Fibroblast activation markers and TGFBI in therapy

Abstract

The present invention relates to a method for detecting markers of fibroblast activity in patients by sandwich immunoassay in order to identify patients, particularly cancer patients, who are expected to benefit from anti-TGFβI therapy. The markers of fibroblast activity may be selected from the N-terminal propeptide of collagen III (Pro-C3), the N-terminal propeptide of collagen VI (Pro-C6), the N-terminal propeptide of collagen I (Pro-C1), and / or the internal sequence (P3NP) within the N-terminal region of type 3 collagen. The present invention also provides anti-TGFβI therapy for use in the treatment of cancer and / or fibrosis.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting markers of fibroblast activation in patients by immunoassay in order to identify patients, particularly cancer patients, who are expected to benefit from anti-TGFβI therapy. The markers of fibroblast activation may be selected from the N-terminal propeptide of type III collagen (Pro-C3), the N-terminal propeptide of type VI collagen (Pro-C6), the N-terminal propeptide of type I collagen (Pro-C1), and / or the internal sequence (P3NP) within the N-terminal region of type III collagen. The present invention also provides anti-TGFβI therapy for use in the treatment of cancer and / or fibrosis. [Background technology]

[0002] Many studies have been conducted, for example in idiopathic pulmonary fibrosis (IPF), to understand the changes in extracellular matrix (ECM) dynamics associated with physiological disorders and decreased lung function [1-4]. In a healthy state, the ECM forms a thin basement membrane layer in the small airways, isolating capillaries from the alveolar space and preventing obstruction of gas diffusion. In fibrosis, the ECM expands, and such diffusion is restricted. The fibrotic ECM in the lung contains high levels of type I, type III, and type VI collagen. In a highly simplified explanation, the basement membrane, which in a normal state is differentiated into a porous structure composed of type IV collagen and laminin, is replaced in pulmonary fibrosis by a densely packed interstitial ECM composed of collagen with a completely different composition and function [5].

[0003] Type III collagen, along with type I collagen, constitutes a major structural protein in the human body. Among structural proteins excluding bone, type III collagen is crucial for the formation of type I collagen fibrils, and structural proteins consist almost entirely of type I collagen [6,7]. During fibril assembly, the N-terminal propeptide of type III procollagen is cleaved by a specific N-protease before it can be incorporated into the extracellular matrix (ECM) as mature type III collagen, resulting in its release from the ECM into circulation. This N-terminal propeptide molecule, also known as “PIIINP,” consists of three identical α-chains and has a total molecular weight of 42 kDa. Removal of PIIINP can be incomplete, and if the propeptide remains attached to the collagen molecule, it can lead to the formation of thin fibrils with abnormal cross-bonding, resulting in a rapid metabolic turnover [8,9]. Therefore, this propeptide can serve as a marker for both the formation and degradation of type III collagen.

[0004] WO2014 / 170312A1 discloses a monoclonal antibody that has specific reactivity to the C-terminal neo-epitope of PIIINP contained in the C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 1) (where X is Gly or Pro) (the neo-epitope is also referred to herein as "Pro-C3"). The document also discloses a competitive immunoassay for detecting the level of the C-terminal neo-epitope of PIIINP in a biological sample by contacting the sample with the monoclonal antibody and determining the amount of antibody bound (also referred to herein as the "Pro-C3 assay"). P3NP is a common biomarker of fibroblast activity containing an internal sequence within the N-terminal region of type 3 collagen, and its sequence is: 114 PGIPGRNGDP 123 It has (Uniprot P02461)(Sequence ID 2).

[0005] Type VI collagen is a unique extracellular collagen capable of forming independent microfibrillary networks within the cell's basement membrane. It can interact with other matrix proteins, including collagen, biglycans, and proteoglycans. Within muscle, type VI collagen forms part of the sarcodiae and is involved in force transmission by anchoring muscle fibers within the extracellular matrix. Mutations in type VI collagen can also cause Vethlem's myopathy and Ulrich's congenital muscular dystrophy. The C-terminal amino acid sequence of the type VI collagen α3 chain has been reported to be cleaved after the secretion of mature type VI microfibrillaries. However, type VI collagen is not only involved in muscle and muscle loss.

[0006] Type VI collagen in microfibrillary stroma (a triple-helical molecule composed of α1(VI), α2(VI), and α3(VI) chains) is prominently expressed in most connective tissues, particularly adipose tissue, and anchors cells in these tissues by interacting with other ECM proteins. During microfibril formation, the triple-helical core of type VI collagen is released from the propeptide by proteolytic reactions, and the cleavage of the C-terminal propeptide of the α3(VI) chain generates an adipokine, which is an endotrophin.

[0007] PRO-C6 is a biomarker related to type VI collagen formation and endotrophin release, containing the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen, which is cleaved when a novel type VI collagen molecule is assembled in the extracellular matrix. This C-terminal epitope is also the C-terminal epitope of endotrophin, a bioactive fragment. The PRO-C6 biomarker and PRO-C6 assay (in particular, PRO-C6 ELISA) are described in WO2016 / 156526. This assay utilizes a monoclonal antibody that specifically binds to the 10-amino acid sequence of the C-terminal of the C5 domain of the α3 chain of type VI collagen. The role of endotrophin as a pro-fibrotic, pro-inflammatory, and pro-tumorogenic molecule has been observed in preclinical models of breast and liver cancer. PRO-C6 has been established as a prognostic biomarker for mortality and disease progression in patients with chronic kidney disease and diabetic nephropathy, and as a predictive marker for responsiveness to hypoglycemic therapy in diabetic patients.

[0008] Type I collagen is the most abundant collagen in the body because it is the main structural protein of bone. PRO-C1 is a biomarker related to the formation of type I collagen and contains the N-terminal propeptide of type I collagen, and its sequence 96 PDGSESPTDQETTGV 110 It has (UniProt P02452)(Sequence ID 3).

[0009] Cancer fibrosis, its associated extracellular matrix, and collagen synthesis are becoming increasingly clear as crucial components for defining the rich stromal response in cancer and for clarifying the prognosis and responsiveness to anti-cancer therapies. Cancer-associated fibroblasts (CAFs) are the main driving force behind this so-called desmoplastic reaction and collagen synthesis. To best develop novel and promising therapies targeting the excessive accumulation of ECM and collagen in the tumor microenvironment, it is essential to elucidate the essence of the CAF-driven collagen synthesis.

