WO2019100111A1

WO2019100111A1 - Methods of treating and diagnosing conditions

Info

Publication number: WO2019100111A1
Application number: PCT/AU2018/051247
Authority: WO
Inventors: Marcel NOLD; Claudia NOLD
Original assignee: Monash University; Hudson Institute of Medical Research
Priority date: 2017-11-21
Filing date: 2018-11-21
Publication date: 2019-05-31

Abstract

The present invention relates to methods and compositions for the treatment or prevention of bronchopulmonary dysplasia (BPD) in an individual. The invention also provides methods and compositions for the treatment of BPD-associated pulmonary hypertension. The invention also provides methods for determining whether an individual is at risk of developing BPD. The association between type-2 immune activation, particularly IL-4, IL-5 and IL-13, and BPD is used for determination of risk and inhibition of the activity of these cytokines is used as treatment.

Description

Methods of treating and diagnosing conditions

Field of the invention

The present invention relates to methods and compositions for treating and preventing bronchopulmonary dysplasia, and bronchopulmonary dysplasia-associated pulmonary hypertension, in neonates. In addition, the invention provides methods for determining whether a neonate is at risk of the development of bronchopulmonary dysplasia.

Related application

This application claims priority from Australian provisional application AU 2017904712, the contents of which are hereby incorporated by reference in their entirety.

Background of the invention

Infants born at a very early stage of development commonly suffer respiratory failure because of their immature lungs, primitive respiratory drive, and vulnerability to infection.

Modern perinatal medicine has dramatically improved the prognosis for infants who are born very prematurely. Although survival of neonates has improved significantly, this has also created a population of infants with substantial morbidity, the most prominent of which is a serious lung disease known as bronchopulmonary dysplasia (BPD).

Bronchopulmonary dysplasia (BPD) is a severe lung condition that is common in premature infants, particularly those born before 30 weeks of gestation. While most prevalent in the tiniest newborn infants, affecting 35-65% of those born at 0.5-1 kg, BPD still occurs in 10% of infants born at 1.2-1.5 kg. Thus, a significant challenge facing neonatologists, is to ensure that the premature infants that are saved, remain healthy.

When first identified, BPD was described as a condition that occurred primarily in infants who were of sufficient size and maturity to survive the stress of prolonged exposure to high oxygen and positive-pressure ventilation. The clinical course and lung pathology of these infants reflected the consequences of severe pulmonary oxygen toxicity and lung overexpansion. The initial pathologic descriptions of BPD noted airway injury, inflammation, interstitial fibrosis, smooth muscle cell hyperplasia, and squamous metaplasia in the distal airways. Mortality among these infants was high, and long-term ventilator-dependent respiratory failure was common among survivors.

With the advent of surfactant therapy, the widespread use of antenatal steroid therapy, and the use of advanced ventilator and nutritional therapies, the epidemiology and pathophysiology of BPD have changed considerably. Now, almost two-thirds of infants who acquire BPD weigh less than 1 kg at birth and are born before 28 weeks of gestation. In contrast to the days prior to surfactant therapy, when pulmonary oxygen toxicity and lung overexpansion (ventilator-induced lung injury) were considered major contributors to the development of chronic lung injury, premature infants developing BPD now, are exposed to lower levels of oxygen and gentler strategies of mechanical ventilation. The lung pathology of these extremely immature infants with so-called“new BPD” differs from the "classic form" of BPD. BPD is now primarily associated with disrupted terminal lung development resulting from a severe depletion of alveoli. Microscopic inspection of the lungs of babies who have died from BPD reveals a partial to complete arrest in distal lung development.

Lung injury in the preterm infant is triggered by a variety of insults resulting from pre- and post-natal infection, trauma caused by invasive and non-invasive mechanical ventilation, and oxygen toxicity. The most common pathway that leads to tissue damage is pulmonary inflammation, which is increasingly being recognised by neonatologists as the common final pathway and main culprit in the pathogenesis of BPD. However, despite such recognition, no anti-inflammatory strategies are in clinical use, with the exception of corticosteroids (eg dexamethasone), whose powerful anti-inflammatory properties reduce the incidence of BPD. Unfortunately, therapy with dexamethasone cannot be considered safe, with reports indicating that severe side effects commonly occur, including short term problems such as hyperglycaemia, arterial hypertension, gastrointestinal bleeding, intestinal perforation and increased severity of retinopathy of prematurity. In addition, dexamethasone treatment results in a number of devastating long term sequelae such as neurodevelopmental abnormalities and cerebral palsy. Thus, treatment with dexamethasone is not recommended for routine care of infants with BPD, and is generally only used as a last resort in life-threatening situations.

Other than anti-inflammatory medications, drugs in clinical use for protecting the developing lung include exogenous surfactant, caffeine and diuretics such as furosemide, hydrochlorothiazide and spironolactone. Although surfactant administration moderately reduces the risk of BPD or death, this therapy has failed to markedly ameliorate or reduce the occurrence of BPD.

Left untreated, the chronic inflammation associated with BPD can also lead to the development of pulmonary hypertension (PH). PH occurs up to 25% of all BPD patients and represents the most severe complication of the condition, as the pulmonary reserves of affected newborn infants are almost non-existent. Moreover, the right heart, which cannot sustain high pressures for an extended period of time, eventually fails. Infants suffering from BPD-associated PH have a survival of only 53% two years after diagnosis. As dysalveolarisation and dysangiogenesis are inextricably linked in lung disease of prematurity, any medication that ameliorates BPD will also benefit PH.

Even for those infants who do not develop PH, BPD leads to major long term problems that reach far beyond the suffering caused by the lung disease itself. Afflicted infants frequently fail to thrive, fall ill more often with recurrent respiratory disease, and mortality is higher compared to age-matched controls. In addition, rehospitalisation occurs in up to 50% of cases during the first two years of life and infants with BPD are more likely to have significant neurodevelopmental delay, with fine and gross motor skills affected in particular. Thus, the suffering caused by BPD in affected infants and their families is immense, and according to estimates, treating infants with BPD in the United States alone costs at least $3 billion (US) per year, making it the second most expensive childhood disease in that country, after asthma. For all of these reasons, BPD represents one of the greatest unmet therapeutic challenges of neonatology today. Nonetheless, to date, efforts to find a safe and effective therapy for BPD have failed.

Accordingly, there is a need to develop methods for treating and preventing BPD and/or BPD-associated PH in preterm infants. In addition, there is a need to develop methods for determining whether a preterm infant is at risk of BPD, so that appropriate intervention can be taken for treating the condition. Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

The present invention provides a method of treating or preventing bronchopulmonary dysplasia (BPD) in an individual, the method comprising administering a therapeutically effective amount of an inhibitor of type 2 immune activation to an individual in need, thereby treating or preventing BPD in the individual.

Preferably, the individual is a pre-term infant, including an individual born before 35 weeks of gestation, optionally, before 30 weeks of gestation.

The individual may be diagnosed with mild, moderate or severe BPD, as further described herein.

The present invention also provides a method of treating or preventing BPD- associated pulmonary hypertension (PH) in an individual, the method comprising administering a therapeutically effective amount of an inhibitor of type 2 immune activation to an individual in need, thereby treating or preventing BPD-associated PH in the individual. Preferably, the BPD-associated PH is treated during the course of providing treatment for BPD. The individual may have been successfully treated for BPD including by any method described herein.

The inhibitor of type 2 immune activation is any molecule that reduces or antagonises signalling of a signalling molecule that is active in the type 2 immune pathway. For example, the inhibitor may be a type 2 antagonist selected from the group consisting of: an interleukin 4 (IL-4) antagonist, an IL-4 receptor antagonist, an interleukin 5 (IL-5) antagonist, an IL-5 receptor antagonist, an interleukin 13 (IL-13) antagonist, an IL-13 receptor antagonist, an interleukin 33 (IL-33) antagonist, and IL-33 receptor antagonist, a thymic stromal lymphopoietin (TSLP) antagonist and a TSLP receptor antagonist. In any embodiment of the invention, the inhibitor of type 2 immune activation is selected from the group consisting of a small compound, a monoclonal antibody, a peptide, a recombinant protein or an interfering polynucleotide or silencing RNA.

In any embodiment of the invention, the inhibitor of type 2 immune activation is a monoclonal antibody, preferably a monoclonal antibody that inhibits the activity of signalling of one or more of IL-4, IL-5, IL-13, IL-33, IL-25, IgE or TSLP.

The type 2 antagonist may be a monoclonal antibody selected from the group consisting of: tralokinumab, lebrikizumab, anrukinzumab, ASLAN004, pitrakinra, dupilumab, mepolizumab, benralizumab, tezepelumab, omalizumab.

In any embodiment of the invention, a combination of inhibitors of type 2 immune activation may be administered in order to target more than one type 2 cytokine. In one embodiment, the methods of the present invention include administration of at least an inhibitor of IL-13. In a further embodiment, the present invention includes administration of at least an inhibitor of IL-13 and an inhibitor of IL-4. In still a further embodiment, the present invention includes administration of at least an inhibitor of IL-13, an inhibitor of IL-4 and an inhibitor of IL-5. Still further, the present invention includes administration of at least an inhibitor of IL-13, IL-4, IL-5 and of one or more of IL-33, IL-25, IgE and/or TSPL.

Preferably, the type 2 antagonist is a monoclonal antibody that specifically inhibits the signalling of IL-13. More preferably, the type 2 antagonist is a monoclonal antibody that specifically inhibits the signalling of IL-13 and IL-4 (for example, ASLAN004, as herein described, which binds to the IL-13receptoralpha1 ).

In any embodiment of the invention, the inhibitor of type 2 activation may be a protein, including a fusion protein that prevents the binding of a type 2 cytokine to its receptor (and consequently inhibits or reduces the activity of that cytokine). In certain embodiments, the inhibitor may be an agent, for example a monoclonal antibody, that specifically binds to the cytokine receptor and thereby inhibits the binds of the endogenous ligand to the receptor. In preferred embodiments, the inhibitor specifically binds to the IL-13Ralpha 1 receptor and thereby inhibits binding or signalling of IL-13 and/or IL-4. In further embodiments, the protein may be a soluble, decoy receptor that binds to the cytokine and thereby prevents the binding of the cytokine to its endogenous, cell- bound receptor. In certain preferred embodiments, the inhibitor of type 2 activation may be a soluble form of the IL-13Ra2 protein, more preferably, a fusion protein comprising the IL-13Ra2 receptor. In a particularly preferred embodiment, the IL-13Ra2 fusion protein comprises IL-13Ra2-Fc.

In any embodiment of the invention, the inhibition of type 2 immune activation results in a decrease in type 2 polarised inflammation the individual.

In any embodiment, the methods of the present invention include determining whether the individual is at risk of BPD. Determining whether an individual is at risk can be performed using standard methods, or alternatively, by determining whether the individual has an increased level of type 2 activation compared with another pre-term individual, who did not develop BPD. More specifically, determining the risk of an individual of BPD may include:

- determining the level of type 2 immune activation in an individual for whom risk is to be determined;

- comparing the level of type 2 immune activation in the individual with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term individuals who developed BPD; determining that the individual is at risk of BPD if the level of type 2 immune activation in the individual is the same or greater than the level of type 2 immune activation in the reference data set; determining that the individual is not at risk of developing BPD if the level of type 2 immune activation in the individual is less than the level of type 2 immune activation in the reference data set.

The level of type 2 immune activation in the individual can be determined by any known method in the art, including by determining or measuring levels of one or more type 2 cytokines in the individual. In some embodiments the type 2 cytokines are selected from IL-4, IL-5, IL-13, IL-33, IL-25, IgE or TSLP. In a preferred embodiment, the type 2 cytokine is IL-13. Still further, determining the risk of BPD in an individual may include:

- obtaining a test biological sample from the individual for whom risk of BPD is to be determined,

- measuring in the test sample, the level of one or more type 2 cytokines,

- comparing the level of type 2 cytokines in the test sample, with the level of type 2 cytokines in a reference data set in the form of data from one or more pre-term individuals who developed BPD; determining that the individual is at risk of BPD if the level of type 2 cytokines in the test sample is the same or greater than the level of type 2 cytokines in the reference data set; determining that the individual is not at risk of developing BPD if the level of type 2 cytokines in the test sample is less than the level of type 2 cytokines in the reference data set.

Still further, determining the risk of BPD in an individual may include:

- providing a test biological sample from the individual for whom risk of BPD is to be determined,

- measuring in the test sample, the level of one or more type 2 cytokines,

The one or more type 2 cytokines for which the level is determined, are selected from the group consisting of: IL-4, IL-5, IL-13, IL-25, IL-33 and TSLP. Preferably, the level of type 2 activation is determined by reference to the level of IL-13 in the test sample.

The present invention also provides a method of determining whether an individual is at risk of BPD, the method comprising:

- measuring in a test biological sample obtained from the individual, the levels of one or more type 2 cytokines,

- comparing the level of type 2 cytokines in the test sample with the level of type 2 cytokines in a reference data set in the form of data from one or more pre-term individuals who developed BPD; determining that the individual is at high risk of BPD if the level of type 2 cytokines in the test sample is the same or greater than the level of type 2 cytokines in the reference data set; determining that the individual is at low risk of BPD if the level of type 2 cytokines in the test sample is less than the level of type 2 cytokines in the reference data set.

The present invention also provides a method of identifying a pre-term infant for treatment for BPD, the method comprising:

- determining the level of type 2 immune activation in the infant,

- comparing the level of type 2 immune activation in the infant with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term infants who developed BPD; determining to treat the infant for BPD if the level of type 2 immune activation in the infant is the same or greater than the level of type 2 immune activation in the reference data set; determining not to treat the infant for BPD if the level of type 2 immune activation in the infant is less than the level of type 2 immune activation in the reference data set.

The present invention also provides a method of identifying a pre-term infant for treatment for BPD, the method comprising: - providing a test biological sample from a pre-term infant,

- measuring in the test sample, the levels of one or more type 2 cytokines, comparing the level of type 2 cytokines in the test sample, with the level of type 2 cytokines in a reference data set in the form of data from one or more pre-term infants who developed BPD; determining to treat the infant for BPD if the level of type 2 cytokines in the test sample is the same or greater than the level of type 2 cytokines in the reference data set; determining not to treat the infant for BPD if the level of type 2 cytokines in the test sample is less than the level of type 2 cytokines in the reference data set.

