LU504827B1

LU504827B1 - Compounds and compositions for use in the treatment of lung diseases

Info

Publication number: LU504827B1
Application number: LU504827A
Authority: LU
Inventors: Christoph Beisswenger; Timo SPEER; Robert Bals; Felix Ritzmann; Danilo Fliser; Yiwen Yao
Original assignee: Univ Saarland
Priority date: 2023-07-28
Filing date: 2023-07-28
Publication date: 2025-01-28
Also published as: WO2025026919A1

Abstract

The invention pertains to compounds and compositions comprising inhibitors of Dickkopf like protein 3 (DKK3) for use in the treatment of lung diseases, in particular of idiopathic pulmonary fibrosis (IPF). Further provided are methods for the diagnosis, stratification, and monitoring of a therapy response of such lung diseases.

Description

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LU504827

COMPOUNDS AND COMPOSITIONS FOR USE IN THE TREATMENT OF LUNG DISEASES

FIELD OF THE INVENTION

[1] The invention pertains to compounds and compositions comprising inhibitors of Dickkopf like protein 3 (DKK3) for use in the treatment of lung diseases, in particular of idiopathic pulmonary fibrosis (IPF). Further provided are methods for the diagnosis, stratification, and monitoring of a therapy response of such lung diseases.

DESCRIPTION

[2] Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial pneumonia with obscure etiology. IPF has been included in the 2018 National Rare Diseases List, and at present. IPF tends to occur in middle- and old-aged male population and is manifested as progressively worsening dyspnoea, accompanied by restrictive ventilatory dysfunction and gas exchange disorder, eventually leading to hypoxemia and even respiratory failure. The prognosis of IPF is poor, and its lung histology and high-resolution CT (HRCT) of the chest are manifested as a usual interstitial pneumonia (UIP) with diffuse alveolitis and pulmonary interstitial fibrosis as the main pathologic features, and progressive and irreversible lung damage. The loss of alveoli in numbers leads to the decline or even loss of the pulmonary ventilation function, resulting in hypoxia, reduced mobility and even death of patients. As a result, the average survival duration for those diagnosed with IPF is only 2.8 years, the mortality rate is higher than that of most tumors, and thus IPF is called a “tumor-like disease”.

[3] The traditional method for treatment of IPF is mainly administrating anti-inflammatory drugs and anti-fibrotic drugs. Glucocorticoids can suppress the inflammatory response and the immune process. Immunosuppressants (cyclophosphamide, azathioprine, methotrexate, etc.) also have the effect of inhibiting the inflammatory response. Thus, glucocorticoids and immunosuppressants/cytotoxic drugs were used as the basic drugs for the treatment of IPF, but they are not ideally effective in patients with intermediate to advanced fibrosis. The drugs currently approved for IPF treatment in China include Pirfenidone, Nintedanib, etc., which have a delay effect on the decline of pulmonary function of patients. But it is difficult to improve the pulmonary function or reverse the progression of the disease, and the occurrence of fibrosis cannot be really prevented. At present, the fundamental reason for the lack of effective conventional treatment for IPF worldwide, other than whole-lung transplantation, is the lack of effective methods to regenerate and repair the damaged alveolar structures.

[4] Thus, it is an object of the invention is to provide improved diagnostic and therapeutic options to improve the situation for patients of IPF.

BRIEF DESCRIPTION OF THE INVENTION

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[5] Generally, and by way of brief description, the main aspects of the present invention can 94927 be described as follows:

[6] In a first aspect, the invention pertains to a compound or composition for use in treating a subject suffering from a lung disease, and/or for methods of treating a lung disease, wherein the treatment comprises a step of administering to the subject in need of treatment a compound or composition (agent) in an effective amount so that the compound or composition (agent) in the subject elicits an anti-DKK3 therapy that involves any one or a combination of: (i) inhibiting and/or reducing the expression of a DKK3 protein or DKK3 mRNA in the subject; and/or (ii) inhibiting and/or reducing a function and/or stability of a DKK3 protein in the subject.

[7] In a second aspect, the invention pertains to compound or composition for use in the treatment of a lung disease in a subject, wherein the compound or composition induces in the subject inhibiting or reducing the expression of a DKK3 protein or DKK3 mRNA, or knocking down or knocking out the DKK3 gene and/or DKK3 protein or DKK3 mRNA expression in the subject.

[8] In a third aspect, the invention pertains to a pharmaceutical composition comprising the compound and composition of the invention together with a pharmaceutically acceptable carrier and/or excipient.

[9] In a fourth aspect, the invention pertains a method for the diagnosis, risk stratification, disease outcome, disease prognosis or differential severity analysis of a lung disease in a subject that has or is suspected to have the lung disease, comprising the steps of: (i) determining the level of a DKK3 biomarker in a biological sample obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; and wherein the level of one or more DKK3 biomarker is correlated with the diagnosis of the lung disease, a defined risk stratification, a defined disease outcome, a defined disease prognosis, or a differential severity of the lung disease in the subject.

[10] In a fifth aspect, the invention pertains to A method for differentially diagnosing between

COPD and IPF in a subject, comprising the steps of: (i) determining the level of a DKK3 biomarker in a biological sample obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; and wherein if the level of one or more DKK3 biomarker as determined in (i) is decreased compared to a certain cut-off value, reference value or control experiment, the subject is diagnosed with COPD and not with IPF, and wherein if the

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LU504827 level of one or more DKK3 biomarker as determined in (i) is increased compared to a certain cut-off value, reference value or control experiment, the subject is diagnosed with IPF and not with COPD.