[0010] While there is strong interest in drug-induced reduction of fibrosis in conditions such as idiopathic pulmonary fibrosis (IPF), non-alcoholic steatohepatitis (NASH), chronic kidney disease (CKD), and systemic sclerosis (SSC), numerous failures continue in Phase II and Phase III trials. This may be due not only to the lack of optimization in both clinical trial design and robust endpoints, but also to a lack of understanding of the driving forces of the disease. Although numerous new drugs are being actively tested in animal studies for liver and lung fibrosis, it is somewhat concerning that these studies have little to no applicability to human clinical settings. It is necessary to understand the pathways that cause fibrosis in humans. To benefit patients in human clinical settings, it is necessary to understand not only the generally common characteristics of central fibrosis indicators, but also the pathology of fibrosis, which involves a wide range of disease pathways.

[0011] The protein TGFβI (transformed growth factor-β-inducing), also known as BigH3, has been shown to 'reprogram' the tumor microspace in pancreatic cancer regions and is associated with survival outcomes, suggesting that TGFβI is a promising novel target for future anticancer drug development

[10]

[11] . Although the relationship between TGFβI, CAF activity, and collagen turnover has been described

[12] , little is known about the direct effects of TGFβI / BigH3 on fibroblasts and associated collagen synthesis.

[0012] Traditional genome-wide association studies (GWAS) have been conducted based on associations with clinical parameters

[13] . While clinical parameters can be considered disease assessment criteria, biochemical markers are more closely related to acute disease activity. In direct consistency, PRO-C3, a measure of type III collagen formation, is a surrogate measure of fibrosis formation

[14] . PRO-C3 is a quantification of the propeptide of type III collagen chains (also known as PNIIIP). When fibroblasts produce type III collagen, the collagen propeptide is released before the collagen is incorporated into the matrix. Consequently, PRO-C3 can be considered a surrogate biomarker for the rate of fibrosis formation. Type I collagen is the most abundant protein in the body and the most abundant protein in the fibrotic extracellular matrix (ECM), whereas type III collagen is not abundant in the body and is specific to the fibrotic ECM

[15] . PRO-C3 was associated not only with the prognosis of liver fibrosis progression

[16] and the diagnosis of liver fibrosis

[15] , but also with pharmacodynamic repose leading to interventions that reduce fibrosis

[17]

[18] . This invention clarifies the relationship between organ fibrosis, PRO-C3, PRO-C1, PRO-C6, and P3NP, and pathway genes that cause high levels of fibrosis formation. [Overview of the project]

[0013] overview To investigate the potential relationship between the biomarker PRO-C3 and pathway genes responsible for high levels of fibrosis, GWAS analysis was performed based on Pro-C3 levels measured using a Pro-C3 assay. A strong association was revealed between elevated Pro-C3 levels and the TGFβI gene. Such a correlation was not observed between the TGFβI gene and other markers of chronic liver disease.

[0014] Researchers hypothesized that high levels of Pro-C3 (propeptide of type III collagen), as a measure of fibrotic activity, are associated with the TGFβI risk allele rs2073511, and investigated whether TGFβI has a direct effect on fibroblast collagen synthesis by stimulating pancreatic cancer-associated fibroblasts (CAFs) in vitro. It is a misconception that TGFβI is periostin and is also known to play a central and multifaceted role in tumorigenesis

[19] . Both of these matrix cellular proteins contain one emylin (EMI) and four fasiclin-1 (FAS) modules

[20] . Interestingly, the biological action of another protein (emilin-2) (wnt signaling / wnt1 interaction) requires the presence of the EMI domain on that protein, and the action is not obtained in deletion mutants lacking this domain. TGFβI and the EMI domain on periostin are closely related due to their high sequence homology and their generally shorter length compared to other EMI domains (Figure 1)

[21] .

[0015] The inventors have demonstrated that TGFβI induces a dose-dependent increase in fibroblast activity markers, namely Pro-C3, Pro-C1, Pro-C6, and P3NP. Furthermore, administration of the EMI domain of periostin also induces an increase in Pro-C3 levels.

[0016] Therefore, in a first embodiment, the present invention provides a method for identifying patients who are relatively likely to respond to anti-TGFβI therapy, comprising the following steps, using an immunoassay for detecting at least one marker of fibroblast activity in a patient sample: The patient's sample is brought into contact with a monoclonal antibody that specifically reacts to an epitope of a marker for fibroblast activity; To determine the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals known to respond to anti-TGFβI therapy and / or a predetermined cutoff value. The marker for fibroblast activity may be selected from PRO-C3 (PIIINP), PRO-C6, PRO-C1, and / or P3NP. A high binding level indicates an increase in the level of fibroblast activity markers. An increase in the level of fibroblast activity markers indicates that the patient is relatively likely to respond to anti-TGFβI therapy, and therefore is considered suitable for anti-TGFβI therapy.

[0017] The present invention provides a method for identifying patients who are relatively likely to respond to anti-TGFβI therapy, comprising the following steps, using an immunoassay to detect Pro-C3 in patient samples: The patient's sample is brought into contact with a monoclonal antibody that specifically reacts to and binds to the PIIINP epitope; To determine the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals known to respond to anti-TGFβI therapy and / or a predetermined cutoff value. A high binding level indicates an increase in Pro-C3 levels. An increase in Pro-C3 levels indicates that the patient is relatively likely to respond to anti-TGFβI therapy, and therefore is considered suitable for anti-TGFβI therapy.

[0018] In a preferred embodiment, the epitope of PIIINP to which the monoclonal antibody specifically reacts and specifically binds is the C-terminal neo-epitope of PIIINP generated by N-protease cleavage of intact type III procollagen. Preferably, the C-terminal neo-epitope of said PIIINP is contained in the C-terminal amino acid sequence CPTGXQNYSP-COOH (where X is Gly or Pro) (SEQ ID NO: 1). Most preferably, the C-terminal neo-epitope of said PIIINP is contained in the C-terminal amino acid sequence CPTGPQNYSP-COOH (SEQ ID NO: 4).