In a preferred embodiment, the test biological sample is a plasma sample.

Preferably, the one or more type 2 cytokines for which the level is determined, are selected from the group consisting of: IL-4, IL-5, IL-13, IL-25, IL-33 and TSLP.

In any embodiment, the type 2 activation is determined by reference to the level of IL-13 in the test sample.

After identifying an individual for treatment for BPD, the treatment may include administration of a type 2 antagonist as herein described, alone or in combination with other known treatments for BPD, including caffeine; a diuretic; a non-specific phosphodiesterase inhibitor; phosphodiesterase-4 inhibitors; phosphodiesterase-5 inhibitors; exogenous surfactant, a corticosteroid; an inhibitor of type 1 immune activation; an antagonist of interleukin-1 (IL-1 ) or an antagonist of IL-1 signalling pathway; curcumin; inhaled nitric oxide (iNO); azithromycin; a bronchodilator or anti- hypertensive (such as bosentan or sildenafil).

Alternatively, after identifying an individual for treatment of BPD, any method of treatment as described herein may be performed.

Preferably the inhibitor of type 2 immune activation is selected from the group consisting of: an interleukin 4 (IL-4) antagonist, an IL-4 receptor antagonist, an interleukin 5 (IL-5) antagonist, an IL-5 receptor antagonist; an interleukin 13 (IL-13) antagonist, an IL-13 receptor antagonist, an IL-25 antagonist, an interleukin 33 (IL-33) antagonist and a thymic stromal lymphopoietin (TSLP) antagonist.

The inhibitor of type 2 immune activation may be selected from the group consisting of a small compound, a monoclonal antibody, a peptide, a recombinant protein or an interfering polynucleotide or silencing RNA.

The inhibitor of type 2 immune activation is preferably a monoclonal antibody, more preferably a monoclonal antibody that inhibits the activity of signalling of one or more of IL-4, IL-5, IL-13, IL-25, IL-33 or TSLP or IgE.

In a preferred embodiment, the monoclonal antibody is one that binds specifically to the IL-13 receptor antibody, for example, ASLAN004.

The present invention also provides the use of an inhibitor of type 2 immune activation, in the manufacture of a medicament for the treatment or prevention of BPD and/or BPD-associated PH. Preferably, the inhibitor of type 2 immune activation is one as described further herein.

The present invention also provides an inhibitor of type 2 immune activation for use in the treatment or prevention of BPD and/or BPD-associated PH. Preferably, the inhibitor of type 2 immune activation is one as described further herein.

The present invention also provides pharmaceutical compositions comprising, consisting essentially of or consisting of an inhibitor of type 2 immune activation, for use in the prevention or treatment of BPD in a pre-term infant. Preferably, the pharmaceutical composition comprises a inhibitor of type 2 immune activation as described further herein.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings

Figure 1 : BPD infants had more IL-4 in cord and peripheral blood than non- BPD infants. Cord and peripheral blood samples were collected from preterm infants (between 24+0 and 28+6 weeks GA) at birth, day 1 , weeks 1 and 2 and 36 weeks cGA in addition to controls including healthy term infants (born between 37+0 and 41 +0 weeks GA) and adults. Blood cells were treated with Brefeldin A (2 μg/ml) and volume- matched vehicle or PMA (2ng/ml) and ionomycin (250ng/ml) for 12-16 hours, then stained and analyzed by flow cytometry. Percentage of IL-4+ cells among viable

CD45+CD3+CD4+ T cells are shown in all groups with preterm infants separated into BPD and no BPD groups as defined, (a) is for cells treated with vehicle while (b) displays cells with PMA and ionomycin (median ± interquartile range, n = 5-10 for no BPD, 22-41 for BPD, 11 for term CB, 9 for term PN, 5 for adult, *P<0.05 and **P<0.005 for BPD vs. no BPD,□ P<0.05 for vs. term PN, # P<0.05 for vs. adult).

Figure 2: At day 14 of gestation, pregnant C57BL/6 dams were injected with LPS or vehicle. Within 24h after birth, pups were exposed to 65% O2 or 21 % O2 (room air) and daily postnatal s.c. injections of IL-1 Ra or vehicle. On day 3, cytokines and chemokines were determined in the lungs by ELISA. Data are means of cytokine abundance normalized to total protein (t.p.) ± SEM. n= 7-20 per group; *, p < 0.05; **, p

< 0.005 and ***, p < 0.001 for room air vehicle vs. hyperoxia vehicle; #, p < 0.05; ##, p < 0.005 and ###, p < 0.001 for hyperoxia vehicle vs. hyperoxia IL-1 Ra; ¨ , p < 0.05; ¨ ¨ , p

< 0.005 and ¨ ¨ ¨ , p < 0.001 for no antenatal LPS air vehicle vs. hyperoxia vehicle.

Figure 3: Pregnant STAT6-ko and C57BL/6 (WT) dams were injected with 150 μg/kg LPS at day 14 of gestation. After delivery, newborn mice were continuously exposed to an F1O2 of 0.21 (room air) or 0.65. Lungs were assessed after day 28. (a) H&E staining of murine lungs at day 28 of the BPD model. One representative 500 μm x 500 μm image per group is depicted. Scale bars: 100 μm. Whole lungs were analyzed for alveolar size (b), the number of alveoli per square millimeter (c) and the surface area to volume ratio (d). Blood was also analysed for partial pressure of O2 at day 28 (e). Data are shown as mean ± SEM. n = ~20 mice per group; *, p < 0.05 and ***, p < 0.001 for room air WT vs. hyperoxia WT; #, p < 0.05; ##, p < 0.005 and ###, p < 0.001 for hyperoxia vehicle vs. hyperoxia STAT6-ko. Figure 4: ELISA showing that type 2 mediators are increased in the hyperoxia group. These mediators are reduced to steady-state in STAT6-deficient mice.

Figure 5: Following antenatal LPS and postnatal exposure to 65% 0₂ or room air, lungs were obtained from 28 d STAT6-ko and C57BL/6 mice. Various cell types were assessed; the ones that show significant changes are shown here. I also have the other cell types. Individual data points with means +/- SEM are shown. *, P < 0.05 and **, P < 0.01 for Air WT vs any other group; #, P < 0.05 and ##, P < 0.01 for Hyp WT vs Hyp STAT6-ko.

Figure 6: Pups exposed to LPS in utero were reared in hyperoxia (65% 0₂) or room air (21 % 0₂) for 28d. (a) IL-13 staining of murine lungs at day 28 of the BPD model. One representative 500 μm x 500 μm image per group is shown. Whole lungs were subjected to IHC assessment of the abundance of IL-13-positive cells (b), IL-33- postive cells (c) and IL-5-positive cells (d) per square millimeter of tissue. Data are shown as mean ± SEM. n = ~10 per group. **, P < 0.01 and ***, P < 0.001 for room air WT vs. hyperoxia WT; ##, p < 0.005 for hyperoxia vehicle vs. hyperoxia STAT6-ko.

Figure 7: Animals were treated identically as in Fig. 3. except after day 28, all groups were kept in room air until day 60. (a-d) One representative image per group is depicted. Branching analysis of the lung vessels after processing. Vessels with a diameter from 4 μm (blue) to 60μm (red) were included in the analysis. Inset images show 3D rendering of CT scans of the same representative lungs prior to processing and analysis and is not colour coded for diameter (e-g) Quantification of the number of vessels in the lung are grouped by vessel diameter: small vessels (4-7 pm) (e), medium vessels (7-30 pm) (f) and large vessels (30-60 pm) (g). Vessel number was normalized to percent of total vessels in the lung. Data are shown as mean ± SEM. N= ~10 *, p < 0.05 and ***, p < 0.001 for room air WT vs. hyperoxia WT; #, p < 0.05; ##, p < 0.005 and ###, p < 0.001 for hyperoxia vehicle vs. hyperoxia STAT6-ko.

Figure 8: (a) Echocardiography was performed on day 28 of the murine BPD model. TPV/RVET ratio is an estimation of the pulmonary artery pressure and is indicative of pulmonary hypertension (b, c) Abundance of angiogenesis markers (b) VEGF-AA and (c) endothelin-1 was determined by ELISA on day 28 lungs. Data are means of cytokine abundance normalized to total protein (t.p.) ± SEM. N= ~10 *, p < 0.05 and ***, p < 0.001 for room air WT vs. hyperoxia WT; #, p < 0.05 and ###, p < 0.001 for hyperoxia vehicle vs. hyperoxia STAT6-ko.

Figure 9: C576BL/6 pups exposed to antenatal LPS and postnatal 65% 0₂ are protected from BPD when injected every second day with blocking antibodies targeted to type 2 cytokines. Pregnant C57BL/6 dams were injected with 150 μg/kg LPS at day 14 of gestation. After delivery, newborn mice were continuously exposed to an Fi0₂ of 0.21 (room air) or 0.65 and postnatally s.c. injected every second day with either treatment (anti-IL-4, anti-IL-5 or anti-IL-13 monoclonal antibodies) or IgG controls for 28 days. Lungs were assessed after day 28. H&E staining of murine lungs at day 28 of the BPD model were assessed. Whole lungs were analyzed for alveolar size (a), the number of alveoli per square millimeter (b) and the surface area to volume ratio (b). Data are shown as mean ± SEM. n= 3-10.

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. All of the patents and publications referred to herein are incorporated by reference in their entirety.

For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

The present inventors have surprisingly found that pre-term individuals who go on to develop BPD have T helper cells that are polarised toward a type 2 phenotype compared to pre-term individuals who do not develop BPD. Thus, the inventors have identified a means for identifying those pre-term individuals who are at greatest need for treatment or preventative therapies for targeting BPD.

Moreover, the inventors have demonstrated that blockade of the type 2 immune activation pathway in an accepted model of BPD, protects against the remodelling of airways and pulmonary vasculature. Thus, the inventors have identified that type 2 blockade may represent a novel approach to the treatment and prevention of BPD and associated pulmonary hypertension in pre-term infants.

Methods of treatment

The present invention includes methods for treating or preventing BPD in an individual.

Typically, an individual requiring treatment or prevention of BPD will be a pre- term infant. As used herein, the term "preterm infant" refers to a human individual born at less than 37 weeks of gestational age, including less than 35 weeks gestational age, or less than or about 30, less than or about 29, less than or about 28, less than or about 27, less than or about 26, less than or about 25, less than or about 24, less than or about 23, or less than or about 22 weeks of gestational age. Typically, at the time where treatment or prevention of BPD is required, the infant is less than 2 years old, less than 12 months old, less than 6 months old, less than 3 months old, less than 2 months old, or less than 1 month old.

As used herein,“gestational age” refers to the time elapsed between the first day of the last menstrual period of the mother of the infant, and the day of delivery. If pregnancy was achieved using assisted reproductive technology, gestational age is calculated by adding 2 weeks to the conception age.

As used herein, “post-menstrual age” (PMA) refers to gestational age plus chronological age (ie, gestational age, plus the time since birth).

As used herein, “corrected age” refers to chronological age reduced by the number of weeks born before 40 weeks of gestation; the term is generally only used only for children up to 3 years of age who were born preterm.

As used herein, "preventing" or "prevention" is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring BPD (i.e. , causing at least one of the clinical symptoms of the disease not to develop in a patient that may be at risk or predisposed to BPD but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known by clinicians.

The terms "treatment" or "treating" of a subject includes the application or administration of a compound as described herein to a subject (or application or administration of a compound of the invention to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) BPD. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.

In certain methods of the invention, the type 2 antagonist is administered in a therapeutically effective amount to treat an infant at risk of developing BPD, and thereby reduce, minimize, or prevent the development of BPD in the infant. As used herein, the term“treat” is used to mean to act upon to reduce, minimize, or prevent the pulmonary sequelae and clinical signs and symptoms that are associated with BPD. Infants who can be treated with the methods of the invention include any infants at risk of developing BPD. Infants at risk of developing BPD include premature infants, infants at risk for premature delivery, and any other infants at risk of developing BPD for other reasons.

In preferred embodiments of the invention, the treatment for BPD occurs during the perinatal period. As used herein, the“perinatal period” in humans for treatment using the methods of the invention includes both the prenatal period (i.e. before birth) as well as the postnatal period (i.e. after birth), and starts from about 26 weeks gestation and continues to about 18 months after birth. For administration during the prenatal period, the type 2 antagonist can be administered either directly to the fetus in utero, or indirectly by administration to the mother.

Generally, the age of the infants to be treated with the methods of the invention is from at least about 20-22 weeks, preferably at least 24 weeks gestation to about 6 months after birth. In certain embodiments, treatment may be continued beyond 6 months of age, particularly if the BPD is chronic. In such cases, treatment may continue until the infant is at least 12 months of age, or at least 18 months of age or older. The number of weeks gestation (i.e., gestational age) can be determined using any of a number of conventional methods. For example, the gestational age can be calculated from the first day of the last menstruation.

Almost two-thirds of infants who acquire BPD weigh less than 1 ,000 grams and are less than 28 weeks gestation at birth. Accordingly, in one embodiment, the infants to be treated with the type 2 antagonists, as herein defined, can be identified by their weight and/or age at birth. In one embodiment, the infant to be treated is a premature infant born at about 32 weeks gestation or younger. In one embodiment, the infant to be treated is a premature infant born at about 28 weeks gestation or younger. In one embodiment, the infant to be treated weighs about 1500 grams or less at birth. In another embodiment, the infant weighs about 1000 grams or less at birth.

Infants at risk of developing BPD also include yet to be born infants (i.e. fetuses) at risk for premature delivery (and therefore at risk for BPD). Such infants may be treated during the prenatal period, either by administration to the mother or direct administration to the fetus in utero. Any pregnant woman at about 23 or more weeks gestation with clinically intact membranes and having one or more risk factors for preterm delivery or preterm delivery markers may be a candidate for treatment.