[11] In a sixth aspect, the invention pertains to method of medical decision making for individual patient therapy related to the severity of a lung disease, preferably a lung disease as recited in any of claims 22 to 32, by monitoring therapy response to a certain drug in a subject, comprising the steps of (i-1) - (i-3): (i-1) providing for at least two biological samples from said subject at various time points selected from the group of e before initiation of therapy, e after initiation of therapy, and/or e at one or more further time point; (i-2) determining the level of a DKK3 biomarker in the at least two biological samples obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; (i-3) associating the level of the DKK3 biomarker in said samples with a positive or a negative response to said certain drug; wherein said positive response is given if the level of the DKK3 biomarker decreases or increases during drug treatment.

DETAILED DESCRIPTION OF THE INVENTION

[12] In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[13] In a first aspect, the invention pertains to a compound or composition for use in treating a subject suffering from a lung disease, and/or for methods of treating a lung disease, wherein the treatment comprises a step of administering to the subject in need of treatment a compound

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LU504827 or composition (agent) in an effective amount so that the compound or composition (agent) in the subject elicits an anti-DKK3 therapy that involves any one or a combination of: (i) inhibiting and/or reducing the expression of a DKK3 protein or DKK3 mRNA in the subject; and/or (ii) inhibiting and/or reducing a function and/or stability of a DKK3 protein in the subject.

[14] “Dickkopf-related protein 3” or “DKK3” is known for its function to antagonize canonical

Wht signaling by inhibiting LRP5/6 interaction with Wnt and by forming a ternary complex with the transmembrane protein KREMEN that promotes internalization of LRP5/6. DKKs play an important role in vertebrate development, where they locally inhibit Wnt regulated processes such as antero-posterior axial patterning, limb development, somitogenesis and eye formation.

In the adult, Dkks are implicated in bone formation and bone disease, cancer and Alzheimer disease (by similarity). Pertinent information on the human DKK3 protein is accessible on

UniProt: Q9UBP4 (Entry version 17-Jul-2023), and more preferably comprises an amino acid sequence shown in SEQ ID NO: 1. The human DKK3 protein is a secreted protein, and its gene is located on chromosome 11: location 11p15.3. The term DKK3 in some embodiments of the invention may also pertain to variants of the human DKK3 protein having an amino acid sequence that is substantially identical to, or of at least 80%, preferably 85%, more preferably 90, 95, 96, 97, 98, 99, or 100% sequence identity to, the amino acid sequence shown in any of

SEQ ID NO: 1 or 2, as determined using, e.g., the “Blast 2 sequences” algorithm described by

Tatusova & Madden 1999 (FEMS Microbiol Lett 174: 247-250), and which (preferably) retain biological activity identical or substantially identical to the respective reference DKK3. Preferred variants of DKK3 protein comprise sequence variants thereof due to sequence polymorphism between and within populations of the respective species, as well as mutations compared to the wild-type sequence of DKK3.

[15] An “inhibitor of DKK3” (or “DKK3 inhibitor”) is any moiety that inhibits DKK3, which can mean inhibition of the expression (eg the amount), function, activity and/or stability of DKKS3, especially of mRNA and/or protein of DKK3. A DKK3 inhibitor may impair, suppress, reduce and/or lower the expression of DKK3 (eg DKK3 mRNA or protein) in a cell. The term “expression” means in this context the cellular process of transcribing a gene into an mRNA and the following translation of the mRNA into a protein (and in certain embodiment, the subsequent transport and localization of such protein). “Gene expression” therefore may thus refer only to the generation of MRNA, irrespectively from the fate of the so produced mRNA, or alternatively/additionally to the translation of the expressed mRNA into a protein (or transport and localization of such protein). The term “protein expression” on the other hand may refer to the complete cellular process of synthesis of proteins and/or transport/localization thereof into certain cellular compartments. A DKK3 inhibitor may impair (eg, induces a decrease or reduction in) the efficiency, effectiveness, amount or rate of one or more activities of DKK3 (for

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LU504827 example, by impairing the expression of DKK3 protein and/or DKK3 mRNA), such as one or more of those activities described herein, for example. À DKK3 inhibitor may have a negative effect towards the stability of DKK3 (eg DKK3 mRNA or protein), which shall be understood in its broadest sense, and shall include inhibitors which, for example, interfere with and reduce the

DKK3 protein half-life or interfere with and disturb DKK3 protein folding, protein presentation or transport/localisation within the cell.

[16] Such a DKK3 inhibiting moiety can act directly, for example, by binding to DKK3 and decreasing the amount or rate of one or more of the properties of DKK3 such as its expression, function and/or stability. A DKK3 inhibitor may also decrease the amount or rate of DKK3 function or activity by impairing its expression, stability, for example, by binding to DKK3 protein or mRNA and modifying it, such as by removal or addition of a moiety, or altering its three- dimensional conformation; and by binding to DKK3 protein or mRNA and reducing its stability or conformational integrity. A DKK3 inhibitor may, alternatively, act indirectly, for example, by binding to a regulatory molecule or gene region to modulate such regulatory protein or gene region function and hence consequentially affect a decrease in the amount or rate of DKK3 expression (eg amount), function/activity and/or stability, in particular by impairing one or more activity of DKK3 protein or mRNA (such as by changing the amount or rate of expression and/or stability of DKK3 protein or mRNA). Thus, a DKK3 inhibitor can act by any mechanisms that impair, such as result in a decrease in, the amount or rate of DKK3 expression (eg amount), function/activity and/or stability. Non-limiting examples of DKK3 inhibitors that act directly on

DKK3 include: (i) siRNA or shRNA molecules that bind to and reduce expression of DKK3

MRNA; (ii) small molecule moieties that bind to the catalytic domain of DKK3 and reduce the kinase activity of DKK3, (iii) gene editing components such as guide nucleic acids (gRNA or gDNA), and (iv) antibodies which bind DKK3 protein and inhibits one or more functions of

DKKS3.

[17] General and specific examples of DKK3 inhibitors are described elsewhere herein, including those as may be characterized by the applicable functional and/or structural features set out herein.