[0019] Preferably, the monoclonal antibody does not specifically recognize or bind to CPTGXQNYSPQ-COOH (where X is Gly or Pro) (SEQ ID NO: 5), which is an extension of the C-terminal amino acid sequence. Preferably, the ratio of the affinity of the antibody for the amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 1) to the affinity of the antibody for the extended amino acid sequence CPTGXQNYSPQ-COOH (SEQ ID NO: 5) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, still more preferably at least 10,000 to 1, still more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1. Preferably, the monoclonal antibody does not specifically recognize or bind to CPTGXQNYS-COOH (SEQ ID NO: 6), which is a truncated form of the C-terminal amino acid sequence. Preferably, the ratio of the affinity of the monoclonal antibody for the amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 1) to the affinity of the monoclonal antibody for the truncated amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 6) is at least 10 to 1, preferably at least 100 to 1, more preferably at least 1,000 to 1, still more preferably at least 10,000 to 1, still more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1. Preferably, the monoclonal antibody is produced against a synthetic peptide comprising or consisting of the C-terminal amino acid sequence CPTGXQNYSP-COOH (where X is Gly or Pro.) (SEQ ID NO: 1). Most preferably, the synthetic peptide has the C-terminal amino acid sequence CPTGPQNYSP-COOH (SEQ ID NO: 4).

[0020] As used herein, the term "neo-epitope" refers to an epitope generated by cleavage of a polypeptide. "C-terminal neo-epitope" refers to a neo-epitope at the tip of the C-terminal of a cleaved polypeptide, i.e., a peptide sequence at the C-terminal end of the polypeptide, and should not be construed to mean its general direction. Similarly, "C-terminal amino acid sequence" refers to the C-terminal peptide sequence at the tip of a polypeptide, i.e., a peptide sequence at the C-terminal end of the polypeptide, and should not be construed to mean its general direction. As used herein, the terms "peptide" and "polypeptide" are used synonymously.

[0021] As used herein, the term "monoclonal antibody" refers to both a whole antibody and fragments thereof that retain the binding specificity of the whole antibody, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, a whole antibody typically has a "Y-shaped" structure in which two identical groups of polypeptide chains pair, and each of the paired elements is composed of one "light" chain and one "heavy" chain. The N-terminal regions of each of the light and heavy chains contain variable regions, and the C-terminal portions of each of the heavy and light chains constitute constant regions. The variable regions contain three complementarity-determining regions (CDRs), which are mainly responsible for antigen recognition. The constant regions enable the antibody to mobilize cells and molecules of the immune system. Antibody fragments that retain binding specificity include at least the CDRs and a sufficient portion of the remainder of the variable region to retain binding specificity.

[0022] In the method of the present invention, any monoclonal antibody containing any constant region known in the art can be used. The constant light chain of humans is classified as either a kappa light chain or a lambda light chain. The constant heavy chain is classified as mu, delta, gamma, alpha, or epsilon, defining IgM, IgD, IgG, IgA, and IgE as antibody isotypes, respectively. The IgG isotype has several subclasses in humans, including IgG1, IgG2, IgG3, and IgG4, and in mice, it has several subclasses including IgG1, IgG2a, IgG2b, IgG2c, and IgG3, but is not limited thereto. The monoclonal antibody may preferably belong to an IgG isotype containing any one of IgG1, IgG2, IgG3, or IgG4.

[0023] The CDR of an antibody can be determined using methods known in the art, such as those described by Kabat et al. The antibody can be generated from B cell clones as described in the examples. The antibody isotype can be determined by ELISA specific to human or mouse IgM, IgG, or IgA isotypes, or to human IgG1, IgG2, IgG3, or IgG4 subclasses, or to mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3. The amino acid sequence of the generated antibody can be determined using standard methods. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA can then be PCR-treated using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to sequences located in the constant region of a predetermined isotype. The light chain can be amplified using primers that bind to the 3' end of the kappa or lambda chain, together with primers that anneal to the V-kappa or V-lambda leader sequence. The full-length heavy and light chains can be generated, and their sequences can be determined.

[0024] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence CPTGXQNYSP-COOH (where X is Gly or Pro) (SEQ ID NO: 1) can be produced by any suitable method known in the art. For example, the monoclonal antibody may be produced in opposition to a synthetic peptide containing or consisting of the amino acid sequence CPTGPQNYSP-COOH (SEQ ID NO: 4), for example, by the following method: Immunize rodents (or other suitable mammals) with a synthetic peptide consisting of the sequence CPTGPQNYSP-COOH (SEQ ID NO: 4), which may optionally be linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin); isolate and clone single antibody-producing cells; and assay the resulting monoclonal antibodies to determine that they have the desired specificity.

[0025] As used herein, “specifically binding” means that antibody binding is specific to the antigen and can be distinguished from unnecessary or nonspecific interactions. The ability of a monoclonal antibody to bind to a specific epitope or epitope sequence can be measured by either the enzyme-linked immunosorbent assay (ELISA) described herein, or by other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analyzed with BIAcore instruments) and conventional binding assays. The degree of binding of a monoclonal antibody to an unrelated protein is less than approximately 10% of the binding of a monoclonal antibody to an epitope or peptide, as measured, for example, by ELISA. “Affinity” refers to the total strength of all non-covalent interactions between a single binding site of a molecule (e.g., the epitope-binding region of an antibody) and its binding partner (e.g., an epitope or antigen). Unless otherwise indicated, “binding affinity” as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between the components of a binding pair (e.g., the antigen-binding portion and the antigen). The affinity of a molecule to its binding partner is generally expressed by the constants of the dissociation rate and the association rate (K, respectively). off , K onIt can be represented by the dissociation constant (Kd), which is the ratio of ( ). Therefore, equivalent affinity may consist of different rate constants as long as the ratio of the rate constants is the same. The dissociation constant represents the concentration of antigen in a state where it occupies half of the binding sites on the antibody. A lower Kd indicates a higher binding affinity between the antibody and the antigen, while a higher Kd indicates weaker binding. To measure the Kd of an antibody, several methods can be utilized, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and assays based on fluorescence emission principles. In certain embodiments, the dissociation constant (KD) of a monoclonal antibody that binds to an epitope or antigen is <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 8 M or less, e.g., 10 8 M to 10 13 M, e.g., 10 9 M to 10 13 M).