A large number of factors are known to be associated with the risk of preterm delivery. These factors include, but are not limited to, multiple fetus gestations; incompetent cervix; uterine anomalies; polyhydramnios; nulliparity; previous preterm rupture of membranes or preterm labor; preeclampsia; vaginal bleeding associated with placental previa; little or no antenatal care; and symptoms such as abdominal pain, low backache, passage of cervical mucus and contractions.

In addition to these risk factors, a number of preterm delivery markers have been identified. As used herein, a“preterm delivery marker” is a marker that, when present at or beyond a threshold level, indicates an increased risk of preterm delivery. Examples of preterm delivery markers include, but are not limited to, fetal restricted antigens such as fetal fibronectin and estriol (U.S. Patent Application Publication No. 2004/0266025), insulin-like growth factor binding protein 1 (a marker for the likelihood of membrane rupture; see U.S. Patent Application Publication No. 2004/0053838), and MMP-8 and MMP-9 (U.S. Patent Application Publication No. 2004/0029176). Methods to detect the level of preterm delivery markers are well known in the art, and described, for example, in U.S. Patent Application Publication No. 2004/0266025.

Although BPD is seen mostly among premature babies, it can also occur in full- term babies who have respiratory problems during their first days of life. Accordingly, the methods of the invention can be used to treat these infants as well.

More particularly, the present invention is directed to the prevention or treatment of BPD, including, for example, BPD induced by, or associated with, pulmonary inflammation; especially perinatal inflammation in combination with oxygen toxicity (eg hyperoxia), perinatal inflammation associated with chorioamnionitis (inflammation of the foetal membranes (amnion and chorion) due to a bacterial infection), perinatal inflammation characterised by, or in combination with, up-regulation of one or more of IL-1 b, TREM-1 and the chemokines MIP-1 a/CCL3, BLC/CXCL13 and KC/CXCL8 (and, especially, up-regulation of one or more of IL-1 b, TREM-1 and MIP-1 a), and optionally, IL-1 b and IL-6, or pulmonary inflammation caused by postnatal bacterial infection and/or invasive or noninvasive mechanical ventilation.

The skilled person will be familiar with methods for determining whether an individual is at risk of BPD, or is displaying early signs or symptoms of BPD. For example, BPD may be diagnosed at 28 days of age or thereabout, 36 weeks post- menstrual age (PMA).

In some instances, the clinical diagnosis of BPD may be made in any prematurely born infant who, at 36 weeks gestation, has lung disease requiring continuous or continual supplemental oxygen and who has had an abnormal chest X- ray. For example, diagnosis may be made according to the following criteria:

• positive pressure ventilation during the first 2 weeks of life for a minimum of 3 days.

• clinical signs of abnormal respiratory function.

• requirements for supplemental oxygen for longer than 28 days of age to maintain Pa02 above 50 mm Fig.

• chest radiograph with diffuse abnormal findings characteristic of BPD.

Alternatively, BPD can be diagnosed using the criteria set forth by The National Institute of Health (US) criteria for BPD (for neonates treated with more than 21 % oxygen for at least 28 days), as shown in Table 1 below from Jobe and Bancalari, Am J Respir Crit Care Med, 2001 , 163:1723-1729, incorporated herein by reference in its entirety. An infant is said to have BPD if they remain on mechanical ventilation or require supplemental oxygen in order to maintain oxygen saturation levels (“Sa02”) greater than or equal to 90% (with the exception of infants requiring supplemental oxygen during feedings) at 36 weeks PMA or at 28 days of age. Generally, chest x-rays will also be performed for infants at 28 days of age in order to confirm the BPD diagnosis. The X-ray of lungs with BPD often have a bubbly, sponge-like appearance. Table 1 : Diagnostic criteria for BPD

As used herein,“BPD” also includes all alternative clinical diagnosis definitions, such as a diagnosis in infants older than four weeks from birth who have had persistent lung disease requiring continual supplemental oxygen and who have had abnormal chest X-rays. BPD is also sometimes referred to in the literature and by pediatric caretakers as“chronic lung disease” (Jobe et al. , Early Hum. Devel. 53:81-94 (1998)), or as“neonatal chronic lung disease” or“chronic lung disease of infancy”.

Still further, the skilled person will be familiar with methods for determining the success of treatment for BPD, including whether treatment or prevention of BPD, using a type 2 antagonist as described herein, has been successful. For example, success of treatment can be determined by measuring for a change or improvement of any one of the BPD symptoms described herein.

Still further, the success of treatment for BPD can be determined by measuring for a reduction in type 2 immune activation in the individual who has received treatment. For example, determining a reduction in type 2 immune activation after treatment, as compared to before treatment will be indicative of the success of treatment. The skilled person will appreciate that determining type 2 immune activation can be performed by any number of conventional methods in the art. The skilled person will be familiar with kits and other methods available for measuring cytokine levels in a biological sample obtained from the individual who has received treatment.

In certain embodiments, an improvement (or reduction in) type 2 immune activation can be by measuring for a reduction in levels of one or more cytokines associated with the type 2 pathway. Examples of type 2 cytokines which can be measured include IL-4, IL-5, IL-13, IL-25, IL-33 and TSLP. IgE can also be measured as an indicator of type 2 activation.

In a preferred embodiment, the success of any treatment for BPD can be determined by measuring for a reduction in the level of IL-13 in the individual who as received treatment for BPD.

In a further aspect, the present invention is directed to the prevention or treatment of BPD-associated PH.

As used herein, the term "BPD-associated pulmonary hypertension" will be understood by those skilled in the art as referring to PH in a subject (especially an infant or young child of < 10 years of age or, more preferably, < 2 years of age) who has experienced or is presently experiencing BPD. In any embodiment of the invention, BPD-associated PH may also refer to BPD-associated PAH (pulmonary arterial hypertension).

BPD-associated PH is typically characterised by abnormally high pulmonary blood pressure, for example a systolic pulmonary arterial pressure (sPAP) being > 40 mmHg (as calculated from the velocity of the tricuspid regurgitation jet measured by trans-thoracic echocardiogram (TTE); see PM Mourani at al. Clinical Utility of Echocardiography for the Diagnosis and Measurement of Pulmonary Vascular Disease in Young Children with Chronic Lung Disease, Pediatrics 121 (2):317-325 (2008), which is hereby incorporated by reference in its entirety).

In embodiments directed to the prevention or treatment of BPD-associated PH, the individual requiring treatment may be a newly born infant (particularly, an infant less than about 3 weeks of age) that has been delivered pre-term, or a young child (eg a child< 5 years of age), and which may, in a particular embodiment, be treated for the prophylaxis (ie prevention) of BPD-associated PH or an improvement of BPD- associated PH symptoms (eg an improvement in pulmonary blood pressure to a level at or closer to the normal range). The skilled person will be familiar with methods for determining an amelioration in any symptoms of BPD-associated PH, including by reference to any of the signs or symptoms of the condition described above.

Type 2 antagonists

As used herein, a“type 2 antagonist” will be understood to encompass any molecule which antagonises, inhibits, or blocks the activation of the type 2 immune pathway of an individual. As used herein, the terms“type 2 antagonist”,“type 2 pathway inhibitor”, “type 2 inhibitor” can be used interchangeably. The skilled person will appreciate that the term “type 2 inhibitor” encompasses inhibitors or antagonists of activated Th2 cells (type 2 helper cells). Further, the skilled person will appreciate that a type 2 inhibitor or antagonist can be a molecule or agent which is an activator of a negative regulator of the type 2 pathway.

The skilled person will be familiar with conventional methods for determining whether the type 2 immune pathway is activated in an individual, including whether that activation is decreased following treatment or administration of a composition as described herein.

It will be understood that blockade of the type 2 pathway as herein described includes inhibition or antagonism of the activity of any one of the targets selected from the group consisting of: ITK, BTK, IL-9 (e.g., MEDI-528), IL-5 (e.g., Mepolizumab, CAS No. 196078-29-2; resilizumab), IL-13 (e.g., IMA-026, IMA-638 (also referred to as, anrukinzumab, INN No. 910649-32-0; QAX-576; IL4/IL13 trap), IL-4 (e.g., AER-001 , IL4/IL13 trap), OX40L, TSLP, IL-25, IL-33 and IgE (e.g., XOLAIR, QGE-031 ; MEDI- 4212); and receptors such as: IL-9 receptor, IL-5 receptor (e.g., MEDI-563 (benralizumab, CAS No. 1044511-01 -4), IL-4 receptor alpha (e.g., AMG-317, AIR-645), IL-13 receptor alphal (e.g., R-1671 ) and IL-13 receptor alpha2, 0X40, TSLP-receptor, IL-7 receptor alpha (a co-receptor for TSLP), IL17 receptor B (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2 (e.g., AMG-853, AP768, AP-761 , MLN6095, ACT129968), Fc epsilon receptor I, Fc epsilon Receptor II/CD23 (receptors for IgE), Flap (e.g., GSK2190915), Syk kinase (R-343, PF3526299); CCR4 (AMG-761 ), TLR9 (QAX-935) and multi-cytokine inhibitor of CCR3, IL5, IL3, GM-CSF (e.g., TPI ASM8). Examples of inhibitors of the aforementioned targets are disclosed in, for example, W02008/086395; W02006/085938; U.S. Pat. No. 7,615,213; U.S. Pat. No. 7,501 ,121 ; W02006/085938; WO 2007/080174; U.S. Pat. No. 7,807,788; W02005007699; W02007036745; W02009/009775; W02007/082068; WO2010/073119;

W02007/045477 ; W02008/134724; U.S. Pat. No. 2009/0047277; and W02008/127271 ).

In preferred embodiments of the invention, the type 2 antagonist is an antagonist of any one or more of the cytokines that are typically activated or which elicit a signalling cascade in the type 2 pathway. Type 2 cytokines which are preferably targeted in the methods of the present invention include but are not limited to IL-4 (interleukin 4), IL-5 (interleukin 5), IL-13 (interleukin 13), interleukin-25 (IL-25), IL-33 (interleukin 33) and TSLP (thymic stromal lymphopoietin).

A therapeutic agent a provided herein includes an agent that can bind to the target identified herein above, such as a recombinant or isolated polypeptide(s) (e.g., an antibody, an immunoadhesin or a peptibody (peptide-Fc fusion) or protein mimetic), an aptamer or a small molecule that can bind to a protein or a nucleic acid molecule that can bind to a nucleic acid molecule encoding a target identified herein (i.e. , siRNA).

The term “small molecule” refers to an organic molecule having a molecular weight between 50 Daltons to 2500 Daltons.

The term“antibody” is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single chain antibodies, multi-specific antibodies and fragments of antibodies. Such antibodies can be chimeric, humanized, human and synthetic.

The term“antisense compound” refers to a nucleic acid molecule that binds to a nucleic acid encoding a cytokine as described herein, and reduces or prevents the expression of the cytokine. Alternatively, the antisense compound may bind to a nucleic acid encoding a type 2 cytokine receptor and inhibit the expression of a receptor for binding to a type 2 cytokine. In any method or composition of the invention, an antisense compound may inhibit expression of human IL-4Ra, IL-13Ra proteins and/or expression of functional IL-4 and IL-13 receptors. Methods for designing and formulating such antisense compounds are described in W02010120511 , the entire contents of which are hereby incorporated by reference in their entirety.

In preferred embodiments, the type 2 antagonist is a monoclonal antibody which specifically binds to a target in the type 2 pathway. For example, the antibody may specifically bind to a target with an affinity which is preferably greater than 10⁶ M^-1, 10⁷ M^-1, 10⁸ M^-1, 10⁹ M^-1, 10¹° M^-1, 10¹¹ M^-1, or 10¹² M^-1.

Preferably, the antagonist inhibits the activity of its target by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%.

In the case of an antagonist that decreases expression of a gene encoding a relevant component of the type 2 pathway, preferably the expression of the gene is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%.

The term "isolated" in relation to a protein or polypeptide means that by virtue of its origin or source of derivation is not associated with naturally-associated components that accompany it in its native state; is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art. By“substantially purified” is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents. The term “recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising or consisting of a protein, this term does not encompass the equivalent protein naturally-occurring within a subject’s body. However, if such a protein is isolated, it is to be considered an isolated protein for use according to the methods or in the compositions described herein. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

In still further methods and compositions of the present invention, the type 2 antagonist utilised can be one which is commercially available, and can be conveniently obtained by the skilled person.

As used herein“an anti-IL-13/IL-4 antagonist” refers to a therapeutic agent that inhibits IL-13 and/or IL-4 signalling. (An IL-13/IL-4 antagonist may be appropriate given the overlapping actions of the IL-13 and IL-4 cytokines. For example, and without wishing to be bound by theory, the inventors understand that IL-4 and IL-13 signalling is mediated via a common receptor, IL-13Ra1. Accordingly, targeting of IL-13Ra1 would inhibit the signalling of both IL-13 and IL-4). In certain examples, anti-IL-13/IL-4 pathway inhibitors include inhibitors of the interaction of IL-13 and/or IL-4 with their receptor(s). Such inhibitors include, but are not limited to, anti-IL-13 binding agents, anti-IL-4 binding agents, anti-IL-13/IL-4 bispecific binding agents, anti-IL-4receptoralpha binding agents, anti-IL-13receptoralpha1 binding agents and anti-IL-13 receptoralpha2 binding agents. Antibodies, including single domain antibodies that can bind IL-13, IL-4, (including bispecific antibody with a single domain binding IL-13 and a single domain binding IL-4), IL-13Ralpha1 , IL-13Ralpha2 or IL-4Ralpha are specifically included as inhibitors. It should be understood that molecules that can bind more than one target are included.

As used herein,“anti-IL-4 binding agents” refers to agents that bind to human IL- 4. Such binding agents can include a small molecule, an aptamer or a polypeptide. Such polypeptide can include, but is not limited to, a polypeptide(s) selected from the group consisting of an immunoadhesin, an antibody, a peptibody and a peptide. According to one embodiment, the binding agent binds to a human IL-4 sequence with an affinity between 1 mM - 1 pM. Specific examples of anti-IL-4 binding agents can include soluble IL-4 receptor alpha (e.g., extracellular domain of IL-4 receptor fused to a human Fc region), anti-IL-4 antibody, and soluble IL-13 receptoralphal (e.g., extracellular domain of IL-13 receptoralphal fused to a human Fc region).

