high concentration of IgG 43RCA-G1 in the airways for at least 6h after local deposition, which is consistent with the therapeutic window and limited passage into the bloodstream.

Effect of formulation on the stability and aerosol performance of a nebulized antibody.

Most monoclonal antibodies (mAbs) are administered to patients intravenously to ensure high bioavailability as rapidly as possible. The airways, however, are an attractive delivery route for mAbs for the treatment of lung diseases, making it possible to increase their concentration in the target organ while limiting their systemic passage. Several challenges must be overcome for translation into clinical practice. For example, the drug and device must be paired for the efficient and reliable deposition of a pharmacologically active and safe mAb in the lung region of interest. Mesh nebulizers appear to be the most effective aerosol-producing devices for delivering large amounts of biopharmaceutical while limiting protein instability during nebulization. We used metrological and analytic methods to analyze the effect of both antibody concentration and surfactant addition on aerosol performance and antibody integrity. These two factors had a limited effect on aerosol performance, but affected antibody aggregation. The addition of surfactants to antibody formulations at concentrations appropriate for lung administration markedly reduced the formation of medium or large aggregates, as shown by dynamic light scattering and fluorescence microscopy. Aggregation was also dependent on the type of mesh nebulizer, highlighting the need to optimize drug and device together.

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.

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.

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.

Inhibition of Wnt/ß-catenin signaling by a soluble collagen-derived frizzled domain interacting with Wnt3a and the receptors frizzled 1 and 8.

The Wnt/ß-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/ß-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/ß-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/ß-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced ß-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth.

The airways, a novel route for delivering monoclonal antibodies to treat lung tumors.

PURPOSE: Lung cancer is the leading cause of cancer-related death worldwide. The efficacy of current systemic treatments is limited, with major side effects and only modest survival improvements. Aerosols routinely used to deliver drugs into the lung for treating infectious and inflammatory lung diseases have never been used to deliver monoclonal antibodies to treat lung cancer. We have shown that cetuximab, a chimeric anticancer anti-EGFR mAb, is suitable for airway delivery as it resists the physical constraints of aerosolization, and have evaluated the aerosol delivery of cetuximab in vivo. METHODS: We developed an animal model of lung tumor sensitive to cetuximab by injecting Balb/c Nude mice intratracheally with A431 cells plus 10 mM EDTA and analyzed the distribution, pharmacokinetics and antitumor efficacy of cetuximab aerosolized into the respiratory tract. RESULTS: Aerosolized IgG accumulated durably in the lungs and the tumor, but passed poorly and slowly into the systemic circulation. Aerosolized cetuximab also limited the growth of the mouse tumor. Thus, administering anticancer mAbs via the airways is effective and may limit systemic side effects. CONCLUSION: Delivery of aerosolized-mAbs via the airways deserves further evaluation for treating lung cancers.