Andrew I. Bayliffe

Selective inhibition of intra-alveolar p55 TNF receptor attenuates ventilator-induced lung injury

Background Tumour necrosis factor (TNF) is upregulated in the alveolar space early in the course of ventilator-induced lung injury (VILI). Studies in genetically modified mice indicate that the two TNF receptors play opposing roles during injurious high-stretch mechanical ventilation, with p55 promoting but p75 preventing pulmonary oedema. Aim To investigate the effects of selective inhibition of intra-alveolar p55 TNF receptor on pulmonary oedema and inflammation during ventilator-induced lung injury using a newly developed domain antibody. Methods Anaesthetised mice were ventilated with high tidal volume and given an intratracheal bolus of p55-specific domain antibody or anti-TNF monoclonal antibody (‘pure’ VILI model). As a model of enhanced inflammation, a subclinical dose of lipopolysaccharide (LPS) was included in the intratracheal antibody bolus (LPS+VILI model). Development of lung injury was assessed by respiratory mechanics and blood gases and protein levels in lavage fluid. Flow cytometry was used to determine leucocyte recruitment and alveolar macrophage activation, while lavage fluid cytokines were assessed by ELISA. Results The ventilation protocol produced deteriorations in respiratory mechanics and gas exchange with increased lavage fluid protein levels in the two models. The p55-specific domain antibody substantially attenuated all of these changes in the ‘pure’ VILI model, while anti-TNF antibody was ineffective. In the LPS+VILI model, p55 blockade prevented deteriorations in respiratory mechanics and oxygenation and significantly decreased neutrophil recruitment, expression of intercellular adhesion molecule 1 on alveolar macrophages, and interleukin 6 and monocyte chemotactic protein 1 levels in lavage fluid. Conclusions Selective inhibition of intra-alveolar p55 TNF receptor signalling by domain antibodies may open new therapeutic approaches for ventilated patients with acute lung injury.

Specificity of human anti‐variable heavy (VH) chain autoantibodies and impact on the design and clinical testing of a VH domain antibody antagonist of tumour necrosis factor‐α receptor 1

During clinical trials of a tumour necrosis factor (TNF)‐R1 domain antibody (dAb™) antagonist (GSK1995057), infusion reactions consistent with cytokine release were observed in healthy subjects with high levels of a novel, pre‐existing human anti‐VH (HAVH) autoantibody. In the presence of HAVH autoantibodies, GSK1995057 induced cytokine release in vitro due to binding of HAVH autoantibodies to a framework region of the dAb. The epitope on GSK1995057 was characterized and dAbs with reduced binding to HAVH autoantibodies were generated; pharmacological comparability was determined in human in‐vitro systems and in‐vivo animal experiments. A Phase I clinical trial was conducted to investigate the safety and tolerability of the modified dAb (GSK2862277). A significant reduction in HAVH binding was achieved by adding a single alanine residue at the C‐terminus to create GSK2862277. Screening a pool of healthy donors demonstrated a reduced frequency of pre‐existing autoantibodies from 51% to 7%; in all other respects, GSK2862277 and the parent dAb were comparable. In the Phase I trial, GSK2862277 was well tolerated by both the inhaled and intravenous routes. One subject experienced a mild infusion reaction with cytokine release following intravenous dosing. Subsequently, this subject was found to have high levels of a novel pre‐existing antibody specific to the extended C‐terminus of GSK2862277. Despite the reduced binding of GSK2862277 to pre‐existing HAVH autoantibodies, adverse effects associated with the presence of a novel pre‐existing antibody response specific to the modified dAb framework were identified and highlight the challenge of developing biological antagonists to this class of receptor.

Inhibition of TNF Receptor p55 By a Domain Antibody Attenuates the Initial Phase of Acid-Induced Lung Injury in Mice

Background Tumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator-induced lung injury. In the current study, we explored the efficacy of this antibody in mouse models of acid-induced lung injury to investigate the longer consequences of treatment. Methods We employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting “dummy” dAb, 1 or 4 h before acid instillation. Results Acid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation, and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase. Conclusion These results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 h. Together with our previous data, the current study lends support toward the clinical targeting of p55 for patients with, or at risk of ARDS.