[0026] Monoclonal antibodies that specifically bind to PRO-C3 may preferably comprise one or more complementarity-determining regions (CDRs) selected from the following. CDR-H1: NYVIH (SEQ ID NO: 7) CDR-H2: YMNPYNDVPKNNAKFRG (SEQ ID NO: 8) CDR-H3: GGFFGPLSY (SEQ ID NO: 9) CDR-L1: RSSQNIVYSNGDTYFE (SEQ ID NO: 10) CDR-L2: KVSQRFS (SEQ ID NO: 11) CDR-L3: FQGAHDPPA (SEQ ID NO: 12) Preferably, the monoclonal antibody may comprise all at least 2, 3, 4, 5, or 6 of the above CDR sequences.

[0027] Preferably, the monoclonal antibody has a light chain variable region comprising the following CDR sequence. CDR-L1: RSSQNIVYSNGDTYFE (SEQ ID NO: 10) CDR-L2: KVSQRFS (Sequence ID 11), and, CDR-L3: FQGAHDPPA (Sequence ID 12)

[0028] Preferably, the monoclonal antibody may have a light chain containing a framework sequence between CDRs, the framework sequence being substantially identical or substantially similar to the framework sequence between CDRs in the light chain sequence below (CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0029] [ka]

[0030] Preferably, the monoclonal antibody has a heavy chain variable region containing the following CDR sequence. CDR-H1: NYVIH (Sequence ID 7) CDR-H2: YMNPYNDVPKNNAKFRG (Sequence ID 8), and, CDR-H3: GGFFGPLSY (Sequence No. 9)

[0031] Preferably, the monoclonal antibody may have a heavy chain containing a framework sequence between CDRs, the framework sequence being substantially identical or substantially similar to the framework sequence between CDRs of the heavy chain sequence described below (CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0032] [ka]

[0033] In this specification, the amino acid sequences of the framework present between the CDRs of an antibody are substantially identical or substantially similar to the amino acid sequences of the framework present between the CDRs of another antibody if they have at least 70%, 80%, 90%, or at least 95% similarity or identity with the amino acid sequences of the framework present between the CDRs of another antibody. Similar or identical amino acids may be consecutive or discontinuous.

[0034] A framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions may be conservative substitutions, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties in terms of size, charge, polarity, etc.: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe, Thy, Trp.

[0035] Programs like the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting spaces within either sequence. For optimal alignment, it is possible to calculate amino acid identity or similarity (in addition to identity, conservation of amino acid types). Programs like BLASTx align the longest segments of similar sequences and assign values ​​to the matching sites. In this way, a comparison can be obtained, and several similar regions with different scores can be discovered. In this invention, it is conceivable to use these two types of analysis. Identity or similarity is preferably calculated over the entire length of the framework sequence.

[0036] In certain preferred embodiments, the monoclonal antibody that specifically binds to PRO-C3 is a light chain variable region sequence:

[0037] [ka]

[0038] and / or heavy chain variable region sequence:

[0039] [ka]

[0040] (CDR is in bold and underlined; framework array is in italics) It may include.

[0041] Suitable immunoassay kits for measuring PRO-C3 levels are commercially available, such as those from Nordic BioScience (cat# 1700AF06).

[0042] The present invention provides a method for identifying patients who are relatively likely to respond to anti-TGFβI therapy, comprising the following steps, using an immunoassay to detect Pro-C6 in patient samples: The patient's sample is brought into contact with a monoclonal antibody that specifically reacts to the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen; To determine the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals known to respond to anti-TGFβI therapy and / or a predetermined cutoff value. A high binding level indicates an increase in Pro-C6 levels. An increase in Pro-C6 levels indicates that the patient is relatively likely to respond to anti-TGFβI therapy, and therefore is considered suitable for anti-TGFβI therapy.

[0043] Preferably, a monoclonal antibody that specifically reacts to and specifically binds to the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen specifically binds to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO: 17) (which may also be referred to herein as the "Pro-C6 sequence" or simply "Pro-C6"). Preferably, the monoclonal antibody does not specifically bind to KPGVISVMGTA (SEQ ID NO: 18), which is an elongated version of the C-terminal amino acid sequence, or to KPGVISVMG (SEQ ID NO: 19), which is a truncated version of the C-terminal amino acid sequence.

[0044] Preferably, the ratio of the affinity of the antibody to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO: 17) to the affinity of the antibody to the extended C-terminal amino acid sequence KPGVISVMGTA (SEQ ID NO: 18) and / or the truncated C-terminal amino acid sequence KPGVISVMG (SEQ ID NO: 19) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0045] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO: 17) can be produced by any suitable method known in the art. For example, the monoclonal antibody may be produced in opposition to a synthetic peptide containing or consisting of the amino acid sequence KPGVISVMGT (SEQ ID NO: 17), for example, by the following method: Immunize rodents (or other suitable mammals) with a synthetic peptide consisting of the sequence KPGVISVMGT (SEQ ID NO: 17) and optionally linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin); isolate and clone single antibody-producing cells; and assay the resulting monoclonal antibodies to determine that they have the desired specificity.

[0046] A monoclonal antibody that specifically binds to the PRO-C6 sequence may preferably contain one or more complementarity-determining regions (CDRs) selected from the following: CDR-L1: RSSQRIVHSNGITFLE (Sequence ID 20) CDR-L2: RVSNRFS (Sequence ID 21) CDR-L3: FQGSHVPLT (Sequence ID 22) CDR-H1: DFNMN (Sequence ID 23) CDR-H2: AINPHNGATSYNQKFSG (Sequence ID 24) CDR-H3: WGNGKNS (Sequence ID 25) Preferably, the monoclonal antibody contains at least two, three, four, five, or six of the above-mentioned CDR sequences.

[0047] Preferably, the light chain variable region of the monoclonal antibody contains the following CDR sequence. CDR-L1: RSSQRIVHSNGITFLE (Sequence ID 20) CDR-L2: RVSNRFS (Sequence ID 21), and, CDR-L3: FQGSHVPLT (Sequence ID 22)

[0048] Preferably, the light chain of the monoclonal antibody contains a framework sequence between CDRs, which is substantially identical or substantially similar to the framework sequence between CDRs in the light chain sequence described below (CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0049] [ka]

[0050] Preferably, the heavy chain variable region of the monoclonal antibody contains the following CDR sequence. CDR-H1: DFNMN (Sequence ID 23) CDR-H2: AINPHNGATSYNQKFSG (Sequence ID 24), and, CDR-H3: WGNGKNS (Sequence ID 25)

[0051] Preferably, the heavy chain of the monoclonal antibody includes a framework sequence between CDRs, which is substantially identical or substantially similar to the framework sequence between CDRs of the heavy chain sequence described below (CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0052] [ka]

[0053] In certain preferred embodiments, the monoclonal antibody that specifically binds to the PRO-C6 sequence is the light chain variable region sequence:

[0054] [ka]

[0055] and / or heavy chain variable region sequence:

[0056] [ka]

[0057] (CDR is in bold and underlined; framework array is in italics) It may include.

