WO2016087582A1 - Methods and pharmaceutical compositions for the prevention and/or treatment of acute exacerbations of chronic obstructive pulmonary disease - Google Patents

Abstract

The present invention concerns an inhibitor of serine proteases for use in the prevention and/or treatment of acute exacerbations of chronic obstructive pulmonary disease (AE- COPD). It also relates to a biological agent which is able to prevent the cleavage of the IL22 receptor by a serine protease.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE PREVENTION AND/OR TREATMENT OF ACUTE EXACERBATIONS OF CHRONIC OBSTRUCTIVE

PULMONARY DISEASE The present invention concerns an inhibitor of serine proteases for use in the prevention and/or treatment of acute exacerbations of chronic obstructive pulmonary disease (AE-COPD). It also relates to a biological agent which is able to prevent the cleavage of the IL22 receptor by a serine protease. Background

Chronic obstructive pulmonary disease (COPD) is a major and increasing global health issue. It is now considered one of the main cause of death worldwide and has no efficient treatment (Barnes PJ. Cellular and molecular mechanisms of chronic obstructive pulmonary disease., Clin Chest Med 2014; 35:71-86., Diaz-Guzman E, Mannino DM. Epidemiology and prevalence of chronic obstructive pulmonary disease. Clin Chest Med 2014; 35:7-16). It is characterized by persistent inflammation in the airways and lung parenchyma and is associated to increased numbers of leukocytes such as macrophages, T lymphocytes as well as polymorphonuclear neutrophils (Hoenderdos K, Condliffe A., The neutrophil in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2013; 48:531 -539, Meijer M, Rijkers GT, van Overveld FJ., Neutrophils and emerging targets for treatment in chronic obstructive pulmonary disease., Expert Rev Clin Immunol 2013;9:1055-10).

Accumulation and activation of neutrophils in COPD result in excessive secretion of inflammatory mediators. An extensive body of evidence implicates neutrophil-derived proteases such as elastase and cathepsin G, as key mediators of lung deterioration (Hoenderdos K, Condliffe A. The neutrophil in chronic obstructive pulmonary disease., Am J Respir Cell Mol Biol 2013; 48:531-539, Meijer M, Rijkers GT, van Overveld FJ., Neutrophils and emerging targets for treatment in chronic obstructive pulmonary disease., Expert Rev Clin Immunol 2013; 9:1055-1068, Stockley RA., Alphal -antitrypsin review. Clin Chest Med 2014; 35:39-50). Those neutrophil proteases can cause degradation of soluble mediators or matrix components as well as alteration of cell surface receptors (Hoenderdos K, Condliffe A., The neutrophil in chronic obstructive pulmonary disease., Am J Respir Cell Mol Biol 2013; 48:531-539).

It has been previously demonstrated that elastase and cathepsin G can deactivate receptors known to be involved in host defense and inflammation, including the lipolysaccharide (LPS) co-receptor CD14 (Le-Barillec K, Pidard D, Balloy V, Chignard M. Human neutrophil cathepsin G down-regulates LPS-mediated monocyte activation through CD14 proteolysis. J Leukoc Biol 2000; 68:209-215, Le-Barillec K, Si-Tahar M, Balloy V, Chignard M. Proteolysis of monocyte CD14 by human leukocyte elastase inhibits lipopolysaccharide-mediated cell activation. J Clin Invest 1999;103:1039-1046) and distinct protease-activated receptors (Pham CTN. Neutrophil serine proteases: specific regulators of inflammation. Nat Rev Immunol 2006; 6:541 -550, Chignard M, Pidard D. Neutrophil and pathogen proteinases versus proteinase-activated receptor-2 lung epithelial cells: more terminators than activators. Am J Respir Cell Mol Biol 2006; 34:394-398).

However, defects were reported in innate defense mechanisms of patients suffering from COPD, despite the presence of abundant innate immune cells such as neutrophils which predominate in the COPD airway wall and lumen (Hoenderdos K, Condliffe A., The neutrophil in chronic obstructive pulmonary disease., Am J Respir Cell Mol Biol 2013; 48:531 -539, Meijer M, Rijkers GT, van Overveld FJ., Neutrophils and emerging targets for treatment in chronic obstructive pulmonary disease., Expert Rev Clin Immunol 2013; 9:1055-1068). Those immune defects ultimately increase the susceptibility of those patients to viral or bacterial infection-driven exacerbations and disease severity.

"Acute exacerbation" refers to worsening of a subject's COPD symptoms from his or her usual state that is beyond normal day-to-day variations, and is acute in onset. Acute exacerbations of COPD greatly affect the health and quality of life of subjects with COPD.

As of today, the mechanisms underlying COPD exacerbations remain poorly understood.

The inventors of the present invention here revealed an unsuspected proteolytic regulation of IL-22-mediated antimicrobial signaling by serine proteases, a process that enhances pathogens burden and results in COPD exacerbations.

Description of the invention

As mentioned above, the inventors of the present invention have established for the first time that serine proteases, including cathepsin G, cleave the IL-22R1 receptor subunit and inhibit IL-22-mediated epithelial cell activation.

The IL22 receptor or lnterleukin-22 receptor is a type II cytokine receptor. It binds to lnterleukin-22. The cell surface-standing IL-22 receptor complex consists of the receptor chains IL-22R1 and IL-10R2 which is expressed on lung epithelial cells.

The human IL22R1 gene encodes a protein of 574 amino acids of SEQ ID N°1 : MRTLLTILTVGSLAAHAPEDPSDLLQHVKFQSSNFENILTWDSGPEGTPDTVYSIEYKTYG ERDWVAKKGCQRITRKSCNLTVETGNLTELYYARVTAVSAGGRSATKMDRFSSLQHTTL KPPDVTCISKVRSIQMIVHPTPTPIRAGDGHRLTLEDIFHDLFYHLELQVNRTYQMHLGGK QREYEFFGLTPDTEFLGTIMICVPTWAKESAPYMCRVKTLPDRTWTYSFSGAFLFSMGFL VAVLCYLSYRYVTKPPAPPNSLNVQRVLTFQPLRFIQEHVLIPVFDLSGPSSLAQPVQYS QIRVSGPREPAGAPQRHSLSEITYLGQPDISILQPSNVPPPQILSPLSYAPNAAPEVGPPS YAPQVTPEAQFPFYAPQAISKVQPSSYAPQATPDSWPPSYGVCMEGSGKDSPTGTLS SPKHLRPKGQLQKEPPAGSCMLGGLSLQEVTSLAMEESQEAKSLHQPLGICTDRTSDPN VLHSGEEGTPQYLKGQLPLLSSVQIEGHPMSLPLQPPSRPCSPSDQGPSPWGLLESLVC PKDEAKSPAPETSDLEQPTELDSLFRGLALTVQWES. After removal of 15 amino acids corresponding to the signal peptide, the protein is secreted as a protein of 559 amino acids with a molecular mass of 63 kDa. The extra portion of the membrane protein has 213 amino acids (amino acid 16-228 - dimmed in the peptide sequence), the transmembrane portion is a 21 amino acid helix (data from http://www.uniprot.org/).