[18] In certain embodiments of the invention the compound or composition (or agent) is a nucleic acid or nucleic acid construct comprising a sequence complementary to or identical to a

DKK3 mRNA or DKK3 gene sequence.

[19] In one particular set of embodiments, the DKK3 inhibitor is a nucleic acid. [200 The terms “nucleic acid”, “polynucleotide” and “oligonucleotide” are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., MRNA), analogues of the DNA or RNA generated using nucleotide analogues

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LU504827 (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogues), and hybrids thereof. The nucleic acid molecule can be single-stranded or double-stranded.

[21] In the case of DKK3 inhibitors being CRISPR/Cas9 constructs and/or guide RNA/DNAs (gRNA/gDNA) and/or tracrRNAs, the basic rules for the design of CRISPR/Cas9 mediated gene editing approaches are known to the skilled artisan and for example reviewed in Wiles MV et al (Mamm Genome 2015, 26:501) or in Savié N and Schwank G (Transl Res 2016, 168:15).

[22] In particular embodiments, the DKK3 inhibitor may be an inhibitory nucleic acid molecule, such as antisense nucleotide molecule including a siRNA or shRNA molecule, for example as described in detail herein below.

[23] In more particular of such embodiments, the inhibitory nucleic acid (such as siRNA or shRNA) can bind to, such as specifically bind to, a nucleic acid (such as mRNA) that encodes or regulates the expression, amount, function, activity or stability of: (i) DKK3; or (ii) a gene that controls the expression, amount, function and/or stability of DKK3 and, for example, thereby modulates the expression, amount function, activity and/or stability of DKK3.

[24] An inhibitor of DKK3 that is a nucleic acid can be, for example, an anti-sense nucleotide molecule, an RNA, DNA or PNA molecule, or an aptamer molecule. An anti-sense nucleotide molecule can, by virtue of it comprising an anti-sense nucleotide sequence, bind to a target nucleic acid molecule (eg based on sequence complementarity) within a cell and modulate the level of expression (transcription and/or translation) of DKK3, or it may modulate expression of another gene that controls the expression, function and/or stability of DKK3. Similarly, an RNA molecule, such as a catalytic ribozyme, can bind to and alter the expression of the DKK3 gene, or it can bind to and alter the expression of other genes that control the expression, function and/or stability of DKK3, such as a kinase molecule, interacting protein, a transcription factor for or repressor protein of DKK3. An aptamer is a nucleic acid molecule that has a sequence that confers it an ability to form a three-dimensional structure capable of binding to a molecular target.

[25] An inhibitor of DKK3 that is a nucleic acid can be, for example, can further be a double- stranded RNA molecule for use in RNA interference. RNA interference (RNAI) is a process of sequence-specific gene silencing by post-transcriptional RNA degradation or silencing (prevention of translation). RNAI is initiated by use of double-stranded RNA (dsRNA) that is homologous in sequence to the target gene to be silenced. A suitable double-stranded RNA (dsRNA) for RNAI contains sense and antisense strands of about 21 contiguous nucleotides corresponding to the gene to be targeted that form 19 RNA base pairs, leaving overhangs of two nucleotides at each 3’ end (Elbashir et al, Nature 411:494-498 (2001); Bass, Nature 411:428-429 (2001); Zamore, Nat. Struct. Biol. 8:746-750 (2001)). dsRNAs of about 25-30

U31172LU - 7 of 18 - nucleotides have also been used successfully for RNAi (Karabinos et al., Proc. Natl. Acad. sa. 904827

USA 98:7863-7868 (2001). dsRNA can be synthesised in vitro and introduced into a cell by methods known in the art.

[26] A particularly preferred example of an antisense molecule of the invention is a small interfering RNA (siRNA) or endoribonuclease- prepared siRNA (esiRNA). An esiRNA is a mixture of siRNA oligos resulting from cleavage of a long double-stranded RNA (dsRNA) with an endoribonuclease such as Escherichia coli RNase Ill or dicer. esiRNAs are an alternative concept to the usage of chemically synthesised siRNA for RNA Interference (RNAI). An esiRNAs is the enzymatic digestion of a long double stranded RNA in vitro.

[27] As described above, a modulator of the invention that is an RNAi molecule (such as an siRNA) may bind to and directly inhibit or antagonise the expression of mRNA of DKK3.

However, a modulator of the invention that is an RNAi molecule (such as an siRNA) may bind to and inhibit or antagonise the expression of mRNA of another gene that itself controls the expression (or function or stability) of DKK3.

[28] The sequence identity of the antisense molecule according to the invention in order to target a DKK3 mRNA (or to target mRNA of a gene controlling expression, function and/or stability of DKK3), is with increasing preference at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% and 100% identity to a region of a sequence encoding the DKK3 protein, as disclosed herein. Preferably, the region of sequence identity between the target gene and the modulating antisense molecule is the region of the target gene corresponding to the location and length of the modulating antisense molecule. For example, such a sequence identity over a region of about 19 to 21bp of length corresponding to the modulating siRNA or shRNA molecule). Means and methods for determining sequence identity are known in the art. Preferably, the BLAST (Basic Local Alignment Search Tool) program is used for determining the sequence identity with regard to one or more DKK3 RNAs as known in the art. On the other hand, preferred antisense molecules such as siRNAs and shRNAs of the present invention are preferably chemically synthesised using appropriately protected ribonucleoside phosphoramidites and a conventional RNA synthesiser. Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO,

USA), Pierce Chemical (part of Perbio Science, Rockford, IL (USA), Glen Research (Sterling,

VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).