[0058] Suitable immunoassay kits for measuring PRO-C6 levels are commercially available, such as those from Nordic BioScience (cat# 4000AF02).

[0059] The present invention provides a method for identifying patients who are relatively likely to respond to anti-TGFβI therapy, comprising the following steps, using an immunoassay to detect Pro-C1 in patient samples: The patient's sample is brought into contact with a monoclonal antibody that specifically reacts with the N-terminal epitope of the propeptide of type I collagen; To determine the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals known to respond to anti-TGFβI therapy and / or a predetermined cutoff value. A high binding level indicates an increase in Pro-C1 levels. An increase in Pro-C1 levels indicates that the patient is relatively likely to respond to anti-TGFβI therapy, and therefore is considered suitable for anti-TGFβI therapy.

[0060] Preferably, a monoclonal antibody that specifically reacts to and specifically binds to the N-terminal epitope of the propeptide of type I collagen specifically binds to the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID NO: 3) (which may also be referred to herein as the “Pro-C1 sequence” or simply “Pro-C1”). Preferably, the monoclonal antibody does not specifically bind to PDGSESPTDQETTGVE (SEQ ID NO: 30), which is an extension of the N-terminal amino acid sequence, or to DGSESPTDQETTGV (SEQ ID NO: 31), which is a truncated version of the N-terminal amino acid sequence.

[0061] Preferably, the ratio of the affinity of the antibody to the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID NO: 3) to the affinity of the antibody to the extended N-terminal amino acid sequence PDGSESPTDQETTGVE (SEQ ID NO: 30) and / or the truncated N-terminal amino acid sequence DGSESPTDQETTGV (SEQ ID NO: 31) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0062] As used herein, the term “N-terminal” refers to the N-terminal peptide sequence at the tip of a polypeptide, i.e., the peptide sequence at the N-end of the polypeptide, and should not be interpreted as referring to its general orientation.

[0063] Monoclonal antibodies that specifically bind to the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID NO: 3) can be produced by any suitable method known in the art. For example, the monoclonal antibody may be produced in opposition to a synthetic peptide containing or consisting of the amino acid sequence PDGSESPTDQETTGV (SEQ ID NO: 3), for example, by the following method: Immunize rodents (or other suitable mammals) with a synthetic peptide consisting of the sequence PDGSESPTDQETTGV (SEQ ID NO: 3), which may optionally be linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin); isolate and clone single antibody-producing cells; and assay the resulting monoclonal antibodies to determine that they have the desired specificity.

[0064] Suitable immunoassay kits for measuring PRO-C1 levels are commercially available, such as those from Nordic BioScience (cat# 2800A0E51).

[0065] The present invention provides a method for identifying patients who are relatively likely to respond to anti-TGFβI therapy, comprising the following steps, using an immunoassay to detect P3NP in patient samples: The patient's sample is brought into contact with a monoclonal antibody that specifically reacts with the internal sequence of the N-terminal region of type 3 collagen; To determine the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals known to respond to anti-TGFβI therapy and / or a predetermined cutoff value. A high binding level indicates elevated P3NP levels. Elevated P3NP levels suggest that the patient is relatively likely to respond to anti-TGFβI therapy and is therefore considered suitable for anti-TGFβI therapy.

[0066] Preferably, a monoclonal antibody that specifically reacts to and specifically binds to the internal sequence of the N-terminal region of type 3 collagen specifically binds to the amino acid sequence PGIPGRNGDP (SEQ ID NO: 2) (which may also be referred to herein as the "P3NP sequence" or simply "P3NP"). Preferably, the monoclonal antibody does not specifically bind to the extended amino acid sequence PPGIPGRNGDP (SEQ ID NO: 32) or PGIPGRNGDPG (SEQ ID NO: 33), or to the truncated amino acid sequence GIPGRNGDP (SEQ ID NO: 34) or PGIPGRNGD (SEQ ID NO: 35).

[0067] Preferably, the ratio of the affinity of the antibody to PGIPGRNGDP (SEQ ID NO: 2) to the affinity of the antibody to the extended amino acid sequence PPGIPGRNGDP (SEQ ID NO: 32) or PGIPGRNGDPG (SEQ ID NO: 33), and / or the truncated amino acid sequence GIPGRNGDP (SEQ ID NO: 34) or PGIPGRNGD (SEQ ID NO: 35) is at least 10:1, more preferably at least 50:1, at least 100:1, at least 500:1, at least 1,000:1, at least 10,000:1, at least 100,000:1, or at least 1,000,000:1.

[0068] Monoclonal antibodies that specifically bind to the amino acid sequence PGIPGRNGDP (SEQ ID NO: 2) can be produced by any suitable method known in the art. For example, the monoclonal antibody may be produced in opposition to a synthetic peptide containing or consisting of the amino acid sequence PGIPGRNGDP (SEQ ID NO: 2), for example, by the following method: Immunize rodents (or other suitable mammals) with a synthetic peptide consisting of the sequence PGIPGRNGDP (SEQ ID NO: 2), which may optionally be linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin); isolate and clone single antibody-producing cells; and assay the resulting monoclonal antibodies to determine that they have the desired specificity.

[0069] Suitable immunoassay kits for measuring P3NP levels are commercially available, such as those from Nordic BioScience (cat# 1090BD01).

[0070] The above method may measure one, two, three, or four markers of fibroblast activity. The above method may measure the levels of Pro-C3 and optionally one, two, or three other markers of fibroblast activity. The above method may measure the levels of Pro-C3 and Pro-C6, Pro-C3 and Pro-C1, or Pro-C3 and P3NP. The above method may measure the levels of Pro-C3, Pro-C6, and Pro-C1, or Pro-C3, Pro-C6, and P3NP. The above method may measure the levels of Pro-C6 and optionally one, two, or three other markers of fibroblast activity. The above method may measure the levels of Pro-C6 and Pro-C1, or Pro-C6 and P3NP. The above method may measure the levels of Pro-C6, Pro-C1, and P3NP. The above method may measure the levels of Pro-C1 and optionally one, two, or three other markers of fibroblast activity. The above method may also measure the levels of Pro-C1 and P3NP. The above method may also measure the levels of P3NP and optionally one, two, or three other markers of fibroblast activity. The above method may also be used to measure the levels of Pro-C3, Pro-C6, Pro-C1, and P3NP.