The cleavage domain comprising SEQ ID N°2: TEFLGTIMICVPTWAKESAPY has been identified by the inventors as described in the experimental part below.

The present invention thus relates to an inhibitor of serine proteases for use in the prevention and/or treatment of acute exacerbations of chronic obstructive pulmonary disease (AE-COPD).

The present invention also relates to a biological agent which is able to prevent the cleavage of the IL22 receptor by a serine protease.

Chronic obstructive pulmonary disease (COPD) also known as chronic obstructive lung disease (COLD), and chronic obstructive airway disease (COAD), among others, is a type of obstructive lung disease characterized by chronically poor airflow. It typically worsens over time. The main symptoms include shortness of breath, cough, and sputum production.

Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) also known as acute exacerbations of chronic bronchitis (AECB) is a sudden worsening of COPD symptoms (shortness of breath, quantity and color of phlegm) that typically lasts for several days. Typically, the acute exacerbation of COPD is manifested by one or more symptoms selected from worsening dyspnea, increased sputum production, increased sputum purulence, change in color of sputum, increased coughing, upper airway symptoms including colds and sore throats, increased wheezing, chest tightness, reduced exercise tolerance, fatigue, fluid retention, and acute confusion, and said method comprises reducing the frequency, severity or duration of one or more of said symptoms. It may be triggered by an infection with bacteria or viruses or by environmental pollutants. Typically, infections cause 75% or more of the exacerbations; bacteria can roughly be found in 25% of cases, viruses in another 25%, and both viruses and bacteria in another 25%. Airway inflammation is increased during the exacerbation resulting in increased hyperinflation, reduced expiratory air flow and worsening of gas transfer.

In one embodiment of the present invention, AE- COPD is caused by a bacterial infection, by a viral infection or by air pollution.

In particular, AE-COPD is caused by a bacterial infection, more particularly by the bacteria Streptococcus pneumoniae or Haemophilus influenzae.

In some embodiments, the subject experienced AE-COPD or is at risk of experiencing AE-COPD. In one embodiment, the subject has experienced at least one AE-COPD in the past 24 months. In another embodiment, the subject has experienced at least one AE-COPD in the past 12 months. In still another embodiment, the subject is a frequent exacerbator. As used herein the term "frequent exacerbator" refers to a subject who suffers from or is undergoing treatment for COPD and who experiences at least 2, and more typically 3 or more, AE-COPD during a 12 month period.

By "subjects", it is meant a human, a male or female, which is afflicted, or has the potential to be afflicted with one or more disorders described herein.

The serine proteases (or serine endopeptidases) are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes.

Examples of serine proteases are trypsin-like proteases: trypsin-like proteases cleave peptide bonds following a positively charged amino acid (lysine or arginine). This specificity is driven by the residue which lies at the base of the enzyme's S1 pocket (generally a negatively charged aspartic acid or glutamic acid); chvmotrvpsin-like proteases: the S1 pocket of chymotrypsin-like enzymes is more hydrophobic than in trypsin-like proteases. This results in specificity for medium to large sized hydrophobic residues, such as tyrosine, phenylalanine and tryptophan; elastase-like proteases: elastase-like proteases have a much smaller S1 cleft than either trypsin- or chymotrypsin- like proteases. Consequently, residues such as alanine, glycine and valine tend to be preferred; or subtilisin-like proteases: a serine protease in prokaryotes. Subtilisin is evolutionarily unrelated to the chymotrypsin-clan, but shares the same catalytic mechanism utilizing a catalytic triad, to create a nucleophilic serine.

In one embodiment of the present invention, serine proteases are neutrophil-derived mediators: the neutrophil proteases, and more particularly elastase and/or cathepsin G and/or proteinase 3 and/or microbial pathogen serine proteases, and more particularly protease IV such as, for example, protease IV of pseudomonas aeruginosa.

Protease IV is a 26 kDa serine protease secreted by the type II secretion system of pseudomonas aeruginosa. Pseudomonas aeruginosa is a Gram negative bacterium responsive for respiratory infection, such as, for example, AE-COPD of severe COPD patients and nosocomial pneumonia in intensive care units.

In one embodiment, the present invention relates to an inhibitor of serine proteases for use in the prevention and/or treatment of acute exacerbations of chronic obstructive pulmonary disease (AE-COPD), wherein said inhibitor is directed against both serine proteases secreted by inflammatory cells of the subject and serine proteases secreted by pathogens.

By "pathogens" is meant, in the context of the invention, pathogens that can be responsive for AE-COPD in the subject such as those previously mentioned.

Thus, the inventors of the present invention propose an innovative therapeutic strategy to prevent the cleavage of the IL22 receptor and restore appropriate epithelial immune defense, by targeting simultaneously host and pathogen serine proteases.

By "an inhibitor of serine proteases", it is meant, in the context of the invention, any compound able to inhibit the activity of said serine proteases, such as, for example, maspin, camostat, serpin, plasminogen activator inhibitor, antithrombin, a1 - antichymotrypsin, a1 -protease inhibitor and Biotin-FPR-CMK (chloromethyl ketone).

In particular, said inhibitor is able to inhibit neutrophil proteases, and more particularly elastase and/or cathepsin G and/or proteinase 3 and/or microbial pathogen serine proteases, and more particularly protease IV.

Even more particularly, said inhibitor is chosen from a1 -antichymotrypsin and a1 - protease inhibitor.

a1 -antichymotrypsin (a1 AC) is an alpha globulin glycoprotein that is a member of the serpin superfamily. In humans, it is encoded by the SERPINA3 gene. a1 -antichymotrypsin inhibits the activity of serine proteases such as cathepsin G by cleaving it into a different shape or conformation.

a1 -Proteinase inhibitor (a1 -PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells.

The present invention also relates to a pharmaceutical composition comprising an inhibitor of serine proteases. In particular, said pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

As used herein, a "pharmaceutically acceptable excipient" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human. A pharmaceutically acceptable excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The definitions of "serine proteases" and "inhibitor of sereine proteases" previously given apply here.

As previously mentioned, the invention also relates to a biological agent which is able to prevent the cleavage of the IL22 receptor by a serine protease.

"Serine protease" is as herein defined.

The biological agent in the scope of the present invention is represented by any compound able to prevent the cleavage of the IL22 receptor by a serine protease, that is to say any compound able to block the interaction between the IL22 receptor and a serine protease.

In particular, said biological agent is an antibody or a fragment thereof, such as a Fab or Fab'.

An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (I) and kappa (k). There are five main heavy chain classes (or isotopes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

"Framework Regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1 -L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively.

As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

As used herein, the term "antibody" denotes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanised, bispecific or multispecific antibodies.

As used herein, antibody or immunoglobulin also includes "single domain antibodies" which have been more recently described and which are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit and bovine. Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibody devoid of light chains. In particular, Camelidae species, for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain. Camelid heavy chain antibodies also lack the CH1 domain.