Novel anti-tumour necrosis factor receptor-1 (TNFR1) domain antibody prevents pulmonary inflammation in experimental acute lung injury

Background Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine with both injurious and protective functions, which are thought to diverge at the level of its two cell surface receptors, TNFR1 and TNFR2. In the setting of acute injury, selective inhibition of TNFR1 is predicted to attenuate the cell death and inflammation associated with TNF-α, while sparing or potentiating the protective effects of TNFR2 signalling. We developed a potent and selective antagonist of TNFR1 (GSK1995057) using a novel domain antibody (dAb) therapeutic and assessed its efficacy in vitro, in vivo and in a clinical trial involving healthy human subjects. Methods We investigated the in vitro effects of GSK1995057 on human pulmonary microvascular endothelial cells (HMVEC-L) and then assessed the effects of pretreatment with nebulised GSK1995057 in a non-human primate model of acute lung injury. We then tested translation to humans by investigating the effects of a single nebulised dose of GSK1995057 in healthy humans (n=37) in a randomised controlled clinical trial in which subjects were subsequently exposed to inhaled endotoxin. Results Selective inhibition of TNFR1 signalling potently inhibited cytokine and neutrophil adhesion molecule expression in activated HMVEC-L monolayers in vitro (P<0.01 and P<0.001, respectively), and also significantly attenuated inflammation and signs of lung injury in non-human primates (P<0.01 in all cases). In a randomised, placebo-controlled trial of nebulised GSK1995057 in 37 healthy humans challenged with a low dose of inhaled endotoxin, treatment with GSK1995057 attenuated pulmonary neutrophilia, inflammatory cytokine release (P<0.01 in all cases) and signs of endothelial injury (P<0.05) in bronchoalveolar lavage and serum samples. Conclusion These data support the potential for pulmonary delivery of a selective TNFR1 dAb as a novel therapeutic approach for the prevention of acute respiratory distress syndrome. Trial registration number ClinicalTrials.gov NCT01587807.

A Randomized Dose-Escalation Study of the Safety and Anti-Inflammatory Activity of the p38 Mitogen-Activated Protein Kinase Inhibitor Dilmapimod in Severe Trauma Subjects at Risk for Acute Respiratory Distress Syndrome.

Objectives:There are no current pharmacological therapies for the prevention or treatment of acute respiratory distress syndrome. Early dysregulated inflammation likely plays a role in acute respiratory distress syndrome development and possibly acute respiratory distress syndrome outcomes. p38 mitogen-activated protein kinase is central to the regulation of multiple inflammatory mediators implicated in acute organ dysfunction and is the target for a novel class of cytokine-suppressive anti-inflammatory drugs. In preclinical models, p38 inhibitors reduce lung injury following pancreatitis and burn injury. Design:We conducted a phase IIa, randomized, double-blind, placebo-controlled, parallel-group study to evaluate the safety and tolerability of dilmapimod, a novel p38 mitogen-activated protein kinase inhibitor, in patients at risk for developing acute respiratory distress syndrome admitted with an Injury Severity Score more than 16, excluding head trauma. Enrolled patients received 4- or 24-hour IV dilmapimod infusions at different doses or placebo, daily for 3 days, in four separate cohorts. Setting:Multicenter randomized clinical trial of large, academic trauma centers. Measurements and Main Results:Seventy-seven patients were enrolled. Although adverse events were common in this critically ill population, dilmapimod was well tolerated, with no clinically relevant safety findings. Pharmacokinetic models indicated that the higher dose of 10 mg given as continuous infusion over 24 hours had the most favorable plasma concentration profile. Likewise, measures of soluble inflammatory markers including interleukin-6, C-reactive peptide, interleukin-8, and soluble tumor necrosis factor receptor 1 were most different between this dosing arm and placebo. Although the study was not specifically designed with acute respiratory distress syndrome as an outcome, the number of patients who developed acute respiratory distress syndrome was small (2/77). Conclusions:The novel p38 mitogen-activated protein kinase inhibitor dilmapimod appears well tolerated and may merit further evaluation for prevention of acute respiratory distress syndrome and other organ injury in larger clinical trials. Furthermore, results of this early-phase trial may aid in design of future studies aimed at prevention of acute respiratory distress syndrome and other organ injury.