[0071] In a preferred embodiment, the patient's sample is a biological fluid. The biological fluid may be, but is not particularly limited to, serum, plasma, urine, amniotic fluid, tissue supernatant, or cell supernatant. Preferably, the biological fluid is blood, serum, or plasma.

[0072] The above immunoassay may be a competitive assay or a sandwich assay, but is not particularly limited. The above immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are known to those skilled in the art. In a preferred embodiment, the immunoassay is a competitive immunoassay. In a preferred embodiment, the competitive immunoassay is a radioimmunoassay, a fluorescent immunoassay, or an enzyme-linked immunosorbent assay (ELISA). The competitive immunoassay includes a competitive immunoassay performed as a radioimmunoassay, a fluorescent immunoassay, or an ELISA, which are methods and techniques well known to those skilled in the art.

[0073] In a preferred embodiment, the immunoassay is a sandwich immunoassay. In a preferred embodiment, the sandwich immunoassay is a radioimmunoassay, a fluorescence immunoassay, or an enzyme-linked immunosorbent assay (ELISA). The sandwich immunoassay includes sandwich immunoassays performed as radioimmunoassays, fluorescence immunoassays, or ELISAs, which are methods and techniques well known to those skilled in the art. As is well known, a sandwich immunoassay uses two or more antibodies to detect an antigen in a sample, including at least one antibody that acts as a capture antibody and is typically immobilized on a solid support, and at least one antibody that acts as a detection antibody. In the present invention, a monoclonal antibody may function as either a capture antibody or a detection antibody. As described above, the monoclonal antibody may be immobilized on a solid support. Any suitable form of solid support and conjugation method known in the art can be used. For example, in certain exemplary embodiments, the monoclonal antibody may be biotinylated and conjugated to a streptavidin-coated solid support.

[0074] In another embodiment, the monoclonal antibody may be labeled, which allows for the detection of the monoclonal antibody and the determination of the amount of binding between the monoclonal antibody and a marker of fibroblast activity, such as PRO-C3, PRO-C6, PRO-C1, or P3NP, which is bound to a first monoclonal antibody present on a solid support. For example, in one preferred embodiment, the monoclonal antibody may be an enzyme-conjugated antibody. The enzyme may be, but is not limited to, horseradish peroxidase (HRP). In yet another embodiment, the monoclonal antibody may be radiolabeled or conjugated to a fluorescent dye molecule. These are preferred labels used in the present invention, but any suitable labeling method, such as a DNA reporter or electrochemiluminescence labeling, can be applied, and the invention is not particularly limited.

[0075] Alternatively, a second monoclonal antibody that is labeled and recognizes the monoclonal antibody may be used, thereby enabling the detection of the monoclonal antibody and the determination of the amount of binding between the second monoclonal antibody and the fibroblast activity marker bound to the first monoclonal antibody present on the solid support. A second monoclonal antibody can be labeled using the labeling method described above.

[0076] As used herein, the term “binding amount” refers to the quantification of binding between a monoclonal sample and a fibroblast activity marker such as PRO-C3(PIIINP), PRO-C6, PRO-C1, or P3NP derived from the sample. This quantification may be determined, for example, by comparing the measured binding amount of the fibroblast activity marker derived from the patient sample with a calibration curve created using the measured binding amount of the fibroblast activity marker derived from a standard sample containing the supernatant obtained from a “scar-in-a-jar” (SiaJ) model in which healthy primary human lung fibroblasts are cultured in the presence of Ficol and transforming growth factor-beta (TGF-β). The fibroblast activity marker may be selected from PRO-C3(PIIINP), PRO-C6, PRO-C1, and / or P3NP. In the examples described below, the ELISA method is used, where spectrophotometric analysis is employed to measure both the amount of binding derived from the patient's sample and the amount of binding when a calibration curve is created.

[0077] As used herein, the term “predetermined cutoff value” means a statistically determined binding amount that indicates a high likelihood of a patient responding to anti-TGFβI therapy, where a measured value of the target peptide in a patient sample being greater than or equal to the statistical cutoff value corresponds to a probability of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% that the recipient will be favorably treated with anti-TGFβI therapy.

[0078] As used herein, “values ​​associated with normal healthy individuals” means the standardized binding amount determined by the method described above for samples obtained from patients who are considered to be healthy, i.e., disease-free, e.g., cancer or specific cancers of concern; and “values ​​associated with known responders to anti-TGFβI therapy” means the standardized binding amount determined by the method described above for samples obtained from patients who are known to have clearly responded to anti-TGFβI therapy, i.e., significantly improved after treatment with anti-TGFβI therapy. “Significant improvement” may be a reduction or disappearance of symptoms, a reduction in cancer size, an improvement in prognosis, or a change in disease severity indicator biomarkers indicating a reduction in disease severity.

[0079] Therefore, in these embodiments, the method may further include administering anti-TGFβI therapy to patients identified as likely to respond to anti-TGFβI therapy. Suitable therapy includes the administration of a neutralizing antibody targeting TGFβI. Patients seeking treatment are preferably those suffering from cancer and / or fibrosis. The cancer may be bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer. The cancer may be stage I, stage II, stage III, or stage IV. The tumor may be a primary tumor or a metastatic tumor.