The variable heavy chain of these single domain antibodies devoid of light chains are known in the art as "VHH" or "nanobody". Similar to conventional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are about ten times smaller than IgG molecules, and as a consequence properly folded functional nanobodies can be produced by in vitro expression while achieving high yield. Furthermore, nanobodies are very stable, and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov; 77(1 ): 13-22).

The antibody of the invention may be a polyclonal antibody.

The antibody may be a monoclonal antibody. Said monoclonal antibody may be humanized. In another example the antibody may be a fragment selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies and VHH, and more particularly chosen from Fab and Fab'.

The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid composition that is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering.

The term "chimeric antibody" refers to an engineered antibody which in its broadest sense contains one or more regions from one antibody and one or more regions from one or more other antibody(ies). In particular, a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, in association with a CH domain and a CL domain of another antibody, in particular a human antibody. As the non- human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. A chimeric antibody may also denote a multispecific antibody having specificity for at least two different antigens. In an embodiment, a chimeric antibody has variable domains of mouse origin and constant domains of human origin

The term "humanised antibody" refers to an antibody which is initially wholly or partially of non-human origin and which has been modified to replace certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains. In an embodiment, a humanized antibody has constant domains of human origin.

"Fragments" of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

The term "Fab" denotes an antibody fragment having a molecular weight of about

50,000 Da and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.

The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

The term "Fab' " refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.

A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragment of the invention includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)₂. "dsFv" is a VH::VL heterodimer stabilised by a disulphide bond. "(dsFv)2" denotes two dsFv coupled by a peptide linker.

The term "bispecific antibody" or "BsAb" denotes an antibody which combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050 764 A1 .

The term "multispecific antibody" denotes an antibody which combines the antigen- binding sites of two or more antibodies within a single molecule.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.

Typically, antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice- weekly, weekly, twice-monthly or monthly) with the relevant antigenic forms. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG- containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761 , 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761 , and WO 90/07861 also propose four possible criteria which may be used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGI, lgG2, lgG3, lgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., /. Mol. Biol. 294:151 , 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591 ,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

In some embodiments, the antibody is modified to reduce or inhibit the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) functionality (i.e. an antibody with reduced Fc-effector function"). In particular, the antibodies of the present invention have no Fc portion or have an Fc portion that does not bind FcyRI and C1 q. In one embodiment, the Fc portion of the antibody does not bind FcyRI, C1 q, or FcyRI 11. Antibodies with such functionality, in general, are known. There are native such antibodies, such as antibodies with an lgG4 Fc region. There also are antibodies with Fc portions genetically or chemically altered to eliminate the Antibody dependent cell cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) functionality.

In particular, the biological agent is an antibody or Fab or Fab' fragment thereof, as above defined, which binds to the cleavage domain of the IL22 receptor by serine proteases.

More particularly, said cleavage domain comprises the amino acid sequence of SEQ ID N°2 or an amino acid sequence at least 80% identical to said SEQ ID N°2. Even more particularly, said cleavage domain has an amino acid sequence of SEQ ID N°2 or an amino acid sequence at least 80% identical to said SEQ ID N°2.

For example, said cleavage domain comprises or has an amino acid sequence at least 85, 90, 92, 95, 97, 98 or 99% identical to said SEQ ID N°2.

By a cleavage site having an amino acid sequence at least, for example, 95%

"identical" to a query amino acid sequence, it is intended that the amino acid sequence of the subject cleavage domain is identical to the query sequence except that the subject cleavage domain sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.

In the context of the present application, the percentage of identity is calculated using a global alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The « needle » program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is, for example, available on the ebi.ac.uk world wide web site. The percentage of identity in accordance with the invention is preferably calculated using the EMBOSS:: needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

Proteins consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations such as deletions, insertions and/or substitutions compared to the reference sequence. In case of substitutions, the protein consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.

Amino acid substitutions may be conservative or non-conservative. Preferably, substitutions are conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and/or chemical properties. The substitution preferably corresponds to a conservative substitution as indicated in the table below. Conservative

Type of Amino Acid

substitutions

Ala, Val, Leu, He, Met, Pro, Amino acids with aliphatic hydrophobic side Phe, Trp chains

Ser, Tyr, Asn, Gin, Cys Amino acids with uncharged but polar side chains

Asp, Glu Amino acids with acidic side chains

Lys, Arg, His Amino acids with basic side chains

Gly Neutral side chain

In one embodiment, the present invention also relates to a method for reducing the probability of occurrence and/or for the treatment of AE-COPD, comprising administering to a subject in need thereof, an effective amount of an inhibitor of serine proteases as defined herein.

By "subjects", it is meant a human, a male or female, which is afflicted, or has the potential to be afflicted with one or more disorders described herein.

As used herein, a "therapeutically effective amount" refers to an amount which is effective in reducing, eliminating, treating or controlling the symptoms of the herein- described disease. The term "controlling" is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the disease described herein, but does not necessarily indicate a total elimination of all disease, and is intended to include prophylactic treatment and chronic use.

The identification of the subjects who are in need of treatment of herein-described disease is well within the ability and knowledge of one skilled in the art. A clinician skilled in the art can readily identify, by the use of clinical tests, physical examination and medical/family history, those subjects who are in need of such treatment.

A therapeutically effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of subject; its size, age, and general health; the specific disease involved; the degree of involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristic of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. According to another embodiment of the present invention, said agent is a competitive exogenous peptide.

As used herein, a "competitive exogenous peptide" represents any exogenous peptide which is able to bind to serine proteases in order to prevent its interaction with the IL22 receptor.

In particular, the amino sequence of said peptide comprises the amino acid sequence of the cleavage domain of the IL22 receptor by serine proteases.

More particularly, said cleavage domain comprises the amino acid sequence of SEQ ID N°2 or an amino acid sequence at least 80% identical to said SEQ ID N°2. Even more particularly, said cleavage domain has an amino acid sequence of SEQ ID N°2 or an amino acid sequence at least 80% identical to said SEQ ID N°2.

For example, said cleavage domain comprises or has an amino acid sequence at least 85, 90, 92, 95, 97, 98 or 99% identical to said SEQ ID N°2.

The definition previously given of what is encompassed by a cleavage domain having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence, also applies here.

The biological agent according to the invention can be used in the prevention and/or treatment of a respiratory inflammatory disease.

By "respiratory inflammatory disease" is meant the general common definition of this class of diseases, among which chronic lung inflammatory diseases such as COPD, mucoviscidosis and acute lung inflammatory disease such as acute respiratory distress syndrome (ARDS), ventilator associated pneumonia/tracheobronchitis and more particularly AE-COPD, can be cited.

In one embodiment, the biological agent according to the invention is thus used in the prevention and/or treatment of AE-COPD.

The present invention thus also relates to a biological agent as defined herein, for use in the prevention and/or treatment of a respiratory inflammatory disease, in particular AE-COPD.