As used herein,“anti-IL-4 receptor alpha binding agents” refers to an agent that binds to human IL-4 receptor alpha. Such binding agents can include a small molecule, an aptamer or a polypeptide. Such polypeptide can include, but is not limited to, a polypeptide(s) selected from the group consisting of an immunoadhesin, an antibody, a peptibody and a peptide. According to one embodiment, the binding agent binds to a human IL-4 receptor alpha sequence with an affinity between 1 mM -1 pM. Specific examples of anti-IL-4 receptor alpha binding agents can include anti-IL-4 receptor alpha antibodies.

As used herein, an“anti-IL-13 binding agent” refers to agent that binds to human IL-13. Such binding agents can include a small molecule, aptamer or a polypeptide. Such polypeptide can include, but is not limited to, a polypeptide(s) selected from the group consisting of an immunoadhesin, an antibody, a peptibody and a peptide. According to one embodiment, the binding agent binds to a human IL-13 sequence with an affinity between 1 mM -1 pM. Specific examples of anti-IL-13 binding agents can include anti-IL-13 antibodies, soluble IL-13receptoralpha2 fused to a human Fc, soluble IL-4receptoralpha fused to a human Fc, soluble IL-13 receptor alpha fused to a human Fc. Examples of soluble IL-13receptoralpha-Fc fusion proteins are known in the art, and can be purchased commercially including from Creative Biomaterials, Aero Biosystems, Sino Biological and G&P Biosciences.

As used herein, an“anti-IL-13 receptor alphal binding agent” refers to an agent that specifically binds to human IL-13 receptor alpha Such binding agents can include a small molecule, aptamer or a polypeptide. Such polypeptide can include, but is not limited to, a polypeptide(s) selected from the group consisting of an immunoadhesin, an antibody, a peptibody and a peptide. According to one embodiment, the binding agent binds to a human IL-13 receptor alphal sequence with an affinity between 1 mM-1 pM. Specific examples of anti-IL13 receptor alphal binding agents can include anti-IL13 receptor alphal antibodies. As used herein, an“anti-IL-13 receptoralpha2 binding agents” refers to an agent that specifically binds to human IL-13 receptoralpha2. Such binding agents can include a small molecule, an aptamer or a polypeptide. Such polypeptide can include, but is not limited to, a polypeptide(s) selected from the group consisting of an immunoadhesin, an antibody, a peptibody and a peptide. According to one embodiment, the binding agent binds to a human IL-13 receptor alpha2 sequence with an affinity between 1 mM-1 pM. Specific examples of anti-IL-13 receptoralpha2 binding agents can include anti-IL-13 receptor alpha2 antibodies.

As used herein, an“anti-IL-5 binding agent” refers to agent that binds to human IL-5. Such binding agents can include a small molecule, aptamer or a polypeptide. Such polypeptide can include, but is not limited to, a polypeptide(s) selected from the group consisting of an immunoadhesin, an antibody, a peptibody and a peptide. According to one embodiment, the binding agent binds to a human IL-5 sequence with an affinity between 1 mM -1 pM. Specific examples of anti-IL-5 binding agents can include anti-IL-5 antibodies, soluble IL5 receptor fused to a human Fc.

As used herein, an“anti-IL-5 receptor binding agents” refers to an agent that specifically binds to human IL-5 receptor alpha subunit or CSF2RB. Such binding agents can include a small molecule, an aptamer or a polypeptide. Such polypeptide can include, but is not limited to, a polypeptide(s) selected from the group consisting of an immunoadhesin, an antibody, a peptibody and a peptide. According to one embodiment, the binding agent binds to a human IL-5 alpha subunit or CSF2RB with an affinity between 1 mM-1 pM.

In preferred methods or composition of the invention, the type 2 antagonist is an IL-4 (BSF1 ) antagonist. Examples of IL-4 inhibitors or antagonists are known in the art. Suitable examples include, but not limited to:

- pitrakinra (Aeovant ®, Bayer), which is a 15-kDa human recombinant protein of wild-type human interleukin-4 (IL-4). It is an IL-4 and IL-13 antagonist that blocks signalling of IL-4 and interleukin-13 (IL-13) by preventing assembly of IL-4 receptor alpha (IL-4Ra) with either IL-2Ry or IL-13Ra. The targets of pitrakinra action are inflammatory cells (dendritic cells, Th2 cells, B cells) and structural cells (smooth muscle, endothelium, epithelium) that express IL-4Ra. The drug has been applied both as a subcutaneous injection and as an inhalable drug.

- pascolizumab (SB 240683, GSK and Protein Design Laboratories, Inc.

(Fremont, CA, USA), which blocks the interaction of IL-4 with its receptor.

In any method or composition of the present invention, the type 2 antagonist is an IL-4 receptor (CD124) antagonist. Examples of IL-4 receptor antagonists are known in the art and include:

- dupilimab (Dupixent ®, SAR231893, REGN668, produced by Sanofi), which is a monoclonal antibody that binds to the alpha subunit of the interleukin-4 receptor (IL-4Ra). Through blockade of IL-4Ra, dupilumab modulates signalling of both the interleukin 4 and interleukin 13 pathway. This antibody is more thoroughly described in WO2010053751 , the entire contents of which are hereby incorporated by reference in their entirety.

In any method or composition of the invention, the type 2 antagonist is an IL-5 antagonist, including but not limited to:

- mepolizumab (Nucala®, GSK), which is a humanized monoclonal antibody that binds to IL-5 and prevents it from binding to its receptor, more specifically the interleukin 5 receptor alpha subunit, on the surface of eosinophil white blood cells. See Greenfeder, et al. , Respiratory Research, 2(2):71 -79 (2001 ), the entire contents of which are hereby incorporated by reference in their entirety.

- reslizumab (Cinqair®), also known as CTx55700, SCH-55700 or CEP-38072 and which binds specifically to IL-5, and blocks its signalling. Reslizumab is a "humanized" (from rat) divalent monoclonal antibody (mAb) with an lgG4 kappa isotype, with binding affinity for a specific epitope on the human interleukin-5 (IL-5) molecule. Reslizumab is a neutralizing antibody that is believed to block IL-5 dependent cell proliferation and/or eosinophil production. Reslizumab is described in, for example, Walsh, GM (2009) "Reslizumab, a humanized anti-IL-5 mAb for the treatment of eosinophil- mediated inflammatory conditions" Current opinion in molecular therapeutics 11 (3): 329-36; US 6,056,957 (Chou); US 6,451 ,982 (Chou); US RE39,548, (Bodmer), each of which is incorporated herein by reference.

In any method or composition of the invention, the type 2 antagonist is an IL-5 receptor antagonist. IL-5 receptor antagonists are known in the art, and suitable examples include but are not limited to:

- benralizumab ((MEDI-563, Fasenra®), is a humanized monoclonal antibody (mAb) that binds to the alpha chain of the interleukin-5 receptor alpha (IL- 5Ra), which is expressed on eosinophils and basophils. It induces apoptosis of these cells via antibody-dependent cell cytotoxicity. Information regarding benralizumab (or fragments thereof) can be found in U.S. Patent Application Publication No. US 2010/0291073 A1 , the disclosure of which is incorporated herein by reference in its entirety.

In any method or composition of the invention, the type 2 antagonist is an IL-13 antagonist. Examples of IL-13 inhibitors and antagonists are known in the art. Suitable examples include, but are not limited to:

- anrukizumab (IMA-638, PF-05230917), which is a humanised monoclonal antibody which binds to IL-13 and blocks its action. See Gauvreau GM, Boulet LP, Cockcroft DW, et al. Am J Respir Crit Care Med. 2010: Epub 2010/11/09., the entire contents of which are hereby incorporated by reference.

- lebrikizumab (TNX-650, MILR1444A, RG3637, produced by Genentech), a monoclonal antibody which binds specifically to IL-13.

- Tralokinumab (CAT-354, produced by AstraZeneca) which is a human monoclonal antibody which binds to and blocks the action of IL-13.

- RPC4046, (produced by AbbVie), which is a monoclonal antibody that blocked binding of IL-13 to both IL-13 receptors: IL-13Ra1 and IL-13Ra2 and is described in Tripp et al., (2017) Advances in Therapy, 34: 1364-1381 , the entire contents of which are hereby incorporated by reference in their entirety.

- GSK679586 (679586), a monoclonal antibody that inhibits binding of IL-13 to both IL-13Ra1 and IL-13Ra2 and is described in de Boever et al., (2014) Journal of Allergy and Clinical Immunology, 133: 989-96, the entire contents of which are hereby incorporated by reference in their entirety. - QAX576 (produced by Novartis), which is described in WO2015198146 and Rothenberg et al. , (2015) Journal of Allergy and Clinical Immunology, 135: 500-7, the entire contents of which are hereby incorporated by reference in their entirety.

In certain embodiments, an anti-IL-13 antibody is one such as is described in US9684000, the entire contents of which are hereby incorporated by reference.

In any method or composition of the invention, the type 2 antagonist is an IL-13 receptor antagonist, including, but not limited to:

- ASLAN004 (CSL334, produced by CSL/Aslan Pharmaceuticals), a monoclonal antibody which binds specifically to the IL-13 receptor alpha (IL- 13RA1 , CD231A1 ). This antibody has been found to neutralise both interleukin 4 (IL-4) and interleukin 13 (IL-13) by binding to the IL-13Ra1. Methods for making ASLAN004 are described in detail in W02008060813, and W02008060814, the entire contents of which are hereby incorporated by reference.

In any method or composition of the invention, the type 2 antagonist is a bi- specific antibody, including but not limited to:

- SAR-156597 (Sanofi), which is a monoclonal antibody that inhibits the signalling of both IL-4 and IL-13.

In any method or composition of the invention, the type 2 antagonist is a TSLP- antagonist. Examples of such antagonists are known in the art, and include but are not limited to:

- tezepelumab (AMG-157), which is a human monoclonal antibody that blocks the interaction of thymic stromal lymphopoietin (TSLP) with the TSLP receptor.

Methods of determining risk of BPD

Because BPD clinically is not diagnosed in prematurely born infants until some time after birth, e.g. 36 weeks gestation, therapies to treat at-risk infants may be administered before the disease is formally diagnosed. However, not all pre-term infants will develop BPD, and as such, there is benefit in being able to more clearly discriminate between pre-term infants who are at greatest risk of developing BPD, from those who are at low risk, so as to minimise the unnecessary exposure of vulnerable individuals to potentially invasive therapy or unnecessary medication.

As such, the present invention also includes methods for determining whether an individual is at risk of BPD, the method comprising:

- determining the level of type 2 immune activation in the individual,

- comparing the level of type 2 immune activation in individual with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term individuals who developed BPD;

- determining that the individual is at risk of BPD if the level of type 2 activation in the individual is the same or greater than the level of type 2 activation in the reference data set;

- determining that the individual is not at risk of BPD if the level of type 2 activation in the individual is less than the level of type 2 activation in the reference data set.

Further, the present invention includes methods for determining whether an individual is at risk of BPD, the method comprising:

- determining the level of type 2 immune activation in a test biological sample from the individual for whom risk of BPD is to be determined,

- comparing the level of type 2 immune activation in test sample with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term individuals who developed BPD;

- determining that the individual is at risk of BPD if the level of type 2 activation in test sample is the same or greater than the level of type 2 activation in the reference data set;

- determining that the individual is not at risk of BPD if the level of type 2 activation in the test sample is less than the level of type 2 activation in the reference data set. In certain embodiments, the present invention also includes methods for determining whether an individual is at risk of BPD, the method comprising:

- measuring in the test sample, the levels of one or more type 2 cytokines, thereby determining the level of type 2 immune activation in the individual,

- comparing the level of type 2 immune activation in the test sample, with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term individuals who developed BPD;

- determining that the individual is at risk of BPD if the level of type 2 activation in the test sample is the same or greater than the level of type 2 activation in the reference data set;

- determining that the individual is not at risk of BPD if the level of type 2 activation in the test sample is less than the level of type 2 activation in the reference data set.

In addition, the present invention includes methods for determining whether an individual is at risk of BPD, the method comprising:

- determining that the individual is at high risk of BPD if the level of type 2 activation in test sample is the same or greater than the level of type 2 activation in the reference data set;

- determining that the individual is at low risk of BPD if the level of type 2 activation in the test sample is less than the level of type 2 activation in the reference data set.

As used herein, a“high risk” of BPD is determined where the results of the above mentioned test indicate that there is at least a 50% chance of the infant developing BPD, preferably at least a 60% chance, 70% chance, 80% chance, 90% chance or 95% chance.

As used herein, a“low risk” of BPD is determined where the results of the above mentioned test indicate that there is no more than a 50% chance of the infant developing BPD, preferably no more than a 40% chance, 30% chance, 20% chance, 10% chance or 5% chance.

The present methods are also useful for identifying a pre-term infant requiring treatment for BPD, the method comprising:

- providing a test biological sample from a pre-term infant,

- measuring in the test sample, the levels of one or more type 2 cytokines, thereby determining the level of type 2 immune activation in the infant, comparing the level of type 2 immune activation in the test sample, with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term infants who developed BPD;

- determining to treat the infant for BPD if the level of type 2 activation in the test sample is the same or greater than the level of type 2 activation in the reference data set;

- determining not to treat the infant for BPD if the level of type 2 activation in the test sample if less than the level of type 2 activation in the reference data set.

The test sample from the individual can be any biological sample obtained which enables the skilled person to determine whether there is an increase or elevation in the type 2 immune pathway in the individual. The term“biological sample” as used herein includes, but is not limited to, blood (including serum, plasma or cells derived therefrom), sputum, bronchioalveolar lavage fluid, tissue biopsies (e.g., lung samples), and nasal samples including nasal swabs or nasal polyps.