[29] The ability of antisense molecules, siRNA, and shRNA to potently, but reversibly, silence genes in vivo make these molecules particularly well suited for use in the pharmaceutical composition of the invention which will be also described herein below. Ways of administering siRNA to humans are described in De Fougerolles et al, Current Opinion in Pharmacology, 2008, 8:280-285. Such ways are also suitable for administering other small RNA molecules like

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LU504827 shRNA. Accordingly, such pharmaceutical compositions may be administered directly formulated as a saline, via liposome based and polymer-based nanoparticle approaches, as conjugated or complexation pharmaceutical compositions, or via viral delivery systems. Direct administration comprises injection into tissue, intranasal and intratracheal administration.

Liposome based and polymer- based nanoparticle approaches comprise the cationic lipid

Genzyme Lipid (GL) 67, cationic liposomes, chitosan nanoparticles and cationic cell penetrating peptides (CPPs). Conjugated or complexation pharmaceutical compositions comprise PEI- complexed antisense molecules, siRNA, shRNA or miRNA. Further, viral delivery systems comprise influenza virus envelopes and virosomes.

[30] The antisense molecules, siRNAs, shRNAs may comprise modified nucleotides such as locked nucleic acids (LNAs). The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge “locks” the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such oligomers are synthesised chemically and are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organisation. This significantly increases the hybridisation properties (melting temperature) of oligonucleotides. Particularly preferred example of siRNAs is GapmeR (LNA™ GapmeRs (Exigon)). GapmeRs are potent antisense oligonucleotides used for highly efficient inhibition of DKK3 mRNA (or of mRNA of a gene controlling expression, function and/or stability of DKK3). GapmeRs contain a central stretch of

DNA monomers flanked by blocks of LNAs. The GapmeRs are preferably 14-16 nucleotides in length and are optionally fully phosphorothioated. The DNA gap activates the RNAse H- mediated degradation of targeted RNAs and is also suitable to target transcripts directly in the nucleus.

[31] Preferred antisense molecules for targeting DKK3 are antisense molecules or constructs having a sequence complementary to a region (such as one described above) of a nucleic acid sequence of an DKK3 mRNA, preferably a sequence complementary to a region of a sequence encoding the amino acid sequence shown in SEQ ID NO: 1, more preferably, a sequence complementary to a region of between about 15 to 25 bp (such as between about 19 and 21 bp) of a sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2, in particular of

SEQ ID NO: 1.

[32] For example, preferred embodiments of the invention pertain to nucleic acid inhibitors of

DKK3 is capable of binding to a DKK3 gene sequence, and preferably wherein the method involves a gene therapy impairing (reducing or inhibiting) the expression of a DKK3 protein or

DKK3 mRNA. In certain embodiments of the invention, the compound or composition (agent) is capable of selectively binding to a DKK3 gene sequence or to a DKK3 mRNA sequence. In

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LU504827 certain embodiments of the invention, the compound or composition (agent) is an RNA construct binding selectively to a DKK3 mRNA via a complementary DKK3 specific mRNA sequence (not a polyA sequence). For example, the therapy of the invention involves a targeted knock out of the DKK3 gene in lung cells in the subject.

[33] In other embodiment of the invention the compound or composition (agent) is an antigen binding construct specifically binding to a DKK3 protein, or a proteinaceous regulator of DKK3 mediated signaling. In such embodiments it may be preferred that the method involves inhibiting a function or stability of a DKK3 protein, or removing or reducing the amount of a DKK3 protein, preferably immunologically by using one or more antibody or antigen binding fragments thereof.

[34] In embodiments relating to antibodies, it may be preferred that an antibody or antigen binding fragment thereof is an anti-DKK3 antibody. Such antibodies are known in the art (see also WO/2020/081579).

[35] Certain preferred embodiments pertain to a genetic construct for gene editing that is used as an inhibitor of expression, function and/or stability of DKK3 in the context of the herein described invention. By using genome editing constructs it is possible to modulate the expression, stability and/or activity of DKK3. Genome editing approaches are well known in the art and may be easily applied when the respective target genomic sequences are known.

Preferably, such approaches may be used in gene therapy using e.g. viral vectors, which specifically target tumor cells in accordance with the above descriptions.

[36] In case of genome editing, DNA is inserted, replaced, or removed, from a genome using artificially engineered nucleases, or so called “molecular scissors”. The nucleases create specific double- stranded break (DSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by natural processes of homologous re-combination (HR) and non-homologous end-joining (NHEJ). For doing so, engineered nucleases such as zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENSs), the CRISPR/Cas system, and engineered meganuclease re-engineered homing endonucleases are routinely used for genome editing. According to another preferred embodiment, for genome editing approaches for modulating/inhibiting DKK3, the rare-cutting endonuclease is Cas9, Cpfl, TALEN, ZFN, or a homing endonuclease may be used. Also, it may be convenient to engineer using DNA-guided Argonaute interference systems (DAIS). Basically, said Argonaute (Ago) protein is heterologously expressed from a polynucleotide introduced into said cell in the presence of at least one exogenous oligonucleotide (DNA guide) providing specificity of cleavage to said Ago protein to a preselected locus. The TALEN and Cas9 systems are respectively described in WO 2013/176915 and WO 2014/191128. The Zinc-finger nucleases (ZFNs) are initially described in Kim, YG; Cha, J.; Chandrasegaran, S. ("Hybrid restriction enzymes: zinc finger fusions to Fok | cleavage domain" (1996). Proc Natl Acad Sci

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USA 93 (3): 1156-60). Cpfl is class 2 CRISPR Cas System described by Zhang et al. (Cpfl is 127904827 single RNA-guided Endonuclease of a Class 2 CRIPR-Cas System (2015) Cell; 163:759-771).

The argonaute (AGO) gene family was initially described in Guo S, Kemphues KJ. ("par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed" (1995) Cell;81(4):611-20).