In another embodiment, the present invention also relates to a pharmaceutical composition comprising a biological agent as defined herein.

In particular, said pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

A "pharmaceutically acceptable excipient" is defined as previously mentioned. As described herein, an inhibitor of serine protease according to the invention or a biological agent according to the invention can be formulated into pharmaceutical compositions, in particular for a use in the previously mentioned methods of treatment or prevention, by admixture with one or more pharmaceutically acceptable excipients. Such compositions may be prepared for use in oral administration, particularly in the form of tablets or capsules, in particular orodispersible (lyoc) tablets; or parenteral administration, particularly in the form of liquid solutions, suspensions or emulsions; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically or via trans-dermal patches or ocular administration, or intravaginal or intra- uterine administration, particularly in the form of pessaries or by rectal administration. Such compositions may also be prepared for delivery during mechanical ventilation, particularly by aerosol route in the form of powders or liquid particles. For example, it could be administered with a nebulizer or a sprayer during invasive or non-invasive mechanical ventilation.

The tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate. Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatin blends optionally blended with plasticizers, as well as a starch capsule. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colorings, and flavorings. In addition, the inhibitor of serine protease according to the invention or biological agent according to the invention may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bi-modal.

Liquid preparations for administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring and coloring agents, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, acrylate copolymers, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, hydrogels, buffered media, and saline. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the inhibitor of serine protease according to the invention or the biological agent according to the invention. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Alternative modes of administration include formulations for inhalation, which include such means as dry powder, aerosol, or drops. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for buccal administration include, for example, lozenges or pastilles and may also include a flavored base, such as sucrose or acacia, and other excipients such as glycocholate. Formulations suitable for rectal administration are preferably presented as unit-dose suppositories, with a solid based carrier, and may include a salicylate.

In one embodiment, the present invention also relates to a method for reducing the probability of occurrence and/or for the treatment of a respiratory inflammatory disease, in particular AE-COPD, comprising administering to a subject in need thereof, a therapeutically effective amount a biological agent which is able to prevent the cleavage of the IL22 receptor by a serine protease, said biological agent being as herein defined.

The previous definitions of "subjects and "therapeutically effective amount" apply here.

In the scope of the present invention, it has to be understood that "an inhibitor for use, or a biological agent for use" is equivalent to "the use of an inhibitor for, or the use of a biological agent for" and in particular that "an inhibitor for use in the treatment or prevention of, or a biological agent for use in the treatment or prevention of" is equivalent to "the use of an inhibitor for the treatment or prevention of, or the use of a biological agent for the treatment or prevention of" and to "the use of an inhibitor for the manufacture of a medicament intended for the treatment or prevention of, or the use of a biological agent for the manufacture of a medicament intended for the treatment or prevention of".

The invention will be further illustrated by the following figures and exampl FIGURES

Figure 1 : Regulation of IL-22R expression by microbial triggers. First, human bronchial epithelial BEAS-2B cells are infected with H3N2 Influenza A virus (IAV) at multiplicity of infection (MOI) = 1 for 20 hours {A), treated with Poly (l:C) at 5μg/mL for 20 hours (ib ), infected with P. aeruginosa mutant strain PAK ApscF MOI =1 for 4 hours (then bacteria are removed and medium with gentamycin is added for 16 hours) (C) or treated with LPS at 10 μg/mL for 20 hours (D). For each condition, potent cell stimulation is verified by measuring the release of a major inflammatory cytokine, i.e. IL-6 in BEAS-2B supernatants by ELISA (£). Then, H22r1 mRNA copy number is determined by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and normalized to the expression of Hprtl mRNA (A-D, left panels). Cell surface expression of IL-22R protein is also assessed by flow cytometry and expressed in median of fluorescence (A-D, right panels). Besides, BALB/c mice (5-7 mice per group) are infected with a sublethal dose of H3N2 IAV (150 pfu). Mouse lungs are collected 2 days post-infection and H22r1 mRNA copy number is determined by RT-qPCR and normalized to the expression of RplpO mRNA (£).

Values represent the mean ± SEM of at least 3 independent experiments. ^*p < 0.05, Man-Whitney test. Ctl = control, IAV = Influenza A virus, MFI = median of fluorescence, PIC = Poly (l:C), PA = P. aeruginosa, LPS = Lipopolysaccharide.

Figure 2: Cigarette smoke does not modulate IL-22R expression. Human bronchial epithelial BEAS-2B cells are exposed to 5% cigarette smoke extract (CSE) or 5% room air (Ctl) during 6 hours. Potent cell stimulation is verified by measuring IL-8 secretion in BEAS-2B cells supernatants by ELISA (A) and NAD(P)H quinone oxidoreductase 1 (NQ01 ) gene expression by RT-qPCR (B). IL-22R expression secondary to CSE is further analyzed at the mRNA and protein levels (C, D). Besides, BALB/c mice (6-10 mice per group) are exposed to cigarette smoke for 8 to 16 weeks using a whole-body smoke exposure system. Because lungs exposed to cigarette smoke can show loss of alveolar walls, alveolar surface is first measured (£).Then, the expression of the inflammatory markers Cxcl1 and Muc5ac (F,G) as well as of Il-22r1 is analyzed in murine lungs by RT- qPCR (H). Finally, Il-22r1 expression is also assessed by RT-qPCR in 129 human lung tissues from patients undergoing thoracic surgery (/; 14 (1 1 %) non-smokers, 53 (41 %) "healthy" smokers, and 62 (48%) COPD patients, among them 24 (19%) are Gold 1 , 30 (23%) are Gold 2, and 8 (6%) are Gold 3)) or in primary cultures of proximal airway epithelial cells isolated from controls (n=6), "healthy" smokers (n=3), or COPD patients (n=4) (J). All values represent the means ± SEM of at least 3 independent experiments (A- D), or two independent experiments {E-H). Median is drawn on the graph for (/). ^*P < 0.05, Man-Whitney test (A-E) or Kruskal-Wallis test (and Dunn's test for post-hoc comparisons) (F-J). 8w = 8 weeks, 16w = 16 weeks, CS = cigarette smoke, CSE = cigarette smoke extract, Ctl = control, MFI = median of fluorescence.

Figure 3: Effect of neutrophils serine proteinases on IL-22R expression. Cell surface expression of IL22-R is assessed by flow cytometry in BEAS-2B cells infected with Influenza A virus (IAV) at MOI=1 for 20 hours, before incubation with supernatants of either activated neutrophils (A), or with 1 μΜ cathepsin G (CG) (B) or with 1 μΜ neutrophil elastase (NE) (C) during 30 minutes at 37°C. The a1 -antichymotrypsin (ACT) is added to inhibit CG (B), while a1 -protease inhibitor (a1 -Pi) is used to inhibit NE (C). Addition of both inhibitors is used to inhibit proteases in supernatants of activated neutrophils (A). (D and £) represent the effect of CG on the expression of IL22-R at the surface of intact BEAS- 2B cells or on the same cells previously exposed to the cell trafficking inhibitor cytochalasin B (Cyto.B), respectively. BALB/c mice (6-10 mice per group) are exposed for 8 weeks to cigarette smoke using a whole-body smoke exposure system. Then, BALs are collected for neutrophil count by flow cytometry (F) while lungs are collected for immunoblot analysis of IL-22R1 expression (G). IL-22R1 quantification is normalized with β-actin expression signal. Values represent the mean ± SEM of three independent experiments. ^*P < 0.05, Kruskal-Wallis test (and Dunn's test for post-hoc comparisons). MFI = median of fluorescence.