In a preferred embodiment, the test sample is a plasma sample or a sample of cord blood Type 2 immune activation can be determined by any method known to the skilled person, including by testing for the levels of one or more type 2 cytokines (ie, cytokines which are associated with the type 2 immune pathway). Preferably, the type 2 cytokines are selected from the group consisting of: IL-4, IL-5, IL-13, IL-25, IL-33 and TSLP. More preferably, the type 2 cytokine that is measured in the test sample is IL-13. Type 2 activation can also be determined by reference to other physiological parameters, including for example, increased IgE.

The treatment for BPD can be any treatment herein described (ie, any treatment described herein for antagonising or inhibiting type 2 (including Th2) immune activation in the infant. Alternatively, or in conjunction, the infant may be treated with other known treatments for BPD, including treatment with a caffeine; a diuretic (such as furosemide, hydrochlorothiazide and spironolactone); a non-specific phosphodiesterase inhibitor (eg pentoxifylline), phosphodiesterase-4 inhibitors (eg rolipram), phosphodiesterase-5 inhibitors (eg sildenafil); an exogenous surfactant, or an anti-inflammatory agent selected from a corticosteroid (e.g., dexamethasone); an interleukin 1 (IL-1 ) antagonist, an IL-1 receptor antagonist, a transforming growth factor (TGF)-p antagonist, curcumin; inhaled nitric oxide (iNO); azithromycin; a bronchodilator (e.g., salbutamol, albuterol, or levalbuterol) or antihypertensive (such as bosentan).

The skilled person will be familiar with the appropriate dose and route of administration of these therapeutic compounds.

The reference data set will typically contain data from one or more individuals (preferably pre-term infants) who have previously had or currently have BPD. The skilled person will be familiar with methods for determining statically significant differences between the level of type 2 immune activation (including, for example, levels of type 2 cytokines in a biological sample obtained from an individual), for the purposes of determining the risk of development of BPD.

In alternative embodiments, a reference data set from pre-term infants who did not develop BPD may be used. It will be appreciated that in this circumstance, the reference data set is useful for demonstrating a level of type 2 activation (or lack thereof) that is indicative of a low risk of BPD. As such, if the test sample of an infant has a level of type 2 activation that is the same or lower that the reference data set in this scenario, the skilled person will understand that the infant is likely at low risk of BPD.

The reference data set may contain information on the level of type 2 immune activation, for example by way of the level of different type 2 cytokines (including chemokines) in a biological sample from one or more individuals. It will be appreciated, however, that any data which provides an indication of type 2 immune activation will be suitable for inclusion in the reference data set, for the purpose of the methods described herein.

Compositions and formulations

“Administration” is not limited to any particular formulation, delivery system, or route and may include, for example, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection) rectal, topical, transdermal, or oral (for example, in capsules, suspensions, or tablets). Administration to an individual may occur in a single dose, bolus dose, or in repeat administrations, and in any of a variety of pharmaceutical compositions containing physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition. Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are known (see, e.g., Physicians' Desk Reference® 2003, 57th ed., Medical Economics Company, 2002; and Remington: The Science and Practice of Pharmacy, eds. Gennado et al. , 20th ed, Lippincott, Williams & Wilkins, 2000).

In one embodiment, administration of compositions comprising a type 2 modulator (including for example, an inhibitor or antagonist) can be via a pulmonary route, including in the form of an inhalant as a powdered or liquid aerosol. Aerosolized formulations can be droplets or powder particles less than 10 μm in diameter. For example, the antagonist can be solubilized in a micronized hydrophobic/hydrophilic emulsion. Such inhalants may be administered by nebulizer, inhaler, intratracheal routes, intraoral or intranasal routes. Aerosols for the delivery of therapeutic agents to the respiratory tract, including aerodynamically light particles, are described, for example, in U.S. Patent Application Publication No. 2005/0244341. Administration of an antagonist to an infant may also be accomplished by means of gene therapy, wherein a nucleic acid sequence encoding the antagonist is administered to the patient in vivo or to cells in vitro, which are then introduced into a patient, and the antagonist (e.g., antisense RNA, snRNA) is expressed from an appropriate nucleic acid vector sequence. Administration of a type 2 antagonist may be effected by gene transfer using a vector comprising cDNA encoding the antagonist, for example cDNA encoding the antagonist.

The type 2 antagonist can be administered to the infant in combination with any other agents or therapies that are currently used or will be used to treat premature infants at risk for or diagnosed with BPD. Poor vitamin A status during the first month of life significantly increases the risk of developing BPD. Studies have also found that dexamethasone can increase plasma levels of vitamin A; which can help wean infants off oxygen therapy, thereby preventing BPD.

Bronchodilator medications are sometimes used to open the airways of the lungs by relaxing the muscles around the airways. Anti-inflammatory medications are used to reduce airway swelling in more severely ill babies whose wheezing and respiratory distress are occasionally difficult to control with bronchodilators only. Accordingly, treatments for BPD that may be used in combination with the methods of the invention include surfactant, oxygen therapy, ventilator therapy, steroids, vitamin A, inhaled nitric oxide, high calorie nutritional formulations, intravenous feeding, antibiotics, fluid restriction and diuretics to decrease water accumulation in the lungs, and physical therapy to improve muscle performance and to help the lungs expel mucus.

The skilled person will be familiar with methods for determining the appropriate dose of a therapeutic agent as herein described, including determining the appropriate dose based on the age and weight of the individual.

For example, a monoclonal antibody as described herein may be administered at 0.1 mg/kg to 100 mg/kg of the patient's body weight. In one embodiment, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight. In another embodiment, the dose is 1 mg/kg to 10 mg/kg of the patient's body weight. Alternatively, the antibody can be administered as a flat dose. In one embodiment the antibody is administered in as a 125-1000 mg flat dose (i.e. , not weight dependent), by subcutaneous injection or by intravenous injection, at a frequency of time selected from the group consisting of: every day, 2-5 times a week, once every week, every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 1 month, 2 months, 3 month or 4 months.

The type 2 antagonist is preferably administered, adapted and/or formulated in a manner or medicament ensuring that, upon administration to the subject, an effective amount of said agent is delivered to the lungs and airways of the subject. As such, the active agent may be, for example, formulated into any suitable medicament; such as a pharmaceutical composition for oral, buccal, nasal, subcutaneous, intramuscular, pulmonary, endotracheal and intravenous administration.

Typically, the pharmaceutical composition will be administered to the individual requiring treatment, in an amount which is effective to achieve a prophylactic and/or therapeutic effect, and may therefore provide between about 0.1 and about 250 mg/kg body weight per day of the active agent, more preferably between about 0.5 and about 100 mg/kg body weight per day of the active agent and, still more preferably between about 1 and about 25 mg/kg body weight per day of the active agent. Most preferably, the pharmaceutical composition will be administered to the subject in an amount of about 5 to about 10 mg/kg body weight per day of the active agent. A suitable pharmaceutical composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective result. However, notwithstanding the above, it will be understood by those skilled in the art that the administered amount of the active agent, and the frequency of administration for any particular subject, may vary and depend upon a variety of factors including the activity of the active agent, the metabolic stability and length of action of the active agent, the age, body weight, sex, health, gestational age, route and time of administration, rate of excretion of the active agent, and the severity of the BPD and/or B PS-associated PH to be treated. The type 2 antagonist may be used in any of the methods and uses of the present invention in combination with one or more other useful therapeutic compounds or substances. For example, the type 2 antagonist may be used in a combination therapy/treatment with an anti-inflammatory agent(s) such as caffeine; a diuretic (such as furosemide, hydrochlorothiazide and spironolactone); a non-specific phosphodiesterase inhibitor (eg pentoxifylline), phosphodiesterase-4 inhibitors (eg rolipram), phosphodiesterase-5 inhibitors (eg sildenafil), corticosteroids (eg dexamethasone); curcumin; inhaled nitric oxide (iNO); azithromycin; a bronchodilator or antihypertensive. The skilled person will be familiar with the appropriate dose and route of administration of these therapeutic compounds.

Additionally, or alternatively, the type 2 antagonist may be used in combination with an antagonist which blocks any alternative inflammatory pathway, for example, an interleukin-1 receptor antagonist (IL-1 Ra) including that described in WO2014194364, the entire contents of which are hereby incorporated in their entirety.

It will be understood that these examples are intended to demonstrate these and other aspects of the invention and although the examples describe certain embodiments of the invention, it will be understood that the examples do not limit these embodiments to these things. Various changes can be made and equivalents can be substituted and modifications made without departing from the aspects and/or principles of the invention mentioned above. All such changes, equivalents and modifications are intended to be within the scope of the claims set forth herein. Examples

Example 1 Human peripheral and cord blood

Preterm infant recruitment: Extremely preterm infants with a gestational birth age between 24+0 and 28+6 weeks were recruited from three tertiary birthing centres with co-located neonatal intensive care units (NICU) within Victoria, Australia. These centres were Mercy Hospital for Women, Heidelberg, Monash Newborn-Monash Medical Centre, Clayton and the Royal Women’s Hospital, Parkville. Suitable parents, identified once admitted to hospital for impending preterm birth, were approached by a member of the recruitment team. Parents were provided with verbal information about the study and a detailed plain language parent information statement. Parents who expressed interest were invited to sign the consent form. Infants were not approached and excluded if they had any major congenital abnormalities or likely imminent demise shortly after birth.

Term infant controls: For collection of cord blood samples from term infant controls, infants born between 37 and 41 weeks of gestation were recruited from Monash Medical Centre and Jessie McPherson Private Hospital, also a tertiary birthing centre with a NICU. To qualify as a healthy term control, infants had to have no health concerns at birth and no maternal or perinatal medical history that could affect their health, including but not limited to intrauterine growth restriction, pre-eclampsia, chorioamnionitis or other maternal infections, gestational diabetes, pre-existing asthma or thyroid disease.

Postnatal peripheral blood samples of term infants were recruited at the day surgery admission centre of Monash Children’s Hospital. To be included in the study, term infants had to be born between 37 and 41 weeks and be between 3 days and 12 weeks old when undergoing surgery for conditions that do not entail systemic inflammation (eg surgery for hernia repair). Consistent with cord blood samples of term infants, these postnatal controls also had to have no health conditions that implicate immune diseases and no maternal or perinatal medical history that could affect their health. Adult controls: Recruitment of healthy adults aged between 20 and 50 years, with no long-term medications or any anti-inflammatory medication a week prior to the samples being taken. Further healthy adults were not taken into the study when they suffered from any acute illness at the time of blood sampling.

Sample collection and sampling time points: All samples were drawn into sodium citrate tubes to accurately to the pre-marked levels where possible. The following citrate tubes were used in this study:

3.5 ml VACUETTE 3.2% 9NC Sodium citrate coagulation tube (Greiner Bio-One, Austria, Catalogue number (Cat #): 454327)

1 ml VACUETTE 3.2% 9NC Sodium citrate coagulation tube (Greiner Bio-One, Austria, Cat #: 454320)

1 ml MiniCollect 3.2% 9NC Sodium citrate coagulation tube (Greiner Bio-One, Austria, Cat #: 450413)

1.4 ml S-Monovette 3.2% 9NC Sodium citrate tube (Sarstedt, Germany, Cat #: 06.1668.100)

Self-labelled microcentrifuge tube containing 3.2% 0.109M buffered sodium citrate (Medicago, Sweden, Cat #: 12-8480-10, reconstituted with deionised water and sterile filtered)

55.6 pi sodium citrate were added to host 500 mI blood

88.9 mI sodium citrate were added to host 800 mI blood

Preterm infants were studied longitudinally across five time points including cord blood at birth and peripheral blood between 8 and 16 hours (also known as day 1 sample for simplification) and on days 7 and 14 and at 36 weeks corrected gestational age from an indwelling arterial catheter or a peripheral vein.

3.5 ml cord blood were collected from umbilical cord following delivery of infant and placenta when researchers were notified of preterm birth. 0.5 ml to 1 ml of peripheral blood were collected from infants at different time points postnatally depending on infants’ weight and age to minimise the impact on infants’ haemodynamic status (Table 2). Postnatal sampling was coordinated with clinical team to coincide with routine clinical blood draws whenever possible to avoid additional discomfort.

Table 2: Volume-weight table for postnatal blood sampling of preterm infants.

Term infant cord blood: 3.5 ml cord blood samples were collected from umbilical cord following delivery of term infant and placenta.

Peripheral term infant blood: 1 ml peripheral blood samples were collected from peripheral vein via cannula inserted by paediatric anaesthetist following induction of anaesthetics in operating theatre.

Adult controls: 3.5 ml blood were drawn from peripheral veins of healthy volunteers into citrate tubes.

Sample preparation: Citrated blood samples from preterm infants were centrifuged at 300g for 15 minutes at room temperature (RT) onsite in hospital laboratories within 2 hours of sample collection. The plasma layer was transferred into cryotubes and frozen in -80°C freezer for later analysis. The remaining blood was diluted 1 :4 in culture media [RPMI 1640 (Gibco, NY, USA, Cat #: 21870-076) with 1 % human serum (Sigma-Aldrich, MO, USA) and 1 :500 Mycozap Plus-PR (Lonza, Switzerland, Cat #: VZA-2022), an antibiotic and antifungal agent]. Cell stimulation: 200 mI resuspended blood cells were aliquoted to 12ml sterile polypropylene tubes (Greiner Bio-One, Austria, Cat #: 184261 ) and rested in an incubator at 37°C, 5% C02 for 2 hours with caps loosened. Samples were then stimulated with either 2 ng/ml phorbol 12-myristate 13-acetate (PMA) (EMD Chemicals- Merck Millipore, CA, USA, Cat #: 524400-5MG) and 250 ng/ml ionomycin (Sigma- Aldrich, Cat #: 10634-1 MG) or vehicle and incubated for a further 12 to 16 hours (depending on when samples were obtained). Simultaneously, 2 μg/ml brefeldin A (BFA) (Sigma-Aldrich, Cat #: B7651-5MG) was added for inhibiting transport of protein mediated by the Golgi apparatus, leading to accumulation of cytokines inside the endoplasmic reticulum and producing an enhanced cytokine signal for detection by flow cytometry_ENREF_671.