[0080] In a second aspect, the present invention provides a drug that binds to the EMI domain of TGFβI or periostin or its analogues. As used herein, “EMI domain” refers to the EMI domain of periostin, TGFβI, or their analogues. In particular, “EMI domain” refers to the 56-amino acid EMI domain derived from periostin (amino acid groups 40-94 of the protein sequence) or the 56-amino acid EMI domain derived from TGFβI (amino acid groups 45-99 of the protein sequence). Preferably, the EMI domain derived from periostin includes or essentially consists of the following sequence:

[0081] [ka]

[0082] Preferably, the EMI-domain derived from TGFβI contains or essentially consists of the following sequence:

[0083] [ka]

[0084] Analogues of the EMI domain for use in the present invention may include proteins containing sequences similar to the amino acid sequence defined by SEQ ID NO: 36 or SEQ ID NO: 37, and they have the same biological effect, namely increasing markers of fibroblast activity levels, for example, increasing the levels of PRO-C3, PRO-C6, PRO-C1, and / or P3NP. For example, analogues of the EMI domain may have at least 70%, 80%, 90%, or at least 95% similarity to the sequence defined by SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment of the sequence defined by SEQ ID NO: 36 or SEQ ID NO: 37. Alternatively, analogues of the EMI domain may have at least 70%, 80%, 90%, or at least 95% identity to the sequence defined by SEQ ID NO: 36 or SEQ ID NO: 37, or a fragment of the sequence defined by SEQ ID NO: 36 or SEQ ID NO: 37. For example, analogues of the EMI domain may include amino acid sequences in which 50, 51, 52, 53, 54, 55, 56, or 57 amino acids are identical to those defined by Sequence ID No. 36 or Sequence ID No. 37. Similar or identical amino acids may be consecutive or discontinuous.

[0085] Programs like the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting spaces within either sequence. For optimal alignment, it is possible to calculate amino acid identity or similarity (in addition to identity, conservation of amino acid types). Programs like BLASTx align the longest segments of similar sequences and assign values ​​to matching sites. In this way, a comparison can be obtained, and several similar regions with different scores can be discovered. In this invention, it is conceivable to use these two types of analysis. Identity or similarity is preferably calculated over the entire length of SEQ ID NO: 36 or SEQ ID NO: 37.

[0086] Analogues of the EMI domain may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitution means that an amino acid residue is replaced with a substitute amino acid residue at the same position. Amino acid substitution may be a conservative substitution, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties in terms of size, charge, polarity, etc.: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe, Thy, Trp.

[0087] The inserted amino acid residues may be inserted at any position, and some or all of the inserted amino acid residues may be inserted so as to be directly adjacent to each other, or none of the inserted amino acid residues may be inserted so as to be directly adjacent to any other inserted amino acid residues. For example, analogues of the EMI domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 added amino acids at the N- and / or C-terminals of the amino acid sequence defined by SEQ ID NO: 36 or SEQ ID NO: 37.

[0088] One, two, or three amino acids may be deleted from the sequence of SEQ ID NO: 36 or SEQ ID NO: 37. Each deletion may occur at either the position of SEQ ID NO: 36 or SEQ ID NO: 37.

[0089] The inserted and substituted amino acids may be natural or unnatural amino acids, and may, for example, contain unnatural side chains and / or be linked via unnatural peptide bonds. Such modified peptide ligands are known in the art. If more than one amino acid residue is substituted and / or inserted, the substituted / inserted amino acid residues may be the same as or different from each other. Each substituted amino acid may have a different side chain with respect to the amino acid being substituted.

[0090] Analogues of the EMI domain may contain one or more modifying groups, where amino acid residues may be chemically modified. Examples of chemical modifications include those corresponding to post-translational modifications, such as phosphorylation, acetylation, and deamidation. Chemical modifications do not necessarily correspond to those that would likely exist in vivo. For example, the N- or C-terminal region of an EMI domain peptide may be modified to improve the peptide's stability, bioavailability, or affinity. Furthermore, examples of unnatural modifications include the incorporation of unencoded α-amino acids, photo-crosslinkable amino acids, N-methylated and β-amino acids, reduction of the backbone, retroinversion using d-amino acids, N-terminal methylation, and C-terminal amidation, and PEGylation.

[0091] Preferably, the drug is an antibody that specifically binds to the EMI domain or its analogues. Preferably, the antibody specifically binds to the active site of the EMI domain.

[0092] The present invention relates to a method for producing a monoclonal antibody that specifically binds to the amino acid sequence of an EMI domain, and includes a method for producing a monoclonal antibody in opposition to a synthetic peptide containing or consisting of the amino acid sequence of an EMI domain.

[0093] The synthetic peptide may contain 5-15 consecutive amino acids of the EMI domain. The synthetic peptide may contain, or consist of, a sequence of amino acids shared by the EMI domains of periostin and TGFβI. The synthetic peptide may contain, or consist of, a sequence of amino acids that forms a portion derived from a region within the EMI domain of periostin or TGFβI that has a high percentage of homology between the two sequences, i.e., a region with high similarity or identity between the amino acid sequences of both the periostin and TGFβI EMI domains. Preferably, the synthetic peptide has at least 70%, 80%, 90%, or at least 95% similarity or identity with respect to the EMI domains of periostin and / or TGFβI. Similar or identical amino acids may be consecutive or discontinuous. Preferably, the synthetic peptide has 100% similarity or identity with respect to the EMI domains of periostin and / or TGFβI.

[0094] Preferably, the synthetic peptide includes or consists of an active site of the EMI domain. Preferably, the synthetic peptide comprises GPNVCAX1Q (SEQ ID NO: 38) (where X1 is V or L), YECCPGY (SEQ ID NO: 39), or GX2KGCPA (SEQ ID NO: 40) (where X2 is E or M).

[0095] The above method, for example, involves immunizing a non-human mammal with a synthetic peptide containing or consisting of the amino acid sequence of the EMI domain, isolating splenocytes from the immunized mammal that produces antibodies that specifically bind to the amino acid sequence of the EMI domain, fusing the isolated splenocytes with hybridoma cells, and then culturing the resulting hybridoma cells to ensure monoclonal proliferation. The synthetic peptide is preferably linked to the immunogenic carrier protein (via any suitable type of linking group).

[0096] The present invention also relates to a method for screening monoclonal antibodies that specifically bind to the amino acid sequence of an EMI domain, comprising contacting a sample containing one or more antibodies with a synthetic peptide containing or consisting of an EMI domain, and detecting the binding between the antibody and the peptide. In a preferred embodiment, the synthetic peptide is a peptide comprising or consisting of an amino acid sequence of an EMI domain, and the peptide is linked (via any suitable type of linking group) to a solid support and / or to a molecule (such as biotin) that enables attachment to the solid support. In a particularly preferred embodiment, the synthetic peptide is a peptide consisting of an amino acid sequence of an EMI domain, and the peptide is linked (via any suitable type of linking group) to a solid support and / or to a molecule (such as biotin) that enables attachment to the solid support. In a preferred embodiment, binding between the antibody and the peptide is detected by enzyme-linked immunosorbent assay (ELISA).