Figure 4: Functional impact of cathepsin G on IL-22/IL-22R signaling. BEAS-2B cells are first exposed (or not) to 1 μΜ cathepsin G (CG) for 30 minutes at 37°C before stimulation (or not) by 20 ng/mL recombinant IL-22. Then, the serine-phosphorylated, active, form of the transcriptional factor STAT3 (p-STAT3) is analyzed in cell lysates by Western blotting. The corresponding signal is eventually quantified after normalization with β-actin expression signal. Values represent the means ± SEM of three independent experiments. ^*P < 0.05, Kruskal-Wallis test (and Dunn's test for post-hoc comparisons).

Figure 5: Cathepsin G cleaves IL-22R and releases a soluble fragment. (A): IL22-R expression in BEAS-2B cells exposed to 1 μΜ cathepsin G (CG) for 30 minutes at 37°C is assessed by western-blotting in either cell lysates (A, upper panel) or in the corresponding cell supernatants (A, lower panel). a1 -antichymotrypsin (ACT)-treated CG or heat- inactivated CG served as negative controls. {B): BALB/c mice are intranasally challenged with 40 μΙ_ of CG (0.2 μΜ), PBS or aACT-treated CG, and BALs are collected two hours later: CG activity is measured in BAL (S, left panel) while IL-22R expression is determined by immunoblotting (B, right panel). The results of one representative gel (out of 3) are shown (B, lower panel). Data are the means ± SEM of three independents experiments. ^*P < 0.05, Kruskal-Wallis test (and Dunn's test for post-hoc comparisons).

Figure 6: In mice, IL-22R is fragmented in lungs with an infection-driven inflammation. BALB/c mice are infected with a sublethal dose of IAV. Body weight is monitored daily after infection (A). At days 2 or 6 post-infection (dpi), BALs are collected for neutrophil count (B) and IL-22R immunoblotting analysis (C). Data represent the means ± SEM (n=6 mice per group) and are representative of two distinct experiments. The results of one representative gel are shown (C). ^*P < 0.05, Kruskal-Wallis test (and Dunn's test for post-hoc comparisons).

Figure 7: IL-22R is cleaved in human lungs with acute exacerbation of COPD. Sputum of COPD patients with (n=1 1 ) or without (n=14) acute exacerbations (AE-COPD) are collected. (A) shows neutrophils count and (B) IL-22R expression assessed by immunoblotting analysis. A pool of sputum served as calibrator for comparisons. Values represent the means ± SEM. ^*P < 0.05, Man-Whitney test.

Figure 8: Schematic model of alteration of IL-22/IL-22R pathway in COPD. Left panel: in healthy lung mucosa, IL-22 stimulates the production of antimicrobial peptides and promotes maintenance and repair of the epithelial barrier in the respiratory tract, thus reducing pathogen burden and dissemination. IL-22 mediates these effects via the IL-22 receptor expressed at the surface of airway epithelial cells. Right panel: neutrophil-derived proteases cleave the IL-22 receptor and inhibit the downstream STAT3-dependent antimicrobial signaling. This major alteration of the immune response of the lung mucosa may further predispose to infection-triggered exacerbations of COPD.

Figure 9: IL22-R expression in BEAS-2B cells exposed to protease-IV (purified Pseudomonas protease) assessed by western-blotting in cell lysates EXAMPLES

EXAMPLE 1

METHODS

Virus, bacteria, cell cultures and mice

The pathogenic human-origin H3N2 Influenza A virus (IAV) strain Scotland/20/74 and the P. aeruginosa mutant strain PAK ApscF have already been described (Guillot L, Le Goffic R, Bloch S, Escriou N, Akira S, Chignard M, Si-Tahar M. Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus. J Biol Chem 2005; 280:5571 -5580, Jyot J, Balloy V, Jouvion G, Verma A, Touqui L, Huerre M, Chignard M, Ramphal R. Type II secretion system of Pseudomonas aeruginosa: in vivo evidence of a significant role in death due to lung infection. J Infect Dis 201 1 ; 203:1369-1377). The human bronchial epithelial cell line BEAS-2B or the primary human cell cultures (MucilAir™) are used. BALB/c mice (female, 18-20 g) are handled after ethical committee agreement.

Preparation of Cigarette Smoke Extract (CSE)

The smoke of two cigarettes is bubbling into 10 mL of medium, the resulting CSE solution is considered to be 100% CSE. Control is made by room-air (RA) bubbled in medium under the same conditions.

Neutrophil activation, measurement and inhibition of protease activities

Human blood neutrophils are purified as described previously (Dubois AV, Gauthier A, Brea D, Varaigne F, Diot P, Gauthier F, Attucci S. Influence of DNA on the activities and inhibition of neutrophil serine proteases in cystic fibrosis sputum. Am J Respir Cell Mol Biol 2012; 47:80-86). Proteinase activity is measured as described in Korkmaz B, Attucci S, Juliano MA, Kalupov T, Jourdan M-L, Juliano L, Gauthier F. Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates. Nat Protoc 2008; 3:991-1000., with the specific FRET substrates of human neutrophil elastase (NE) or cathepsin G (CG).

Infection and treatment of lung epithelial cells

To investigate the effects of microbial triggers, cells are incubated with IAV at multiplicity of infection (MOI) of 1 , Poly (l:C) at 5μg/mL, LPS at 10 μg/mL or P. aeruginosa MOI =1 . To investigate the effects of CSE, cells are exposed to 5% CSE or 5% RA for 6 hours immediately after CSE or RA preparation. To investigate the effects of neutrophil serine proteases, cells are incubated with either CG or NE for 30 minutes at 37°C. Next, cells are washed with PBS and reactions are stopped by addition of a protease inhibitor cocktail. Infection and treatment of mice

CSE-challenged mice are exposed to the smoke of 18 cigarettes twice daily (5 days/week) for 8 to 16 weeks using a whole-body smoke exposure system. Age-matched control animals are exposed to room air only.

Mice infection by IA V. Mice are infected intranasally with IAV in sterile PBS in a total volume of 40 μΙ_. For sub-lethal infection, 150 plaque-forming units (PFU) of H3N2 IAV are instilled.

CG-challenged mice. CG, oc-antichymotrypsin (ACT)-treated CG or PBS alone is administered intranasally to the mice in a final volume of 40μΙ_. BAL are performed two hours after instillation.