Flow cytometry -Staining protocol: Whole blood phenotyping was performed following the set incubation time. Cells were first resuspended with 1 ml Dulbecco’s phosphate buffered saline (PBS) (Gibco, Cat #: 14200166) and transferred to 1.5 ml Eppendorf tubes. Then cells were pelleted by centrifugation (300g for 15 minutes at RT) with supernatant removed via vacuum aspiration. 10 mI eBioscience Fluman Fc Receptor Binding Inhibitor (Invitrogen, CA, USA, Cat #: 14-9161 -73) were added to every 50 mI of cell pellet and incubated for 20 minutes at RT.

10 mI cells were transferred to corresponding 5 ml round bottom polystyrene tubes (Technoplas, SA, Australia, Cat #: S7512) containing 90 mI housemade flow buffer [PBS + 2% foetal bovine serum (FBS) (Gibco, Cat #: 10437028) + 2 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, Cat #: ED-100G)] to act as unstained and viability only controls. Remaining 50 mI cells were surface stained with a premixed antibody cocktail for 30 minutes at RT in the dark. Antibodies were titrated prior to use to determine the optimum volume required for analysis.

Each tube was fixed and permeabilised with 250 mI of eBioscience FOXP3/ transcription factor staining buffer set (Invitrogen, Cat #: 00-5523-00) for 30-60 minutes at RT in the dark before being washed once with 1 ml of 1X Permeabilization Buffer (Invitrogen, Cat #: 00-8333-56) at 500g for 5 minutes at RT. Supernatant were aspirated via vacuum aspiration to 80 mI residual volume. Premixed antibody cocktail of the intracellular staining antibodies (see Table 3b) were added to cell mixture and incubated for 1 hour at RT. Samples were then washed once with 2 ml flow buffer at 500g for 5 mins at RT with supernatant decanted. Tubes were stored in the dark at 4°C wrapped in foil until acquisition.

Conventionally, single colour controls for compensations were prepared with the same tissue samples. Due to the limited availability of samples from preterm infants, single colour controls were prepared with BD CompBead Anti-Mouse Ig, k / Negative Control (FBS) Compensation Particles Set (BD Biosciences, CA, USA, Cat #: 552843) according to manufacturer’s instructions. Compensation control of viability stain was prepared with ArC amine reactive compensation bead kit (Invitrogen, Cat #: A10346) according to manufacturer’s instructions. In addition, single colour controls and fluorescence-minus-one (FMO) controls for each colour were done in cord blood samples of some term infant and adult controls. Additional isotype controls for IL-4 were stained with Mouse lgG1 k isotype control PE-Cyanine(Cy)7 (Invitrogen, Cat #: 25- 4714-42) for gating.

Instrumentation and data acquisition: Data were acquired on the BD LSR II which was calibrated daily using BD FACSDiva CS&T Research Beads (BD Biosciences, Cat #: 655051 ). We used unstained control and the IL-4 isotype control samples to establish photomultiplier tube (PMT) voltages on cytometer each time before sample acquisition. We acquired 5000 events of single colour compensation bead controls for later compensation, 50000 events for all other controls (unstained, single colour and FMO) and minimum 50000 events for stained samples.

Flow cytometric data analysis: Post-acquisition compensation was performed using single colour controls and data were analysed using FlowJo software (Version: X 10.2; FlowJo, LLC, OR, USA) on a Windows 10 workstation. Any plots with less than 500 events in the parent gate were excluded from analysis.

Results: BPD infants had more IL-4 in cord and peripheral blood than non-BPD infants. Cord and peripheral blood samples were collected from preterm infants (between 24+0 and 28+6 weeks GA) at birth, day 1 , weeks 1 and 2 and 36 weeks cGA in addition to controls including healthy term infants (born between 37+0 and 41 +0 weeks GA) and adults. Blood cells were treated with Brefeldin A (2 μg/ml) and volume- matched vehicle or PMA (2ng/ml) and ionomycin (250ng/ml) for 12-16 hours, then stained and analyzed by flow cytometry. Percentage of IL-4+ cells among viable CD45+CD3+CD4+ T cells are shown in all groups with preterm infants separated into BPD and no BPD groups as defined (a) is for cells treated with vehicle while (b) displays cells with PMA and ionomycin (median ± interquartile range, n = 5-10 for no BPD, 22-41 for BPD, 11 for term CB, 9 for term PN, 5 for adult, *P<0.05 and **P<0.005 for BPD vs. no BPD, # P<0.05 for vs. term PN, # P<0.05 for vs. adult). The type 2 pathway (measured in peripheral blood by determining IL-4 in T cells) is activated strongly and for an extended period of time in those infants that go on to develop BPD. This is not the case for the type 1 , type 3 and Treg pathways (data not shown).

Animal studies: C57BI/6J mice were initially purchased from Jackson Laboratory, bred and housed under specific pathogen free conditions. B6.129S2(C)- Stat6tm1 Gru/J mice were obtained by the method described in Nguyen et al. , (2016) Eur. J. Immunol. 46: 307-318), bred and housed under specific pathogen free conditions. All experimental procedures conformed with the guidelines established by the NHMRC, had the approval of Monash University’s Ethics Committee, and complied with the principles expressed in the Declaration of Helsinki. BPD was induced using a murine model of BPD that has been previously established (Nold et al., (2013), PNAS 110(35): 14384-9). Briefly, pregnant STAT6 KO and C57BL/6 dams were injected at 14 d of gestation with 150 μg/kg of lipopolysaccharide (LPS) intraperitoneally (i.p.). Within 24 h after birth, pups and dams were randomised and exposed to gas at 10 L/min with an F1O2 of 0.65 (hyperoxia) or 0.21 (room air) continuously for either 3, 5 or 28 d. Temperature (22°C) and humidity (50-60%) were kept constant and light was cycled in a 12 h day/night rhythm. Dams were rotated between the room air and hyperoxia groups in a 3 d cycle to protect them from prolonged hyperoxia and limit dam effects on study outcome. At 3, 5 or 28d, mice were anesthetized with isoflurane, and then humanely killed by cervical dislocation or decapitation.

Example 2: Cytokine profile in murine lungs at d3

Murine protein analysis: At day 3 lungs were harvested, washed in ice-cold PBS, snap frozen in liquid nitrogen and stored at -80 °C. For analysis, the lungs were homogenized in lysis buffer (PMID 23946428) using an Ultra Turrax. The homogenate was centrifuged for 10 min at 14,000 ^c g and the supernatant was assayed for protein. All results were normalized to total protein concentration Proteome Profiler Arrays (R&D Systems) were used as described previously (Nold et al. , 2013 supra).

Results: Cytokines were measured using a cytokine profiler from R&D systems characteristic of type 1 and type 2 polarisation in samples from an earlier study. Pronounced increases in IL-4 (4-fold), IL-5 (7-fold) and IL-13 (53-fold), were observed in cytokines central to type 2 signaling, in lung lysates from mouse pups on day 3 of life exposed to perinatal lipopolysaccharide (LPS) and postnatal hyperoxia of 65% compared to no antenatal LPS, air vehicle-controls (Fig. 2a). In contrast, the increase in the type 1 cytokines IFNy and IL 12 (2.4-fold) was markedly weaker.

Example 3 Alveolar structure and lung function at d28

Murine lung preparation & histology. After cervical dislocation, 28d lungs were intubated via the trachea and the left lobe was tied off at the main bronchus and removed for flow cytometry or snap frozen in liquid nitrogen and stored at -80 °C for molecular analysis. The right lung was fixed with 4% paraformaldehyde (PFA) (pH 7.4, instilled at a pressure of 20 cm H20), removed from the thorax, kept in 4% PFA for a minimum of 24 h, then immediately processed for paraffin embedding and sectioning. Lung tissue was cut into 4-μm sections, H&E-stained and scanned for histological analysis on an Aperio Scanscope (ePathology Solutions). Images of H&E stained whole lungs were analysed using ImageJ software (National Institutes of Health) by measuring the alveolar number and size and by calculating the surface area to volume ratio (SVR).

Murine blood analysis: Blood collection was carried out under general anesthesia (isoflurane drop jar method) via retro-orbital bleeding in 28 d old mice. Blood was collected using preheparinised capillaries (safeCLINITUBES), and a sample size of 85microl was immediately analysed for p02, hemoglobin (ctHb g/dl) and hematocrit (Hot %) using ABL800 BASIC (Radiometer Medical, Copenhagen, Denmark). Blood temperature was measured for the automatic temperature correction of the blood gas value.

Results: Blockade of a pathway is the ideal strategy to explore its involvement in a biological process. Thus, mice deficient in STAT6 (STAT6-ko), a central transducer of type 2 signaling, were tested in the context of the established mouse model of BPD. Antenatal inflammation and 28 days of exposure to hyperoxia at 65% O2 caused lung tissue injury characterized by enlarged alveoli and sparse secondary septation (Fig. 3a), which is similar in morphology to human BPD. Quantitative analysis of d28 lung sections revealed that compared to WT mice reared in room air, lungs from wild-type (WT) animals reared in hyperoxia exhibited a 29% increase in alveolar size (Fig. 3b), a 36% reduction in alveolar number (Fig. 3c) and an 18% decrease in the surface area-to- volume ratio, which may be regarded as a surrogate parameter for gas exchange (Fig. 3d). STAT6-ko mice exhibited a markedly attenuated morphological phenotype (Fig. 3a); indeed, tissue injury was almost completely prevented (Fig. 3b-d).

Given the amelioration of lung tissue injury in the STAT6-ko animals, the effect of type 2 blockade on lung function was investigated. A significant 25% decrease in p02 in WT hyperoxia pups was observed compared to the WT air group, whereas this decrease was prevented in STAT6-ko pups (Fig. 3e).

The results demonstrate that blockade of type 2 immune pathways markedly attenuates the morphological phenotype associated with BPD and protects against lung tissue injury.

Example 4

Murine protein analysis: At day 3 lungs were harvested, washed in ice-cold PBS, snap frozen in liquid nitrogen and stored at -80 °C. For analysis, the lungs were homogenized in lysis buffer (PMID 23946428) using an Ultra Turrax. The homogenate was centrifuged for 10 min at 14,000 ^c g and the supernatant was assayed for protein. All results were normalized to total protein concentration Proteome Profiler Arrays (R&D Systems) were used as described previously (PMID 23946428). ELISAs (Elisakit.com, IL-1 b; BD, IL-6) were performed according to the manufacturer’s instructions. Cytokine abundance of Eotaxin, IL-33 and IL-13 was measured by multiplex ELISA using the Quantibody Array (RayBiotech, Norcross, USA), sample protein concentrations were equalized to 230 μg/ml and performed as per manufacturer’s instructions. Quantibody Array slides were scanned using the Genepix 4000B microarray scanner (Molecular Devices, Sunnyvale, USA) and cytokine changes were normalized to 1 mg total protein. Results: WT pups exposed to 65% 02 exhibited increased pulmonary abundance of the type 2 chemokine eotaxin (2-fold, Fig. 4a) and the type 2 cytokines IL- 33 (6-fold, Fig. 4b) and IL-13 (4-fold, Fig. 4c) compared to WT control pups reared in room air. IL-4, IL-5 and IL-17A were not detectable by ELISA, which is likely due to its lower sensitivity compared to the cytokine profiler used in 2013 (Fig. 2). Also in accord with our previous study PMID 23946428, IL-1 b and IL-6 were significantly increased (2- and 5-fold, Fig. 4d & e) between the hyperoxia and room air groups. STAT6-deficiency prevented the hyperoxia-induced increases in eotaxin, IL-33, IL-13, IL-1 b and IL-6; each of these cytokines was at or near steady-state in the lungs of STAT6-ko pups despite hyperoxia exposure (Fig. 4).

Example 5 Murine lung Flow Cytometry at d28.

Flow cytometry was performed on lung cells isolated from mice exposed to prenatal LPS and postnatal hyperoxia, as previously published (Nold et al. , 2013, supra). For lung cell isolation on days 28, the left lung was minced and digested with collagenase D (1 mg/mL) at 37 °C for 30 min. After digestion, lung homogenates were macerated through a 70-mM cell strainer, red blood cells were lysed (BD Pharm Lyse) for 2* 10 min, then centrifuged at 400 ^c g for 10 min at 18 °C. Pellets were washed twice with PBS and cells were stained and acquired using a FACS Canto II flow cytometer (BD). Fc receptors were blocked using anti-CD16/CD32 (eBioscience). Cells were determined by surface expression of CD1 1 c, CD1 1 b, and GR1 for monocytes and macrophages; B220+CD1 1 c+ for plasmacytoid DC; B220-CD1 1 c+ for conventional DC; F4/80+CD1 1 b+ for macrophages; B220+CD1 1 c- for B cells; DX5+CD3- for NK cells; and CD4 or CD8 for T cells. A total of 20,000 CD45+ cells were analyzed per sample. A forward scatter gate was used to eliminate cell debris and gates were set using fluorophore minus one staining gates. Data were analyzed using Flow Jo software. All antibodies were obtained from BD Pharm ingen, except F4/80 (Serotec).