[37] The use of the CRISPR/Cas9, CRISPR/Cpfl or the Argonaute genome-editing systems is particularly adapted to be used in combination with the transfection of guide RNA or guide

DNA sequences. In this context the guide-RNAs and a nucleic acid sequence coding for Cas9 nickase (or similar enzymes), is transfected into a target cell (preferably a tumor cell) so that they form a complex able to induce a nick event in double-stranded nucleic acid targets in order to cleave the genetic sequence between said nucleic acid targets.

[38] In certain embodiments, it may be useful to deliver the guide RNA-nanoparticle formulations separately from the Cas9. In such an instance a dual-delivery system is provided such that the Cas9 may be delivered via a vector and the guide RNA is provided in a nanoparticle formulation, where vectors are considered in the broadest sense simply as any means of delivery, rather than specifically viral vectors. Separate delivery of the guide RNA- nanoparticle formulation and the Cas9 may be sequential, for example, first Cas9 vector is delivered via a vector system followed by delivery of sgRNA-nanoparticle formulation) or the sgRNA-nanoparticle formulation and Cas9 may be delivered substantially contemporaneously (i.e., co-delivery). Sequential delivery may be done at separate points in time, separated by days, weeks or even months. In certain embodiments, multiple guide RNAs formulated in one or more delivery vehicles (e.g., where some guide RNAs are provided in a vector and others are formulated in nanoparticles) may be provided with a Cas9 delivery system. In certain embodiments, the Cas9 is also delivered in a nanoparticle formulation. In such an instance the guide RNA-nanoparticle formulation and the Cas9 nanoparticle formulation may be delivered separately or may be delivered substantially contemporaneously (i.e., co- delivery). As will now be apparent to the person of ordinary skill, the gene target of such genome-editing approaches may be the gene of DKK3. Alternatively, the gene target of such editing may be another gene that controls the expression, function and/or stability of DKK3.

[39] In preferred embodiments of the invention, the compounds for genome editing approaches according to the invention comprise at least the use of a guide RNA or DNA complementary to a region (such as one described above) of a DKK3 sequence. In some additional embodiments, the compounds for use in genome editing approaches of the invention may include donor sequences homologous to such a region of DKK3, as templates for homology directed repair. The donor sequences comprise a mutated sequence of DKK3 that when used in the CRISPR induced repair mechanism in a target cell, is by homologous

U31172LU - 11 of 18 - recombination inserted/copied into the DKK3 genomic locus, and therefore yields into a mutated oo

DKK3 gene which is characterized by a reduced expression, function and/or stability of the expressed DKK3. CRISPR/Cas9 genome editing in cancer therapy is reviewed for example in

Khan FA et al: “CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases.” (Oncotarget. 2016 May 26. doi: 10.18632/oncotarget.9646; incorporated by reference in its entirety).

[40] Preferably, in context of the invention the antibody of antigen binding fragment thereof is administered to the subject, and preferably to the lung of the subject.

[41] In preferred embodiments of the invention, the therapy is administered to, and preferably confined to, the lung, or lung tissue in the subject.

[42] As used herein, a “lung disease” may be selected from a “fibrotic lung disease”. The term “fibrotic lung disease” in context of the present invention refers to a group of diseases characterized by the formation or development of excess fibrous connective tissue (fibrosis) in the lungs. Symptoms of pulmonary fibrosis are mainly: shortness of breath, particularly with exertion; chronic dry, hacking coughing; fatigue and weakness; chest discomfort; and loss of appetite and rapid weight loss. Pulmonary fibrosis may be a secondary effect of other diseases, most of them being classified as interstitial lung diseases, such as autoimmune disorders, viral infections or other microscopic injuries to the lung. Pulmonary fibrosis can also appear without any known cause (“idiopathic”). Idiopathic pulmonary fibrosis is a diagnosis of exclusion of a characteristic set of histologic/pathologic features known as usual interstitial pneumonia (UIP).

Also included are in certain embodiments of the invention fibrotic changes after acute respiratory distress syndrom (ARDS).

[43] A lung disease in context of the invention is preferably a fibrotic lung disease, most preferably the lung disease is idiopathic lung fibrosis (IPF).

[44] In some embodiments of the invention, the method described comprises a step of obtaining a biological sample of the subject either before treatment, concomitant with treatment, or after treatment. Such method may include one or more steps of a diagnostic method as described herein below.

[45] In a second aspect, the invention pertains to a compound or composition for use in the treatment of a lung disease in a subject, wherein the compound or composition induces in the subject inhibiting or reducing the expression of a DKK3 protein or DKK3 mRNA, or knocking down or knocking out the DKK3 gene and/or DKK3 protein or DKK3 mRNA expression in the subject. The specific definitions and embodiments provided herein in context of the first aspect shall equally apply for the second aspect.

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[46] In a third aspect, the invention pertains to a pharmaceutical composition comprising the 004827 compound and composition of the invention together with a pharmaceutically acceptable carrier and/or excipient.

[47] In a fourth aspect, the invention pertains a method for the diagnosis, risk stratification, disease outcome, disease prognosis or differential severity analysis of a lung disease in a subject that has or is suspected to have the lung disease, comprising the steps of: (i) determining the level of a DKK3 biomarker in a biological sample obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; and wherein the level of one or more DKK3 biomarker is correlated with the diagnosis of the lung disease, a defined risk stratification, a defined disease outcome, a defined disease prognosis, or a differential severity of the lung disease in the subject.

[48] Preferably, said diagnosis of the lung disease, defined risk stratification, defined disease outcome, defined disease prognosis, or a differential severity of the lung disease in a subject is given if the level of one or more DKK3 biomarker as determined in (i) is above or below a certain cut-off value, reference value or control experiment.

[49] In some embodiments of this aspect, the lung disease is an obstructive lung disease, such as preferably Chronic obstructive pulmonary disease (COPD). The COPD may be characterized by two conditions: emphysema and chronic obstructive bronchitis.