S94 Tumour Necrosis Factor receptor 1 inhibition using a novel inhaled human antibody reduces inflammation in a human model of lung injury induced by inhaled lipopolysaccharide; a randomised placebo-controlled clinical trial

Introduction and Objectives Tumour Necrosis Factor receptor 1 (TNFR1) transduces the pro-inflammatory activity of TNF-α, whereas signalling through TNFR2 may contribute to tissue repair. Attenuation of TNFR1 signalling, whilst simultaneously preserving the effects of TNFR2 signalling, may be beneficial in management of acute lung injury (ALI). GSK1995057 is a novel, fully human antibody fragment (domain antibody) that selectively binds TNFR1 and antagonises signalling of TNF-α via TNFR1. The aim of this clinical study was to investigate the effect of nebulised GSK1995057 on pulmonary and systemic inflammation and cell injury in an in vivo human model of lung injury induced by inhaled lipopolysaccharide (LPS). Methods Healthy subjects were enrolled in a double-blind, placebo-controlled study and randomised to nebulised GSK1995057 or placebo (1:1) administered 1 hour prior to LPS inhalation. Measurements were performed in bronchoalveolar lavage (BAL) fluid obtained at 6 hours after LPS challenge (7 hours after dosing) and in serum obtained over 24 hours post dosing of GSK1995057. The primary endpoint was BAL neutrophil count at 6 hours post LPS exposure. Data are geometric mean (95% CI). Results Thirty-seven healthy subjects were enrolled. One subject in the placebo group was excluded from the analysis of BAL markers as the BAL was technically poor. Pre-treatment with inhaled GSK1995057 significantly reduced pulmonary and systemic markers of inflammation. In addition, there was a reduction in pulmonary vWF reflecting reduced endothelial cell injury/activation (Table 1). The prevalence of LPS-induced clinical symptoms (e.g. fever, nausea) was also lower in GSK1995057 treated subjects compared with placebo treated subjects. There were no serious adverse events related to study drug. Abstract S94 Table 1. Effect of GSK1995057 on markers of pulmonary and systemic inflammation. BALF Placebo(n = 18) GSK1995057(n = 18) % reduction P-value PMN (104 cells/ml) 6.5 (4.5, 9.4) 4.5 (2.89, 6.89)3.8 (2.8, 5.3)* 31(41)† 0.17(0.03)† IL-1b(pg/ml) 6.8(4.3, 10.4) 1.4 (1.0, 2.1) 79 <0.0001 IL-6 (pg/ml) 386.1(277.5, 537.4) 169.4 (111.2, 257.9) 56 0.003 IL-8 (pg/ml) 332.1(254.6, 433.2) 117.5 (82.5, 167.54) 65 <0.0001 MIP1alpha (pg/ml) 133.7(87.2, 205.1) 15.6 (8.3, 29.1) 88 <0.0001 MCP-1 (pg/ml) 799.4 (591.6, 1080.2) 159.5(101.9, 249.5) 80 <0.0001 vWF (ng/ml) 12.8(9.2, 17.9) 8.1(6.1, 10.8) 37 0.04 Serum Placebo (n = 19) GSK1995057 (n = 18) % reduction P-value CRP (µg/ml)** 55.2(31.0, 98.4) 12.0 (6.6, 21.8) 78 0.0007 OSM (pg/ml)*** 20.7(13.4, 32.0) 7.5(4.8, 11.7) 64 0.002 * PMN data with subject classified as biological outlier (>3 x inter quartile range outside the upper quartile) removed. † % Reduction and statistical significance for BALPMN data with biological outlier excluded. ** CRP data taken at 24h post GSK1995057 dosing. Data are adjusted means with baseline and time effects considered. *** OSM data taken at 6h post LPS inhalation. Data are adjusted means with baseline and time effects considered. Conclusion This is the first report that inhalation of a novel human antibody fragment directed against the TNFR1 receptor attenuates mechanisms implicated in the pathophysiology of ALI. GSK1995057 may be a potential therapy for ALI. ClinicalTrials.gov identifier: NCT01587807. This work was funded by GlaxoSmithKline.