Murine lung Innate lymphoid cell isolation. After cervical dislocation, lungs from 28d old mice were harvested and washed in ice-cold PBS. Single-cell suspensions were obtained using the lung dissociation kit and gentleMACS Octo dissociation with heaters, according to manufacturers’ instructions (Miltenyi). Cells were washed with PBS and were stained for viability using Zombie Aqua (diluted 1 :200, BioLegend) for 15 mins at room temperature. Cells were washed once with FCM buffer (PBS, 2% FCS, 2 mM EDTA) and incubated with an Fc-receptor blocking Ab (eBioscience) for 20 min at 4°C. Cells were surfaced stained with the following antibody mastermix: ebioscience, B220 (RA3-6B2), CD11 b (M1/70), CD11 c (N418), NKp46 (29A1.4), CD3e (145-2C11 ), CD45.2 (104), Gr-1 (RB6-8C5), RORyT (AFKJS-9), TCRp (H57-597), TER-119 (TER- 119); BD, CD4 (RM4.5), KLRG1 (2F1 ); Biolegend, GATA3 (16E10A23) for 30 mins at 4°C. Cells were washed once with FCM buffer and then fixed with Foxp3/Fixation/Permeablisation Concentrate and Diluent (eBioscience) for 30 mins at room temperature. Cells were then washed with permeabilisation buffer (eBioscience) and stained intracellularly for transcription factors T-bet (04-46, eBioscience), RORyT (AFKJS-9, eBioscience) and GATA3 (16E10A23, Biolegend) at room temperature for 30 min. Cells were washed with FCM buffer and then acquired using a LSRFortessa X-20 (BD). Two million events were collected for each condition. Electronic compensation was performed with single-color controls stained separately with the individual Abs and the BD CompBeads anti-mouse Ig, k/negative control (BSA) compensation particle set and anti-rat/hamster Ig, k/negative control (BSA) compensation particle set (BD Biosciences). CS&T beads (BD Biosciences) were used to determine voltage, laser delays, and area scaling and to track those settings over time. A forward scatter gate was used to eliminate cell debris and gates were set using fluorophore minus one staining gates. Data were analyzed using Flow Jo software. Lineage (Lin) included CD11 b, CD11 c, TER119, B220, CD3e, Gr-1. Innate lymphoid cells were determined as CD45+CD4-TCRp-Lin- and were further divided by intracellular staining of T-bet, GATA3 and RORyT.

Results: Figure 5 shows that murine BPD is associated with a decrease in conventional DC, macrophages and T cells. Whereas the reduction in T cells is rescued in STAT6-ko, that in cDC and macrophages is not. There is no change in ILC1 with hyperoxia, but ILC2 are reduced and ILC3 are increased. STAT6-deficiency only has hyperoxia-independent effects (i.e. it does not rescue any hyperoxia-mediated changes). Example 6 Immunohistochemistry d28.

After cervical dislocation, 28d lungs were intubated via the trachea and the left lobe was tied off at the main bronchus and removed for flow cytometry or snap frozen in liquid nitrogen and stored at -80 °C for molecular analysis. The right lung was fixed with 4% paraformaldehyde (PFA) (pH 7.4, instilled at a pressure of 20 cm H₂0), removed from the thorax, kept in 4% PFA for a minimum of 24 h, then immediately processed for paraffin embedding and sectioning. Lung tissue was cut into 4-μm sections and H&E- stained for histology. For IHC, 4-um sections were cut and deparaffinised. Antigen retrieval was performed in 10mM sodium citrate buffer, pH 6.0, in a pressure cooker for 5 (TSLP) or 10 min (IL-13). Endogenous peroxidase was inhibited using a 0.3% H2O2/TBS solution applied to the slides for 15 min at RT. A 30-min RT blocking step using 1.5% rabbit serum in TBS (part of the Vectastain Elite ABC Kit, Vector Laboratories) or DAKO Protein Block, Serum-Free (Agilent Technologies) was performed prior to IL-13, IL-33 and IL-5 staining respectively. Slides were then incubated with goat anti-mouse IL-13, goat anti IL-5 and goat anti-IL-33 antibodies (1 :500, Santa Cruz) overnight at 4 °C. Secondary antibody incubation and diaminobenzidine (Invitrogen) staining were performed according to the instructions of the Vectastain kit. Sections were counterstained with Harris hematoxyline (Amber Scientific) and scanned on an Aperio Scanscope (ePathology Solutions).

Results: Figure 6 IHC of lungs on d28 reveals that IL-13 is still increased, whereas the difference in IL-33 observed on d3 is not detectable on d28. IL-5 also remains moderately elevated in the BPD-mice.

Example 7 Vasculature d28.

Murine lung iodine casts: The CT scans presented in this publication were conducted in the enclosure 3B of the IMBL with the X-ray photon energy tuned to 35keV utilizing‘Ruby’ detector. The detector is build utilizing photo-sensitive device coupled by a bright lens to a suitable X-ray sensitive scintillator. The system was conceived by the IMBL team and designed and fabricated at Monash University in the Division of Biological Engineering at the Laboratory for Dynamic Imaging (LDI). The sensor is the PCO.edge mounted on a vertical motor-driven slide set within a light-tight enclosure. A mirror is used to view a phosphor plate set orthogonally to the direction of the beam. This allows protection of the sensor from direct and scattered beam radiation using suitable high-Z materials. For this experiment the sensor was equipped with a Nikon Micro- Nikkor 105 mm/f 2.8 macro lens allowing the slide to be used as a zoom control. The scintillator was a 20 microns thick terbium-doped gadolinium oxy- sulfide (Gadox, P43) screen with aluminum powder coat as an optical block. During the experiment the system was tuned to produce 2560 x 2160 pixels images giving a field of view of 15 x 12 mm with 6.0μm pixel size with the measured resolution of 20.1 microns. CT data acquisition for the samples consisted of 1800 projections over the ark of 180 degree. The scans included 40 images of each background (no sample in the beam) and dark- field (beam is off) contrasts both before and after the sample acquisition. Under the conditions the exposures were 180ms per projection, and accumulated time taken to scan single sample was approximately 6 minutes. Data post-processing included several steps. In the first step clean background and dark-field images were formed as the median of all 40 repeats. Then they were applied to all raw sample images (sample - dark-field / background - dark-field) and light noise suppression was applied to the resulting images. The reconstruction utilized the Filtered Back-Projection algorithm and also included the phase retrieval and the ring artifact suppression filter as necessary. This processing was performed on MASSIVE high performance cluster in XLI software Ref2. Reconstructed slices were stored as 32-bit float-point TIFF images and further converted into 8-bit integer volumes for rendering and analysis. Images were then rendered using Drishti. The software used in the pipeline included several open-source projects: ImageMagick for noise removal, cropping and image format conversion, CTas for the background and darkfield removal.

Murine Lung Branching Analysis: The original data recorded at the IMBL beamline was saved as 8bit 3D .tif image stacks and displayed as 3D volumes in Imaris (Bitplane AG) for data quality check. The images then underwent several pre- processing steps before the final branching analysis was performed. Pre-processing of the images was necessary to fill the larger diameter blood vessel so the Imaris Filament Tracer could detect the vessel inside rather than the vessel membranes. Edge detection of large vessels was performed in Fiji ref by running two subsequent difference-of- Gaussian (DoG) algorithms with increasing diameter (r1 =2, r2=5), followed by thresholding, binarizing, and despeckling on each plane of the image stack. Then Matlab (MathWorks Inc) was used to fill large vessels by further despeckling, skeletonizing, removing skeleton fragments, then detecting the tips of the skeleton, closing with nearest neighbor algorithm and filling all fully-enclosed areas. Fiji and Matlab scripts can be found in the supplementary data of this manuscript. The original and the filled image stacks were then loaded in Imaris as two channels , a smoothed surface was created around the filled data set and used to remove potential artifacts of the filling algorithm in the red channel. In a final step, Imaris Filament Tracer was used to detect large vessels in the filled channel, followed by the detection of small vessels, in the original image stack (green channel). Overlaying the original volume with the analyzed Filament network provided a visual quality check of the branching analysis .

Results: Given the grave clinical outlook of infants suffering from BPD-PFI and our poor understanding of its pathogenesis, the morphological, functional and molecular changes in the pulmonary vasculature precipitated by perinatal inflammation and hyperoxia, as well as effects of type 2 immune blockade on the blood vessels in the lung were investigated. To investigate pulmonary vascular morphology (Fig. 7), ex vivo micro-CT imaging on iodine-stained lungs from d28 pups was performed, and the slices were then rendered to generate 3D images of the pulmonary vascular bed (Fig. 7a-d). Vessel numbers were quantified and grouped by diameter (Fig. 7e-g). These experiments revealed a marked reduction in the number of small blood vessels in lungs from WT animals reared in hyperoxia compared to WT mice housed in room air for 28d (e.g. -84% for capillaries sized 4-5 and 5-6 μm, -76% for vessels of 6-7 μm diameter, - 67% for 7-10 μm; Fig. 8e and images in Fig. 7a & c). Whereas the numbers of vessels with a diameter of between 10 and 30 μm was similar between the experimental groups (Fig. 7f), up to 8-fold increase in the number of larger blood vessels (30-60 pm, Fig. 7g) was observed in the hyperoxia group. Interestingly, deficiency in type 2 immunity conferred significant partial protection from BPD-associated loss in small vessels (Fig. 7e and images in Fig. 7a-d), but not from the increase in the number of large vessels (Fig. 7g and images in Fig. 7a-d).

Example 8 Echocardiography & angiogenic mediators on d28.

Murine Echocardiography: At 28 d, mice were anaesthetized with isoflurane (3%) via inhalation. Once anaesthetised, a lower concentration of isoflurane (1.5%) was delivered to maintain anaesthesia whilst the echocardiography was performed. During the procedure the mouse was kept on a heated pad. The echoes were imaged using a high frequency echocardiography machine (Vivid 7 GE with 15mHz linear probe). After the imaging, the mice were humanely killed by cervical dislocation.

Murine protein analysis: At day 5 lungs were harvested, washed in ice-cold PBS, snap frozen in liquid nitrogen and stored at -80 °C. For analysis, the lungs were homogenized in lysis buffer (PMID 23946428) using an Ultra Turrax. The homogenate was centrifuged for 10 min at 14,000 ^c g and the supernatant was assayed for protein. All results were normalized to total protein concentration. ELISAs (R&D Systems, VEGF-A, Endothelin-1 ; were performed according to the manufacturer’s instructions.

Results: To confirm that the vascular injury described in Fig.7a-g has a functional correlate, d 28d-old mice were subjected to echocardiography. Pulmonary vascular resistance was measured using the ratio time to peak velocity/right ventricular ejection time (TPV/RVET) as a surrogate parameter. These experiments revealed a significant reduction in the TPV/RVET ratio (from 0.32 in WT pups housed in room air to 0.26 in the WT hyperoxia group, Fig. 8a), which was completely prevented in STAT6-ko pups (0.32 in STAT6-ko pups housed in hyperoxia, Fig. 8a).

To shed light on the molecular pathomechanisms underpinning BPD-PFI in this model, protein abundance of vascular endothelial growth factor (VEGF) and endothelin- 1 (ET-1 ), mediators known to affect pulmonary angiogenesis and vasculogenesis were determined in lysates of lungs on experimental d5. Exposure 65% O2 moderately, but significantly increased VEGF-AA (1.7-fold, Fig. 8b) and ET-1 (1.5-fold, Fig. 8c) in WT pups compared to room air. Blockade of type 2 immunity prevented this increase.

Example 9: In vivo blockade of type 2 cytokines to prevent or treat BPD

The efficacy of type 2 cytokine blockade was tested using the BPD model previously reported in Nold et al PNAS 2013 {supra).

Administration of LPS to the pregnant dam and postnatal exposure to hyperoxia impaired alveolar development. Exposure to 65% O2 for 28d resulted in an increased number of large, simplified alveoli and reduced secondary septation. Immediately after birth pups were injected every second day for 28 days with antagonists of IL-4, IL-5 or IL-13. The antagonists used were: a monoclonal antibody for binding to IL-4 (BioXcell mouse monoclonal anit-IL-4 antibody), a monoclonal antibody for binding to IL-5 (BioXcell mouse monoclonal anti-IL-5 antibody, a monoclonal antibody for binding to IL-13 (RnD systems mouse anti-IL-13 monoclonal antibody). lgG1 or lgG2a were used as controls.

The results indicate a positive trend towards improved lung function when the activity of IL-13 was blocked. More specifically, hyperpoxia results in an increase in alveolar size in this model of BPD, compared to animals not exposed to hyperoxia. Treatment with anti-IL-13 monoclonal antibody resulted in a decrease in the overall alveolar size. A similar, although marginally less significant effect was observed when animals received anti-IL-4 or anti-IL-5 antibody (Figure 9a). Thus, type 2 cytokine blockade prevented the extent of increased alveolar size that is observed in BPD.

In the animal model of BPD, further signs of impaired lung function include reduced alveolar number and reduced alveolar surface area/volume. Treatment with anti-IL-13 antibody resulted in increased alveolar number and surface area/volume. A similar, although less pronounced result was observed following treatment with anti-IL-4 or anti-IL-5 monoclonal antibodies. (Figure 9 b and c).

These data are consistent with the observations obtained when STAT6 KO mice were exposed to the BPD model Fig 3 (a-e). Moreover, based on the observations made for the STAT6 KO mice, it is expected that blockade of type 2 activation by targeting multiple components of type 2 immune activation (such as IL-13 and IL-4, which signal together upstream of STAT6, or targeting of all three of IL-13, IL-4 and IL- 5) would see an increase in clinical outcomes.

Overall, the data show that blockade of type 2 cytokines using type 2 cytokine antagonists prevents the development or reduces the severity of the clinical signs associated with BPD. The results suggest that blockade of a single type 2 cytokine will provide for improved clinical outcomes. It is expected that blockade of multiple type 2 cytokines, (for example, IL-13 and IL-4 or the combination of targeting all 3 of IL-13, IL-4 and IL-5) would provide for superior results.

Claims

1. A method of treating or preventing bronchopulmonary dysplasia (BPD) in an individual, the method comprising administering a therapeutically effective amount of an inhibitor of type 2 immune activation to an individual in need, thereby treating or preventing BPD in the individual.

2. The method of claim 1 , wherein the individual is a pre-term infant.

3. The method of claim 2, wherein the infant is born before 35 weeks of gestation, preferably, before 32 weeks of gestation.

4. The method of any one of the preceding claims, wherein the inhibitor of type 2 immune activation is selected from the group consisting of: an interleukin 4 (IL-4) antagonist, an IL-4 receptor antagonist, an interleukin 5 (IL-5) antagonist, an IL-5 receptor antagonist, an interleukin 13 (IL-13) antagonist, an IL-13 receptor antagonist, an interleukin 33 (IL-33) antagonist, an interleukin-25 (IL-25) antagonist, an immunoglobulin E (IgE) antagonist and a thymic stromal lymphopoietin (TSLP) antagonist.

5. The method of any one of the preceding claims, wherein the inhibitor of type 2 immune activation is selected from the group consisting of a small compound, a monoclonal antibody, a peptide, a recombinant protein, a fusion protein, or an interfering polynucleotide or silencing RNA.