Human samples

Human lung tissue from non-smokers, smokers and patients with COPD is obtained from patients undergoing surgery for lung carcinoma. Lung samples are located at least 3 cm away from the edge of the tumor, and the absence of carcinoma is checked histologically. Sputum are collected prospectively from COPD or AE-COPD patients and analyzed for IL22-R1 receptor by Western-blotting analysis. A pool of sputum served as calibrator for comparisons. This study is approved by French bioethical authorities. Informed written consent is obtained from each participant.

Statistical analysis

Results are expressed as means ± SEM. Statistical significance between the different values is analyzed by Mann-Whitney test or Kruskal-Wallis (and Dunn's test for post-hoc comparisons) according to the number of group to analyze. Statistical analysis is performed using GraphPad Prism^® 5. A p value of less than 0.05 is considered significant.

RESULTS

Viral infection induces an IL-22R up-regulation in lung epithelial cells

It is now well-established that IL-22R1 is expressed only on outer-body barriers such as lung epithelial cells and that IL-22/IL22R1 enhances epithelial host defense and restore epithelial homeostasis (Sabat R, Ouyang W, Wolk K. Therapeutic opportunities of the IL-22-IL-22R1 system. Nat Rev Drug Discov 2013; 13:21 -38). By contrast, no information is available concerning the regulation of IL-22R1 expression in lung epithelial cells challenged by microbial triggers. The inventors have thus assessed the expression of IL-22R1 gene and protein in human bronchial epithelial cells exposed to either (/) replicative influenza A virus (IAV) and Poly(l:C) -a viral pathogen-associated molecular pattern (PAMP) mimetic- on one hand, or (/^'/) the Gram negative bacteria P. aeruginosa and LPS (a major bacterial PAMP), on the other hand. Figures 1 A-D shows that IL-22R1 is constitutively expressed in human bronchial epithelial BEAS-2B cells. Interestingly, IL-22R1 expression is clearly up-regulated in IAV- and Poly(l:C)-infected cells (by 8.7 and 5.6 fold increase, respectively, in three independent experiments, p<0.01 ; Figure 1 A-B). By contrast, neither P. aeruginosa nor LPS regulate IL-22R1 under the same experimental conditions (Figure 1 C-D). This lack of IL-22R1 modulation by P. aeruginosa and LPS is not due to a reduced immunostimulatory activity compared to the viral triggers as the secretion of IL-6 upon exposure of BEAS-2B cells with each of those stimuli, was very similar (always superior to 1000 pg/mL, in three independent experiments; Figure 1 E). Besides, it is observed that IL-22R1 expression was approximately 200-fold higher in mouse trachea compared to the lung compartment per se (p<0.0001 ; data not shown) and it is confirmed that IL-22R1 expression is significantly up-regulated in lung by lAV-challenge (by 3.1 -fold higher, p<0.01 ) (5 to 7 mice/group, three independent experiments, Figure 1 F).

Cigarette smoke does not modulate IL-22 R expression

Recurrent or chronic infections in smokers or COPD patients support the hypothesis that primary defects in host innate immunity defense mechanisms against lung pathogens is present, leading to chronic inflammation (Brusselle GG, Joos GF, Bracke KR. New insights into the immunology of chronic obstructive pulmonary disease. Lancet 201 1 ; 378:1015-1026). As the epithelial cell is the first cell type to be in contact with inhaled smoke and thus susceptible to be altered, it is wondered whether IL-22 R1 expression is impaired by cigarette smoke. To this purpose, epithelial cell culture were exposed to cigarette smoke extract (CSE), and validated CSE-induced cell stimulation by measuring IL-8 production and NAD(P)H quinone oxidoreductase 1 (NQ01 ) gene upregulation (Figure 2A-B). Of note is that a modulation of IL-22R1 expression secondary to CSE is not observed (four independent experiments, p>0.05; Figure 2C-D).

As bronchial epithelial cells directly exposed to CSE constitute a reductionist and acute experimental model, mice chronically challenged by cigarette smoke were also used for 8 to 16 weeks using a whole-body smoke exposure system. Under these conditions, mouse survival was not altered whereas lung CxcH and Muc5ac gene upregulation as well as alveolar tissue destruction are evidenced (two independent experiments with more than six mice per group, Figure 2E-G). By contrast, no modulation of IL-22R1 RNA expression is observed (p>0.05; Figure 2H). Nevertheless, those cellular and animal studies were extended to the analysis of IL22-R1 expression in lungs of COPD patients. Indeed, COPD is a multifactorial pathobiological process and thus cannot be considered solely as a complication of cigarette smoke exposure. 129 human lung tissues from patients undergoing surgery for lung carcinoma were examined. The mean age of the patients is 65.6 ± 1 years. Sample sizes in each group are as followed: 14 (1 1 %) non- smokers, 53 (41 %) "healthy" smokers, and 62 (48%) COPD patients, among them 24 (19%) were Gold 1 , 30 (23%) were Gold 2, and 8 (6%) were Gold 3. Figure 2I shows that lung IL-22R1 expression is modified neither by the smoker status nor by the COPD severity grade. Similar observations are obtained using proximal primary airway epithelial cells from control individuals (n=6), "healthy" smokers (n=3), or COPD patients (n=4), p>0.05; Figure 2J.

Effect of neutrophils serine proteinases on IL-22R expression

First, the effect of supernatants of activated neutrophils on IL-22R1 expressed at the surface of epithelial BEAS-2B cells are examined. Remarkably, IL-22R1 is strongly impaired under this condition while specific inhibitors of serine proteinases prevents such receptor disappearance (Figure 3A). Moreover, the two most important neutrophil serine proteinases, i.e. CG and NE, specifically decrease IL-22R1 (Figure 3B-C). Thereafter, it is focused on CG and found that a progressive decrease of IL22-R1 expression is observed upon exposure of BEAS-2B cells to increasing concentrations of this neutrophil proteinase (0.01 to 2 μΜ), down to 50 % of the control values (Figure 3D). Finally, to exclude an IL- 22R1 shedding resulting from an intracellular pathway triggered by CG acting on another membrane structure, BEAS-2B cells are treated with cytochalasin B before CG treatment. Figure 3E shows that an intact cell metabolism is not required for CG-triggered IL-22R1 alteration as the expression of this receptor is still strongly reduced under these conditions, a value comparable to that observed upon incubation of nonfixed cells with CG. Next, it is investigated whether the alteration of IL-22R1 protein expression could be observed in lungs of a mouse model of cigarette smoke-induced COPD. As expected, mice chronically challenged by cigarette smoke have a higher number of neutrophils in BAL compared to room air controls (>30 fold increase, p<0.01 ; Figure 3F). Interestingly, while IL-22R1 gene expression is not affected by cigarette smoke (Figure 2H), a clear expression decrease of the 64 kDa IL-22R1 protein in the lungs of cigarette smoke- exposed mice is observed (approximatively 2 fold reduction, /x0.02; Figure 3G).