[60] Said diagnosis of COPD, defined risk stratification, defined disease outcome, defined disease prognosis, or a differential severity of the COPD in the subject is preferably given if the level of one or more DKK3 biomarker as determined in (i) is decreased compared to a certain cut-off value, reference value or control experiment.

[51] In some embodiments, the lung disease is a fibrotic lung disease (pulmonary fibrosis), such as a pathological condition associated with a formation or development of excess fibrous connective tissue (fibrosis) in lungs. Such fibrotic lung disease may be selected from idiopathic pulmonary fibrosis (IPF), familial interstitial pulmonary fibrosis idiopathic, an interstitial lung disease such as idiopathic interstitial pneumonia, a connective tissue disease associated interstitial lung disease, and hypersensitivity pneumonitis; and more preferably is idiopathic pulmonary fibrosis (IPF).

[62] A diagnosis of pulmonary fibrosis, defined risk stratification, defined disease outcome, defined disease prognosis, or a differential severity of the pulmonary fibrosis in the subject is in some embodiments given if the level of one or more DKK3 biomarker as determined in (i) is increased compared to a certain cut-off value, reference value or control experiment.

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[63] Yet, in a further embodiment, the DKK3 biomarker is a DKK3 protein, or protein fragment thereof, and wherein the determining the level of the DKK3 biomarker involves and one antibodies, or an antigen binging capture molecule, a DKK3 binding receptor protein, or fragment thereof which selectively binds to the DKK3 protein or protein fragment thereof, antibodies or antigen binding agents that are bound to a solid support such as, for example, a plastic surface or beads or in an array, and may involve a test format for detecting protein levels including an immunoassay such as an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blotting and immunoprecipitation.

[54] Preferably, a DKK3 biomarker is a nucleic acid biomarker, and wherein determining the level of the DKK biomarker comprises the use of a nucleic acid detection assay selected from

DNA sequencing methods, in particular any next generation sequencing (NGS) based methods, probe hybridization methods, enzyme mismatch cleavage methods; polymerase chain reaction (PCR) based assays; branched hybridization methods; rolling circle replication; NASBA, molecular beacon technology; E-sensor technology; cycling probe technology, Dade Behring signal amplification methods; ligase chain reaction; and sandwich hybridization methods, or combinations thereof.

[65] In a fifth aspect, the invention pertains to A method for differentially diagnosing between

COPD and IPF in a subject, comprising the steps of: (i) determining the level of a DKK3 biomarker in a biological sample obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; and wherein if the level of one or more DKK3 biomarker as determined in (i) is decreased compared to a certain cut-off value, reference value or control experiment, the subject is diagnosed with COPD and not with IPF, and wherein if the level of one or more DKK3 biomarker as determined in (i) is increased compared to a certain cut-off value, reference value or control experiment, the subject is diagnosed with IPF and not with COPD.

[66] In a sixth aspect, the invention pertains to method of medical decision making for individual patient therapy related to the severity of a lung disease, preferably a lung disease as recited herein elsewhere, by monitoring therapy response to a certain drug in a subject, comprising the steps of (i-1) - (i-3): (i-1) providing for at least two biological samples from said subject at various time points selected from the group of e before initiation of therapy, e after initiation of therapy, and/or

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LU504827 e at one or more further time point; (i-2) determining the level of a DKK3 biomarker in the at least two biological samples obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; (i-3) associating the level of the DKK3 biomarker in said samples with a positive or a negative response to said certain drug; wherein said positive response is given if the level of the DKK3 biomarker decreases or increases during drug treatment.

[57] In context of the various aspects and embodiments of the invention; the biological sample may be a lung sample, preferably is a lung tissue sample or a liquid lung sample, such as preferably a bronchoalveolar lavage fluid (BALF) sample.

[68] The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments ofthe invention described and/or claimed herein.

[59] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by 220%, +15%, +10%, and for example +5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[60] It is to be understood that application of the teachings of the present invention to a

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LU504827 specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

[61] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[62] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

[63] The figures show:

[64] Figure 1: shows DKK3 expression in COPD and FLD/IPF. A. DKK3 was measured in the

BAL fluid of COPD (obstructive “O”) and FLD patients (restrictive “R”) by ELISA. As compared to individuals without obstruction / restriction (“No O / No R”), DKK3 concentrations were significantly increased in FLD patients and decreased in CODP patients. B.

Immunohistochemistry showed equivalent results with less intensive staining for DKK3 in COPD lungs as compared to control tissue. C. DKK3 immunofluorescence is more prominent in lung section s from IPF lungs as compared to controls patients. D. DKK3 positive cells were quantified in 7 random alveolar regions of healthy and IPF lungs.

[65] Figure 2: shows DKK3 in preclinical murine models: (A) Expression of DKK3 is decreased in smoke-exposed animals, (B) DKK3 is induced in bleomycin-challenged animals. (C) Animalwere subjected to bleomycin treatment to induce pulmonary fibrosis and WT and

DKK3-deficient (DKK3-/-) mice were compared. Pulmonary function parameters such as elastance, resistance and FEF were preserved in the absence of DKK3 (day 21, one-way

ANOVA, statistics levels: *p<0.05; ***p<0.001). (C). Lung sections were stained by H.E. and showed decreased areas of fibrosis in DKK” animals (D). A fibrotic score was determined in random areas and showed significant protection from fibrosis in DKK’ animals (one-way

ANOVA).