T2 Blockade of intraalveolar p55 TNF-receptor signalling by a domain antibody decreases inflammation and oedema in an in vivo mouse model of ventilator-induced lung injury

Introduction and Objectives Tumour necrosis factor (TNF) alpha is transiently up-regulated within the alveolar space during ventilator-induced lung injury (VILI). We previously found that the two TNF receptors play opposing roles during VILI in knock-out mice, with p55 promoting but p75 preventing pulmonary oedema. This suggests that specific blockade of p55 receptor signalling within the alveolar space may be beneficial in VILI. Domain antibodies (dAbs) are the smallest antigen-binding fragments of the IgG molecule, which may have advantages over complete antibodies due to their small size and monovalent binding (mAbs often have agonist activity due to receptor cross-linking). In this study we tested the effects of an intratracheally (i.t.) delivered dAb that binds to and inhibits the murine p55 receptor (Biopharmaceutical R&D, GlaxoSmithKline), on pulmonary oedema and inflammation in mouse models of VILI. Methods C57BL6 mice were ventilated with a high-stretch protocol (standardised by plateau pressure at 12.5–13.5 cm H2O; tidal volume 20–22 ml/kg, PEEP 3 cm H2O, O2 with 2–4% CO2). Mice then received an i.t. bolus of either non-specific ‘dummy’ dAb or p55-specific dAb (25 μg in 50 μl), and were ventilated for up to 4 h (1-hit model). As a 2-hit model, 20 ng LPS were included in the dAb bolus. Development of lung injury was assessed by respiratory elastance and blood gases, and protein level in bronchoalveolar lavage fluid (BALF) at termination. In the 2-hit model, neutrophil infiltration into BALF, the activation state of alveolar macrophages, and neutrophil margination within lung vasculature were all assessed by flow cytometry. Results High stretch ventilation produced deteriorations in elastance and PO2 and high BALF protein in both models. Treatment with the p55-specific dAb substantially attenuated all of these changes in the 1-hit model (Abstract T2 Table 1). In the 2-hit model, p55 blockade prevented deteriorations in elastance and oxygenation, and significantly decreased neutrophil margination, intraalveolar neutrophil infiltration and ICAM-1 expression on alveolar macrophages.Abstract T2 Table 1 dummy dAb p55-dAb After instillation End After instillation End 1-hit model Elastance (ml/cmH2O/kg) 0.91±0.13 1.07±0.21* 0.91±0.16 0.93±0.18 PO2 (mm Hg) 444±72 287±180* 450±26 415±86 BALF protein (mg/ml) 2.9±1.7 1.4±0.4† 2-hit model Elastance (ml/cmH2O/kg) 0.94±0.11 1.08±0.2* 0.88±0.08 0.9±0.11 PO2 (mm Hg) 486±21 305±168* 497±16 434±114 BALF protein (mg/ml) 3.0±1.6 2.1±1.5 Lung neutrophils 2.30±0.57×106 1.25±0.34×106† BALF neutrophils/ml 1.92±1.69×105 0.47±0.41×105† ALveolar macrophage ICAM-1 (MFI) 80±20 56±13† Mean±SD, N=8–10.* p<0.05 vs after instillation.† p<0.05 vs dummy. Conclusions Use of dAbs to selectively inhibit intra-alveolar p55 TNF receptor signalling may open new therapeutic approaches for ventilated patients with acute lung injury. This study was supported by Biopharmaceutical R&D, GlaxoSmithKline.