Inhibition by CG of IL-22-triggered activation of bronchial epithelial cells

Binding of IL-22 to its receptor is known to induce a cascade of downstream signaling pathway that involves the phosphorylation of the transcriptional factor STAT3 at the Tyr705 residue. STAT3 phosphorylation then mediates the biological effects of IL-22 on epithelial cells (Sabat R, Ouyang W, Wolk K. Therapeutic opportunities of the IL-22-IL- 22R1 system. Nat Rev Drug Discov 2013; 13:21 -38). Hence, to assess the functional impact of IL-22R1 alteration by CG, bronchial epithelial BEAS-2B cells are exposed or not to 1 μΜ CG for 30 minutes before the addition of an optimal concentration of recombinant IL-22 (20ng.mL ¹). As shown in Figure 4, the STAT3 phosphorylation signal associated to the control BEAS-2B cells is dramatically reduced in CG-exposed cells.

Cathepsin G cleaves the IL-22R1 and releases a soluble fragment

The foregoing experiments suggest that CG causes neither internalization nor shedding of IL-22R1 through an activation of bronchial epithelial cells. Conversely, those results rather point out to a direct proteolysis of IL-22R1 by this neutrophil proteinase. Experiments are therefore conducted to verify this hypothesis. Thus, cell lysates and supernatants collected from BEAS-2B cells incubated or not with CG are subjected to electrophoresis and immunoblotting with a specific antibody targeted toward the extra- cellular part of IL-22R1 . Considering lysates of untreated cells, a major band of approximately 62 kDa corresponding to the intact IL-22R1 is observed. By contrast, IL- 22R1 is undetectable in CG-challenged BEAS-2B cells (Figure 5A) whereas CG inhibited by a1 -antichymotrypsin does not modify the pattern of IL-22R1 expression and structure. More interestingly, immunoblot analysis of supernatants of CG-challenged cells shows a major band of approximately 25 kDa (Figure 5A). The absence of IL-22R1 expression in CG-treated cells together with the presence of a 25kDa IL-22R1 antigen in supernatants suggest that CG cleaves the IL-22R1 subunit and releases the extracellular part of the receptor into the medium. To further provide evidence that such IL-22R proteolysis also occurs in vivo, mice are intranasally challenged with purified CG (0.2 μΜ), and BAL fluids are collected two hours later for immunoblot analysis (two independent experiments). Neither spontaneous mortality nor lung inflammation is observed under these conditions. For instance, IL-6 concentration is similar in BALs of CG-treated- or control mice (data not shown). CG activity is observed in BAL fluids (7.2 nM; Figure 6A). More important, a 25 kDa fragment of IL-22R is recovered in BAL fluids of CG-exposed mice (p<0.02; Figure 5B).

IL-22R is strongly fragmented upon lAV-triggered acute pneumonia

Next, it is investigated whether the soluble fragment of IL-22R1 observed previously could be detected in a more pathophysiologic context, such as a lung infection by lAV. It is hypothesized that IL-22R1 could be cleaved by serine proteases released in situ from activated neutrophils, those leukocytes being massively recruited in lung tissues infected by lAV ( Si-Tahar M, Blanc F, Furio L, Chopy D, Balloy V, Lafon M, Chignard M, Fiette L, Langa F, Charneau P, Pothlichet J. Protective Role of LGP2 in Influenza Virus Pathogenesis. J Infect Dis 2014; 210:214-223). To this purpose, mice are infected by a sublethal dose of lAV (at least 6 mice per group, two independent experiments) and the body weight is monitored daily after infection (Figure 6A). At days 2 and 6 post-infection (p.i.), BALs are collected for neutrophil count and anti-IL22R1 immunoblotting analysis. At day 2 p.i., IAV induced limit weight lost and neutrophil recruitment in lung tissues (Figure 6B). By contrast, at day 6 p.i, mice lost approximately 20% of their initial body weight and a major neutrophil infiltration into the lungs is observed (3.7 10⁵ neutrophil total count in BAL, p<0.001 ). Concomitantly, immunoblotting reveals that a 25 kDa fragment of IL-22R is released in the airspaces of lAV-infected mice at day 6 p.i. (Figure 6C). It is of note in that regard that a strong positive relationship between neutrophil count and quantification of the soluble fragment of IL-22R1 in BAL fluids is observed (r = 0.53, p<0.01 , Spearman test).

A fragment of IL-22R is released in the lungs of COPD patients with acute exacerbations (AE)

It is then searched for the release of a 25 kDA fragment of IL-22R in sputum of either COPD (n=14) or AE-COPD patients (n=1 1 ). AE-COPD patients have a higher number of neutrophils in sputum compared to COPD patients (Figure 7A, p<0.001 ). Interestingly, a soluble IL-22R1 fragment of 25 kDa is detected in both COPD and AE-COPD sputa (Figure 7B) but with the highest detection in fluids of AE-COPD individuals (p<0.02). The absence of IL-22R immunoblotting is confirmed on purified neutrophils (data not shown) and could not be a confounding factor.

DISCUSSION

Cigarette smoke is the major risk factor for COPD in developed countries (Rennard SI, Daughton DM. Smoking cessation. Clin Chest Med 2014; 35:165-176). Increasing evidence suggests that smoke affects the immune system by several mechanisms, including by enhancing the release of chemoattractants in the lung mucosa that further triggers a neutrophil infiltration (Murugan V, Peck MJ. Signal transduction pathways linking the activation of alveolar macrophages with the recruitment of neutrophils to lungs in chronic obstructive pulmonary disease. Exp Lung Res 2009; 35:439^185, Bauer CMT, Morissette MC, Stampfli MR. The influence of cigarette smoking on viral infections: translating bench science to impact COPD pathogenesis and acute exacerbations of COPD clinically. Chest 2013; 143:196-206).

Here, the inventors establish for the first time the capacity of serine proteases to cleave the immune IL-22 receptor and reveal its harmful impact on the antimicrobial response of the lung mucosa.

IL-22-/IL-22R signaling pathway is pivotal at barrier surfaces where epithelial cells play an active role in the initiation, regulation, and resolution of immune responses. Functional studies in murine model systems indicate that IL-22 has immunoregulatory properties in infection, inflammation but also autoimmunity and cancer (Sabat R, Ouyang W, Wolk K. Therapeutic opportunities of the IL-22-IL-22R1 system. Nat Rev Drug Discov 2013;13:21-38; Sonnenberg GF, Fouser LA, Artis D. Functional biology of the IL-22-IL- 22R pathway in regulating immunity and inflammation at barrier surfaces. Adv Immunol 2010; 107:1 -29.). In these models, IL-22 can be either pathologic or protective, depending on the context in which it is expressed.