6. The method of claim 5, wherein the inhibitor of type 2 immune activation is a monoclonal antibody, preferably a monoclonal antibody that inhibits or blocks the activity or signalling of one or more of IL-4, IL-5, IL-13, IL-25, IL-33, or TSLP.

7. The method of any one of claims 1 to 5, wherein the inhibitor of type 2 immune activation comprises an IL-13 antagonist.

8. The method of claim 7, wherein the IL-13 antagonist is a monoclonal antibody that specifically inhibits the signalling of IL-13.

9. The method of claim 7 or 8, wherein the IL-13 antagonist specifically inhibits or blocks the activity of the IL-13Receptoralpha 1 and/or the activity of IL- 13Receptoralpha 2.

10. The method of claim 7, wherein the IL-13 antagonist is a soluble IL-13 receptor, optionally a soluble IL-13 receptor fusion protein, that acts as a decoy receptor to inhibit the signalling of IL-13.

11. The method of claim 10, wherein the IL-13 antagonist is a soluble IL-13Ralpha2 receptor fusion protein.

12. The method of any one of claims 1 to 6 wherein the inhibitor of type 2 immune activation comprises an IL-4 antagonist.

13. The method of any one of claims 1 to 6, wherein the inhibitor of type 2 immune activation comprises an IL-5 antagonist.

14. The method of claim 6, wherein the inhibitor of type 2 immune activation is a monoclonal antibody selected from the group consisting of: tralokinumab, lebrikizumab, anrukinzumab, ASLAN004, pitrakinra, dupilumab, mepolizumab, benralizumab, tezepelumab, omalizumab.

15. The method of claim 14 wherein the monoclonal antibody is ASLAN004.

16. The method of any one of the preceding claims, wherein administration of the inhibitor of type 2 immune activation results in a decrease in the levels or activity of one or more type 2 cytokines in the individual.

17. The method of claim 16, wherein administration of the inhibitor of type 2 immune activation results in a decrease in the level or activity of IL-4 and IL-13.

18. The method of any one of the preceding claims, wherein the method comprises administering more than one inhibitor of type 2 immune activation.

19. The method of 18, wherein the more than one inhibitor of type 2 immune

activation comprises an IL-4 antagonist and an IL-13 antagonist.

20. The method of claim 18, wherein the more than one inhibitor of type 2 immune activation comprises an IL-4 antagonist, an IL-5 antagonist and an IL-13 antagonist.

21. The method of any one of claims 18 to 20, wherein the more than one inhibitor of type 2 immune activation comprises at least one monoclonal antibody that inhibits or blocks the activity or signalling of a type 2 cytokine.

22. The method of any one of the preceding claims, wherein the treatment includes treating or preventing BPD-associated pulmonary hypertension (BPD-associated PH).

23. The method of any one of the preceding claims, wherein the treatment comprises administration of an additional therapeutic agent for treating BDP, wherein preferably the additional agent is selected from: caffeine; a diuretic; a non- specific phosphodiesterase inhibitor; phosphodiesterase-4 inhibitors;

phosphodiesterase-5 inhibitors; a surfactant, a corticosteroid; an inhibitor of type 1 immune activation; curcumin; inhaled nitric oxide (iNO); azithromycin; a bronchodilator or anti-hypertensive.

24. The method of any one of the preceding claims, wherein the method includes determining whether the individual is at risk of BPD.

25. The method of claim 24, wherein determining the risk of an individual of BPD includes:

- determining the level of type 2 immune activation in the individual, - comparing the level of type 2 immune activation in the individual with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term individuals who developed BPD;

- determining that the individual is at risk of BPD if the level of type 2 immune activation in individual is the same or greater than the level of type 2 immune activation in the reference data set;

- determining that the individual is not at risk of BPD if the level of type 2 immune activation in the individual is less than the level of type 2 activation in the reference data set.

26. The method of claim 25, wherein the level of type 2 immune activation is

determined by measuring the levels of one or more type 2 cytokines in a biological sample obtained from the individual.

27. The method of claim 26, wherein the one or more type 2 cytokines for which the level is determined, are selected from the group consisting of: IL-4, IL-5, IL-13, IL-25, IL-33 and TSLP.

28. The method of claim 26 or 27, wherein the type 2 cytokine is IL-13.

29. A method of treating or preventing BPD-associated pulmonary hypertension (PH) in an individual, the method comprising administering a therapeutically effective amount of an inhibitor of type 2 immune activation to an individual in need, thereby treating or preventing BPD-associated PH in the individual.

30. The method of claim 29, wherein the individual is a pre-term infant, optionally an infant before 35 weeks of gestation.

31. The method of claims 29 or 30, wherein the inhibitor of type 2 immune activation is selected from the group consisting of: an interleukin 4 (IL-4) antagonist, an IL-4 receptor antagonist, an interleukin 5 (IL-5) antagonist, an IK-5 receptor antagonist, an interleukin 13 (IL-13) antagonist, an IL-13 receptor antagonist, an interleukin 33 (IL-33) antagonist and a thymic stromal lymphopoietin (TSLP) antagonist.

32. The method of any one of claims 29 to 31 , wherein the inhibitor of type 2

immune activation is selected from the group consisting of a small compound, a monoclonal antibody, a peptide, a recombinant protein or an interfering polynucleotide or silencing RNA.

33. The method of claim 32, wherein the inhibitor of type 2 immune activation is a monoclonal antibody, preferably a monoclonal antibody that inhibits or blocks the activity or signalling of one or more of IL-4, IL-5, IL-13, IL-25, IL-33 or TSLP.

34. The method of any one of claims 29 to 32, wherein the inhibitor of type 2 immune activation comprises an IL-13 antagonist.

35. The method of claim 34, wherein the monoclonal antibody specifically inhibits the signalling of IL-13.

36. The method of claim 34 or 35, wherein the IL-13 antagonist specifically inhibits or blocks the activity of IL-13Ralpha 1 receptor.

37. The method of claim 34 or 35, wherein the IL-13 antagonist specifically inhibits or blocks the activity of IL-13Ralpha 2 receptor.

38. The method of claim 34, wherein the IL-13 antagonist is a soluble IL-13 receptor, optionally a soluble IL-13 receptor fusion protein, that acts as a decoy receptor to inhibit the signalling of IL-13.

39. The method of claim 33 or 35, wherein the inhibitor of type 2 immune activation is a monoclonal antibody selected from the group consisting of: tralokinumab, lebrikizumab, anrukinzumab, ASLAN004, pitrakinra, dupilumab, mepolizumab, benralizumab, tezepelumab, omalizumab.

40. The method of claim 39, wherein the monoclonal antibody is ASLAN004.

41. The method of any one of claims 29 to 40, wherein the method comprises

administering more than one inhibitor of type 2 immune activation.

42. The method of 41 , wherein the more than one inhibitor of type 2 immune

activation comprises an IL-4 antagonist and an IL-13 antagonist.

43. The method of claim 41 , wherein the more than one inhibitor of type 2 immune activation comprises an IL-4 antagonist, an IL-5 antagonist and an IL-13 antagonist.

44. A method of determining whether an individual is at risk of BPD, the method

comprising:

- determining the levels of type 2 immune activation in an individual for whom risk of BPD is to be determined,

- comparing the level of type 2 immune activation in the individual with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term individuals who developed BPD;

- determining that the individual is at risk of BPD if the level of type 2 immune activation in the individual is the same or greater than the level of type 2 immune activation in the reference data set;

- determining that the individual is not at risk of BPD if the level of type 2 immune activation in the individual is less than the level of type 2 cytokines in the reference data set.

45. The method of claim 44, wherein the level of type 2 immune activation is determined by measuring the levels of one or more of: IL-4, IL-5, IL-13, IL-33, IL- 25, IgE and TSLP in a test biological sample obtained from the individual.

46. A method of determining whether an individual is at risk of BPD, the method

comprising: - providing a test biological sample from the individual for whom risk of BPD is to be determined,

- measuring in the test sample, the levels of one or more type 2 cytokines,

- comparing the level of type 2 cytokines in the test sample, with the level of type 2 cytokines in a reference data set in the form of data from one or more pre-term individuals who developed BPD;

- determining that the individual is at risk of BPD if the level of type 2 cytokines in the test sample is the same or greater than the level of type 2 cytokines in the reference data set;

- determining that the individual is not at risk of BPD if the level of type 2 cytokines in the test sample is less than the level of type 2 cytokines in the reference data set.

47. The method of claim 46, wherein the test biological sample is a plasma sample.

48. The method of claim 46 or 47, wherein the one or more type 2 cytokines for which the level is determined, are selected from the group consisting of: IL-4, IL-

5, IL-13, IL-33, IL-25 and TSLP.

49. The method of any one of claims 46 to 48, wherein the type 2 cytokine is IL-13.

50. A method of identifying a pre-term infant for treatment for BPD, the method

comprising:

- determining the levels of type 2 immune activation in the individual

- comparing the level of type 2 immune activation in the individual with the level of type 2 immune activation in a reference data set in the form of data from one or more pre-term infants who developed BPD;

- determining to treat the infant for BPD if the level of type 2 immune activation in the individual is the same or greater than the level of type 2 immune activation in the reference data set;

- determining not to treat the infant for BPD if the level of type 2 immune activation in the individual is less than the level of type 2 cytokines in the reference data set.

51. The method of claim 50, wherein the level of immune activation is determined by measuring the levels of one or more of IL-4, IL-5, IL-13, IL-25, IL-33, IgE and TSLP in a test biological sample obtained from the individual.

52. The method of any one of claims 50 to 51 , wherein the type 2 activation is

determined by reference to the level of IL-13 in the test sample.

53. The method of any one of claims 50 to 51 , wherein the treatment for BPD is

selected from the group consisting of: an inhibitor of type 2 immune activation, caffeine; a diuretic; a non-specific phosphodiesterase inhibitor;

phosphodiesterase-4 inhibitors; phosphodiesterase-5 inhibitors; an exogenous surfactant, a corticosteroid; an inhibitor of type 1 immune activation; curcumin; inhaled nitric oxide (iNO); azithromycin; a bronchodilator or an anti-hypertensive.

54. The method of claim 53, wherein the inhibitor of type 2 immune activation is

selected from the group consisting of: an interleukin 4 (IL-4) antagonist, an IL-4 receptor antagonist, an interleukin 5 (IL-5) antagonist, an IL-5 receptor

antagonist, an interleukin 13 (IL-13) antagonist, an IL-13 receptor antagonist, an interleukin 33 (IL-33) antagonist and a thymic stromal lymphopoietin (TSLP) antagonist.

55. The method of claim 53 or 54, wherein the inhibitor of type 2 immune activation is selected from the group consisting of a small compound, a monoclonal antibody, a peptide, a recombinant protein or an interfering polynucleotide or silencing RNA.

56. The method of claim 55, wherein the inhibitor of type 2 immune activation is a monoclonal antibody, preferably a monoclonal antibody that inhibits the activity of signalling of one or more of IL-4, IL-5, IL-13, IL-25, IL-33 or TSLP.

57. The method of claim 56, wherein the monoclonal antibody that binds specifically to the IL-13 receptor antibody.

58. The method of claim 57, wherein the monoclonal antibody is ASLAN004.

59. Use of an inhibitor of type 2 immune activation in the manufacture of a

medicament for the treatment or prevention of BPD in an individual.

60. The use of claim 59, wherein the individual is a pre-term infant, optionally an infant born before 35 weeks of gestation, preferably, before 32 weeks of gestation.

61. The use of claim 59 or 60, wherein the inhibitor of type 2 immune activation is selected from the group consisting of: an interleukin 4 (IL-4) antagonist, an IL-4 receptor antagonist, an interleukin 5 (IL-5) antagonist, an IL-5 receptor antagonist, an interleukin 13 (IL-13) antagonist, an IL-13 receptor antagonist, an interleukin 33 (IL-33) antagonist, an interleukin-25 (IL-25) antagonist, an immunoglobulin E (IgE) antagonist and a thymic stromal lymphopoietin (TSLP) antagonist.

62. The use of claim 61 , wherein the inhibitor of type 2 immune activation is an

antagonist of IL-13, IL-4, IL-5, or a combination thereof.

63. The use of claim 62, wherein the inhibitor of type 2 immune activation is a

monoclonal antibody that specifically inhibits or blocks the signalling of IL-13.

64. The use of claim 62, wherein the inhibitor of type 2 immune activation is an IL-13 receptor antagonist, preferably ASLAN004.

65. One or more type 2 immune activation inhibitors for the prevention or treatment of BPD in an individual.

66. The one or more type 2 immune activation inhibitors for the use of claim 65, wherein the individual is a preterm infant, optionally an infant born before 35 weeks of gestation, preferably, before 32 weeks of gestation.

67. The one or more type 2 immune activation inhibitors for the use of claim 65 or 66, wherein the one or more inhibitors specifically inhibit the activity or signalling of IL-13.

68. The one or more type 2 immune activation inhibitors for the use of claim 67,

wherein the inhibitor is an IL-13Receptoralpha 1 or an IL-13Receptoralpha 2 antagonist, preferably wherein the inhibitor is ASLAN004.

69. The one or more type 2 immune activation inhibitors for the use of claim 67,

wherein the one or more inhibitors is a soluble IL-13 receptor, preferably a soluble IL-13 receptor fusion protein, that acts as a decoy receptor for blocking or inhibiting the signalling of IL-13.

70. The one or more type 2 immune activation inhibitors for the use of claim 69,

wherein the one or more inhibitors is a soluble IL-13Receptoralpha2, or a derivative, fusion protein or fragment thereof.

71. The one or more type 2 immune activation inhibitors for the use of any one of claims 65 to 70, wherein the one or more type 2 immune activation inhibitors specifically inhibit the activity or signalling of at least IL-13 and IL-4.

72. The one or more type 2 immune activation inhibitors for the use of any one of claims 65 to 71 , wherein the one or more type 2 immune activation inhibitors specifically inhibit the activity or signalling of at least IL-13, IL-4 and IL-5.