[66] The sequences show:

[67] SEQ ID NOs. 1: shows UniProt database entry of the amino acid sequence of accession number Q9UBP4 relating to human DKK3

[68] MQRLGATLLCLLLAAAVPTAPAPAPTATSAPVKPGPALSYPQEEATLNEMFREVEELMEDTQHKLRSAVEEMEA

EEAAAKASSEVNLANLPPSYHNETNTDTKVGNNTIHVHREIHKITNNQTGQMVFSETVITSVGDEEGRRSHECIIDEDCG

PSMYCQFASFQYTCQPCRGQRMLCTRDSECCGDQAQLCVWGHCTKMATRGSNGTICDNQRDCQPGLCCAFQRGLLFPV

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CTPLPVEGELCHDPASRLLDLITWELEPDGALDRCPCASGLLCQPHSHSLVYVCKPTFVGSRDQDGEILLPREVPDEYEVG

SFMEEVRQELEDLERSLTEEMALREPAAAAAALLGGEEI

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EXAMPLES

[69] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[70] The examples show:

[71] Example 1: Association of Dickkopf 3 (DKK3) Expression in Fibrotic Lungs

[72] DKKS3 is associated with COPD and FLD/IPF: Initially, the inventors aimed to identify biomarkers for COPD and FLD. The inventors performed a screen for markers that are altered in these disease entities. DKK3 was identified in bronchoalveolar lavage fluid (BALF) and showed significantly altered levels between patients with COPD and fibrotic lung disease. DKK3 levels were decreased in COPD samples (obstructive lung function) and increased in the BALF from patients with FLD (restrictive lung function, Fig. 1A). Immunostaining of lungs from patients with COPD, IPF, and controls shows equivalent changes of protein expression (Fig. 1B, C, D).

[73] These expression data of DKK3 are confirmed by published sequencing data from several studies. We analyzed published bulk and single cell sequencing (scRNA-seq) data.

DKK3 was found highly expressed in aberrant basaloid, alveolar type 1 cell (AT1), fibroblast and pericyte of IPF lungs [6]. In contrast, COPD lungs showed decreased overall expression of

DKK3 [7], especially in alveolar fibroblasts and pericytes [8].

[74] Example 2: DKK3 is Necessary for Development of Fibrosis in Murine Models

[75] To obtain mechanistic insight in the role of DKK3 in regeneration, we studied the DKK3 expression in disease models of smoke-induced lung damage (“COPD-like”) [9] and bleomycin- induced fibrosis [10]. Equivalent to the data from human clinical investigation as described above, expression of DKK3 was decreased in smoke-exposed animals (Fig. 2A) and induced in bleomycin-challenged animals and (Fig. 2B).

[76] In a next step, we analyzed whether the development of fibrosis in response to bleomycin-was altered in DKK3-deficient (DKK3”) mice (Fig. 2C-E). DKK3” animals showed significantly preservation of lung function (Fig. 2C) and reduced fibrotic development after bleomycin-exposure as compared to wildtype animals (Fib. 2 D,E).

REFERENCES

[77] The references are: 1. Habuta M, Fujita H, Sato K, Bando T, Inoue J, Kondo Y, Miyaishi S, Kumon H, Ohuchi H.

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla. Cell Tissue Res 2020: 379(1): 157-167.

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LU504827 2. Fang X, Hu J, Chen Y, Shen W, Ke B. Dickkopf-3: Current Knowledge in Kidney

Diseases. Front Physiol 2020: 11: 533344. 3. Busceti CL, Di Menna L, Bianchi F, Mastroiacovo F, Di Pietro P, Traficante A, Bozza G,

Niehrs C, Battaglia G, Bruno V, Fornai F, Volpe M, Rubattu S, Nicoletti F. Dickkopf-3

Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor. Front Cell

Neurosci 2018: 12: 292. 4. Cheng WL, Yang Y, Zhang XJ, Guo J, Gong J, Gong FH, She ZG, Huang Z, Xia H, Li H.

Dickkopf-3 Ablation Attenuates the Development of Atherosclerosis in ApoE-Deficient

Mice. J Am Heart Assoc 2017: 6(2). 5. Federico G, Meister M, Mathow D, Heine GH, Moldenhauer G, Popovic ZV, Nordstrom V,

Kopp-Schneider A, Hielscher T, Nelson PJ, Schaefer F, Porubsky S, Fliser D, Arnold B,

Grone HJ. Tubular Dickkopf-3 promotes the development of renal atrophy and fibrosis.

JCI Insight 2016: 1(1): e84916. 6. Adams TS, Schupp JC, Poli S, Ayaub EA, Neumark N, Ahangari F, Chu SG, Raby BA,

Deluliis G, Januszyk M, Duan Q, Arnett HA, Siddiqui A, Washko GR, Homer R, Yan X,

Rosas 10, Kaminski N. Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis. Sci Adv 2020: 6(28): eaba1983. 7. Xu F, Vasilescu DM, Kinose D, Tanabe N, Ng KW, Coxson HO, Cooper JD, Hackett TL,

Verleden SE, Vanaudenaerde BM, Stevenson CS, Lenburg ME, Spira A, Tan WC, Sin

DD, Ng RT, Hogg JC. The molecular and cellular mechanisms associated with the destruction of terminal bronchioles in COPD. Eur Respir J 2022: 59(5). 8. Sauler M, McDonough JE, Adams TS, Kothapalli N, Barnthaler T, Werder RB, Schupp

JC, Nouws J, Robertson MJ, Coarfa C, Yang T, Chioccioli M, Omote N, Cosme C, Jr.,

Poli S, Ayaub EA, Chu SG, Jensen KH, Gomez JL, Britto CJ, Raredon MSB, Niklason

LE, Wilson AA, Timshel PN, Kaminski N, Rosas 10. Characterization of the COPD alveolar niche using single-cell RNA sequencing. Nat Commun 2022: 13(1): 494. 9. Ritzmann F, Borchardt K, Vella G, Chitirala P, Angenendt A, Herr C, Menger MD, Hoth M,

Lis A, Bohle RM, Bals R, Beisswenger C. Blockade of PD-1 decreases neutrophilic inflammation and lung damage in experimental COPD. Am J Physiol Lung Cell Mol

Physiol 2021. 10. Jenkins RG, Moore BB, Chambers RC, Eickelberg O, Konigshoff M, Kolb M, Laurent GJ,

Nanthakumar CB, Olman MA, Pardo A, Selman M, Sheppard D, Sime PJ, Tager AM,

Tatler AL, Thannickal VJ, White ES, Cell ATSAoR, Molecular B. An Official American

Thoracic Society Workshop Report: Use of Animal Models for the Preclinical Assessment of Potential Therapies for Pulmonary Fibrosis. Am J Respir Cell Mol Biol 2017: 56(5): 667-679.