Remarkably, the present invention reveals that the IL-22/IL22R pathway can be severely impaired by neutrophil proteinases, including cathepsin G, at the surface of lung epithelial cells. The inventors have provided the first evidence of a proteolytic regulation of IL-22R signaling. Of note is that IL-22/IL22R-dependent signaling pathway involves predominantly the transcription factor STAT3. For instance, STAT3 deficiency in epithelial cells mimics that of 1122-/- mice in a model of colitis, implicating a requirement for STAT3 in in vivo IL-22-mediated effects (Pickert G, Neufert C, Leppkes M, Zheng Y, Wittkopf N, Warntjen M, Lehr H-A, Hirth S, Weigmann B, Wirtz S, Ouyang W, Neurath MF, Becker C. STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing. J Exp Med 2009; 206:1465-1472). Thus, it is especially noteworthy that IL-22R1 cleavage by cathepsin G resulted in a major inhibition of STAT3-dependent signaling.

On the basis of the data presented here, it is suggested that neutrophil proteases including cathepsin G, may impair pathogens killing by reducing the expression of IL-22R and the downstream STAT3-dependent antimicrobial effectors; this protease-dependent disarming of IL-22/IL-22R axis may predispose to bacterial or viral replication and spreading and consequently, maybe detrimental in the setting of AE-COPD. The mechanism proposed to better construe how neutrophil proteases enhance the susceptibility of COPD lungs to life-threatening infections is shown in Figure 8.

The underlying mechanism of cathepsin G-dependent down-expression of IL-22R appears to be a direct proteolytic cleavage. Indeed, internalization of this receptor can be excluded as (/) a major 25 kDa fragment of IL-22R1 was detectable in the extracellular milieu after cell exposure to cathepsin G (see Figs. 5 to 7 ), and (/^'/) IL-22R1 was releasable from the surface of epithelial cells whose intracellular trafficking and actin polymerization were inhibited by cytochalasin B. Hence, using both cellular and animal models as well as human fluids samples, evidence of a yet unsuspected way to generate soluble IL-22R1 through neutrophil proteolysis is brought. Interestingly, this enzymatic product of IL- 22R may share similar effects to the soluble IL-22 binding protein (IL-22BP), a 27 kDa secreted single-chain IL-22R which lacks a trans-membrane and intracellular domain (Dumoutier L, Lejeune D, Colau D, Renauld JC. Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived inducible factor/IL-22. J Immunol 2001 ; 166:7090-7095). IL-22BP specifically binds to IL-22 but not to other IL-10 family members and prevents the interaction of IL-22 with its transmembrane receptor complex and so inhibits its effects (Wolk K, Witte E, Hoffmann U, Doecke W-D, Endesfelder S, Asadullah K, Sterry W, Volk H-D, Wittig BM, Sabat R. IL-22 induces lipopolysaccharide-binding protein in hepatocytes: a potential systemic role of IL- 22 in Crohn's disease. J Immunol 2007;178:5973-5981 , Logsdon NJ, Jones BC, Josephson K, Cook J, Walter MR. Comparison of interleukin-22 and interleukin-10 soluble receptor complexes. J Interferon Cytokine Res 2002; 22:1099-1 1 12).

EXAMPLE 2

IL22-R expression in BEAS-2B cells exposed to protease-IV (purified Pseudomonas protease) is assessed by western-blotting in cell lysates.

BEAS-2B cells are seeded into 6-well culture plates and incubated 48 hours at 37°C and 5% C0₂. Cells are treated with 0,1 μΜ of cathepsin G or 0,1 μΜ of protease IV for 30 minutes at 37°C. Next, cells were washed with PBS and reactions are stopped by addition of a protease inhibitor cocktail. Protein lysates are prepared using a lysis buffer (150 mM sodium chloride, 50 mM Tris-HCI, 1 mM ethylenediaminetetraacetic acid, 1 % Triton100, 1 % sodium deoxycholic acid, 0.1 % sodium dodecyl sulphate) and a protease inhibitor cocktail (diluted 1/200). SDS-PAGE is thereafter performed using 100 μg of total proteins loaded per well on 4/12 % acrylamide gels. Proteins are subsequently transferred to nitrocellulose membranes, probed with anti-IL-22Ra1 antibody diluted 1/2000, and bound antibodies are revealed with an anti-rabbit IgG (HRP linked) diluted 1/15000 and ECL detection reagents. The automated imaging system (MF ChemiBis 3.2, DNR Biolmaging Systems) is used for detection, and the FUJI FILM MultiGauge software is subsequently used.

The results shown in Figure 9 reflect that the IL22 receptor is cleaved by protease IV, a 26 kDa serine protease secreted by Pseudomonas aeruginosa.

Conclusions

The present invention shows that serine proteases contribute to AE-COPD by impairing the antimicrobial IL-22/IL-22R signaling pathway, leading to consider said serine proteases as key therapeutic targets in AE-COPD.

Claims

1 . An inhibitor of serine proteases for use in the prevention and/or treatment of acute exacerbations of chronic obstructive pulmonary disease (AE-COPD).

2. A pharmaceutical composition comprising an inhibitor of serine proteases.

3. An inhibitor of serine proteases for use according to claim 1 or a pharmaceutical composition according to claim 2, wherein said serine proteases are neutrophil proteases and/or microbial pathogen proteases.

4. An inhibitor of serine proteases for use according to any of claims 1 or 3 or a pharmaceutical composition according to any of claims 2 or 3, wherein said serine proteases are elastase and/or cathepsin G and/or proteinase 3 and/or protease IV.

5. An inhibitor of serine proteases for use according to any of claims 1 and 3 to 4 or a pharmaceutical composition according to any of claims 2 to 4, wherein said inhibitor is chosen from a1 -antichymotrypsin and a1 -protease inhibitor.

6. A biological agent which is able to prevent the cleavage of the IL22 receptor by a serine protease.

7. A biological agent according to claim 6, wherein said agent is an antibody or a Fab or Fab' fragment thereof.

8. A biological agent according to claim 7, wherein said antibody or Fab or Fab' fragment thereof binds to the cleavage domain of the IL22 receptor by serine proteases.

9. A biological agent according to claim 8, wherein said cleavage domain comprises an amino acid sequence of SEQ ID N°2 or an amino acid sequence at least 80% identical to said SEQ ID N°2.

10. A biological agent according to claim 6, wherein said agent is a competitive exogenous peptide.

1 1 . A biological agent according to claim 10, wherein the amino acid sequence of said peptide comprises the amino acid sequence of the cleavage domain of the IL22 receptor by serine proteases.

12. A biological agent according to claim 1 1 , wherein said cleavage domain comprises an amino acid sequence of SEQ ID N°2 or an amino acid sequence at least 80% identical to said SEQ ID N°2.

13. A biological agent according to any of claims 6 to 12, for use in the prevention and/or treatment of a respiratory inflammatory disease, in particular AE-COPD.

14. A pharmaceutical composition comprising a biological agent as defined in any of claims 6 to 12.

15. An inhibitor of serine proteases for use according to any of claims 1 and 3 to 5 or a biological agent for use according to claim 13, wherein the AE-COPD is caused by a bacterial infection or by a viral infection.

16. A method for the treatment of AE-COPD, comprising administering to a subject in need thereof, an effective amount of an inhibitor of serine proteases.