Claims

U31172LU - Tof4- LU504827 CLAIMS

1. A compound or composition for use in treating a subject suffering from a fibrotic lung disease, wherein the treatment comprises a step of administering to the subject in need of treatment a compound or composition (agent) in an effective amount so that the compound or composition (agent) in the subject elicits an anti-DKK3 therapy that involves any one or a combination of: () inhibiting and/or reducing the expression of a DKK3 protein or DKK3 mRNA in the subject; and/or (i) inhibiting and/or reducing a function and/or stability of a DKK3 protein in the subject.

2. The compound or composition for use of claim 1, wherein in (i) the compound or composition (agent) is a nucleic acid or nucleic acid construct comprising a sequence complementary to or identical to a DKK3 mRNA or DKK3 gene sequence.

3. The compound or composition for use of claim 1 or 2, wherein the fibrotic lung disease is idiopathic pulmonary fibrosis. (IPF)

4. The compound or composition for use of claim 2, wherein the nucleic acid is capable of binding to a DKK3 gene sequence, and preferably wherein the method involves a gene therapy impairing (reducing or inhibiting) the expression of a DKK3 protein or DKK3 MRNA.

5. The compound or composition for use of claim 4, wherein the nucleic acid or nucleic acid construct induces a knock-out the DKK3 gene, introduces a genetic mutation into the DKK3 gene, or knocking down the DKK3 mRNA or DKK3 mRNA translation (DKK3 protein expression).

6. The compound or composition for use of any one of claims 1 to 5, wherein the method involves the use of any of the following nucleic acid based targeted genetic perturbation approaches: RNAi, siRNA, shRNA, microRNA, dsRNA, sgRNA, CRISPR/Cas9 gene editing, or any DNA or other equivalent thereof, and preferably wherein the compound or composition (agent) is a nucleic acid that comprises sequence complementary to a DKK3 gene or DKK3 mRNA.

7. The method of claim 6, wherein the compound or composition (agent) is capable of

U31172LU -2of4- selectively binding to a DKK3 gene sequence or to a DKK3 mRNA sequence. LU504827 8 The compound or composition for use of any one of claims 1 to 7, wherein in (ii) the compound or composition (agent) is an antigen binding construct specifically binding to a DKK3 protein, or a proteinaceous regulator of DKK3 mediated signaling.

9. The compound or composition for use of any one of claims 1 to 8, wherein the therapy is administered to, and preferably confined to, the lung, or lung tissue in the subject.

10. A compound or composition for use in the treatment of a lung disease in a subject, wherein the compound or composition induces in the subject an inhibition or reduction of the expression of a DKK3 protein or DKK3 mRNA, or involves knocking down or knocking out the DKK3 gene and/or DKK3 protein or DKK3 mRNA expression in the subject.

11. The compound or composition for use in claim 10, in a treatment of any one of claims 1 to 9.

12. A method for the diagnosis, risk stratification, disease outcome, disease prognosis or differential severity analysis of a lung disease in a subject that has or is suspected to have the lung disease, comprising the steps of: () determining the level of a DKK3 biomarker in a biological sample obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; and wherein the level of one or more DKK3 biomarker is correlated with the diagnosis of the lung disease, a defined risk stratification, a defined disease outcome, a defined disease prognosis, or a differential severity of the lung disease in the subject; and wherein said lung disease in the subject is Chronic Obstructive Pulmonary Disease (COPD), if the level of one or more DKK3 biomarker as determined in (i) is decreased compared to a certain cut-off value, reference value or control experiment; and wherein said lung disease in the subject is IPF if the level of one or more DKK3 biomarker as determined in (i) is increased compared to a certain cut-off value, reference value or control experiment.

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13. A method for differentially diagnosing between COPD and IPF in a subjedt/°0#827 comprising the steps of: () determining the level of a DKK3 biomarker in a biological sample obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 mRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation; and wherein if the level of one or more DKK3 biomarker as determined in (i) is decreased compared to a certain cut-off value, reference value or control experiment, the subject is diagnosed with COPD and not with IPF, and wherein if the level of one or more DKK3 biomarker as determined in (i) is increased compared to a certain cut-off value, reference value or control experiment, the subject is diagnosed with IPF and not with COPD.

14. A method of medical decision making for individual patient therapy related to the severity of IPF, by monitoring therapy response to a certain drug in a subject, comprising the steps of (i-1) - (i-3):

i-1. providing for at least two biological samples from said subject at various time points selected from the group of (i) before initiation of therapy, (ii) after initiation of therapy, and/or (iii) at one or more further time point;

i-2. determining the level of a DKK3 biomarker in the at least two biological samples obtained from the subject, wherein the DKK3 biomarker is selected from one or more of: DKK3 protein or fragments thereof, stable- or functional DKK3 protein, DKK3 MRNA transcript or fragments thereof, DKK3 gene-expression/gene-activation and DKK3 chromosomal DNA methylation;

i-3. associating the level of the DKK3 biomarker in said samples with a positive or a negative response to said certain drug; wherein said positive response is given if the level of the DKK3 biomarker decreases or increases during drug treatment.

15. The method of any one of claims 12 to 14, wherein the biological sample is a lung sample, preferably is a lung tissue sample or a liquid lung sample, such as preferably a

U31172LU -4of4- bronchoalveolar lavage fluid (BALF) sample.

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