[0097] In a preferred embodiment, hybridoma cells that produce antibodies that specifically bind to the amino acid sequence of the EMI domain are produced by immunizing a non-human mammal with a synthetic peptide containing or consisting of the amino acid sequence of the EMI domain, isolating splenocytes from the immunized mammal that produces antibodies that specifically bind to the amino acid sequence of the EMI domain, fusing the isolated splenocytes with hybridoma cells, and then culturing the resulting hybridoma cells to ensure monoclonal proliferation.

[0098] The present invention relates to a monoclonal antibody that specifically binds to the amino acid sequence of the EMI domain, preferably the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, the monoclonal antibody is a monoclonal antibody produced in opposition to a synthetic peptide containing or consisting of the amino acid sequence of the EMI domain.

[0099] The present invention relates to the amino acid sequence GPNVCAX1Q (SEQ ID NO: 38) (where X1 is V or L), or YECCPGY (SEQ ID NO: 39), or GX2KGCPA (SEQ ID NO: 40) (where X2 is E or M). A method for producing a monoclonal antibody that specifically binds to a particular substance, wherein the monoclonal antibody is produced by counteracting a synthetic peptide containing or consisting of the amino acid sequence GPNVCAX1Q (SEQ ID NO: 38), YECCPGY (SEQ ID NO: 39), or GX2KGCPA (SEQ ID NO: 40).

[0100] The above method may include, for example, immunizing a non-human mammal with a synthetic peptide containing or consisting of the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40), isolating splenocytes from the immunized mammal that produces antibodies that specifically bind to the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40), fusing the isolated splenocytes with hybridoma cells, and culturing the resulting hybridoma cells to ensure monoclonal proliferation. The synthetic peptide is a synthetic peptide comprising or consisting of the amino acid sequence GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40), and it is preferable that the peptide be linked to an immunogenic carrier protein (via any suitable linking group).

[0101] The present invention also relates to a method for screening antibodies that specifically bind to the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 4) and / or GX2KGCPA (SEQ ID NO: 40), comprising contacting a sample containing one or more antibodies with a synthetic peptide according to the first embodiment, and detecting the binding between the antibody and the peptide. In a preferred embodiment, the synthetic peptide is a peptide comprising or consisting of the amino acid sequence GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40), and the peptide is linked (via any suitable type of linking group) to a solid support and / or to a molecule (such as biotin) that enables attachment to the solid support. In a particularly preferred embodiment, the synthetic peptide is a peptide consisting of the amino acid sequence GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40), and the peptide is linked (via any suitable type of linking group) to a solid support and / or to a molecule (such as biotin) that enables attachment to the solid support. In a preferred embodiment, binding between the antibody and the peptide is detected by enzyme-linked immunosorbent assay (ELISA).

[0102] In a preferred embodiment, hybridoma cells that produce antibodies specifically binding to the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40) are produced by immunizing a non-human mammal with a synthetic peptide containing or consisting of the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40), isolating spleen cells from the immunized mammal that produces antibodies specifically binding to the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40), fusing the isolated spleen cells with hybridoma cells, and then culturing the resulting hybridoma cells to ensure monoclonal proliferation.

[0103] The present invention relates to a monoclonal antibody that specifically binds to the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40). Preferably, the monoclonal antibody is a monoclonal antibody produced in opposition to a synthetic peptide containing or consisting of the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40).

[0104] Monoclonal antibodies may be produced in opposition to synthetic peptides containing or consisting of EMI domain active sites. Monoclonal antibodies can be produced by suitable methods known to those skilled in the art, such as immunizing mice or other non-human mammals, isolating splenocytes (B cells) from the immunized mammals and fusing them with hybridoma cells, and then culturing the resulting hybridoma cells to ensure monoclonal proliferation, but are not particularly limited.

[0105] For example, monoclonal antibodies can be produced from B cell clones, as described in the examples. Preferably, the monoclonal antibody is a humanized antibody containing the human framework and / or constant region sequence. Such antibodies may be produced, for example, from genetically transformed mice (or other non-human mammals) into which human immunoglobulin genes have been introduced. Alternatively, they may be produced by generating chimeric antibodies, in which case a monoclonal antibody from a mouse (or other non-human mammal) is first produced, the antigen-binding sequence (including at least CDRs) of the antibody is determined, and then a recombinant antibody containing an antibody-binding sequence appended to the domain sequence of the human framework and / or constant region is produced.

[0106] Furthermore, this specification also provides a method for screening antibodies that specifically bind to EMI domain active sites. A method for screening antibodies that specifically bind to EMI domain active sites may include contacting a sample containing one or more antibodies with a synthetic peptide containing or consisting of EMI domain active sites, and then detecting the binding between the antibody and the peptide. The binding may be detected by enzyme-linked immunosorbent assay (ELISA). Various suitable types of ELISAs are known in the art and can be suitably used. For example, the ELISA method described in the examples may be used, in which a liquid sample containing one or more antibodies is brought into contact with a solid support on which a peptide containing or consisting of an EMI domain active site is immobilized, thereby inducing binding between the peptide immobilized on the support and the antibody specific to the EMI domain active site. After removing unbound antibodies, an enzyme-conjugated antibody can be added that binds to antibodies immobilized on a solid support via a peptide containing or consisting of an EMI domain active site. The enzyme activity can then be evaluated by incubation with a substrate that generates a measurable product, thereby enabling quantitative detection of antibodies bound to peptides containing or consisting of an EMI domain active site that are bound to the solid support.

[0107] The monoclonal antibodies described above, which target the EMI domain 2 active site, can suppress the fibrillation-inducing effect of TGFβI by binding to the EMI active site and blocking its access (thus eliminating TGFβI-induced fibrosis). In addition to this direct neutralizing effect, such monoclonal antibodies also possess an Fc region, and can exert a function of eliminating fibrosis through Fc-mediated purification of EMI / TGFβI to which the antibody is bound. Therefore, these antibodies can be used in the treatment of fibrotic diseases and general pathological fibrosis. However, monoclonal antibodies that target other parts of EMI can also be used to eliminate the action of TGFβI. In particular, such antibodies can be used to purify TGFβI via Fc, even if they do not block access to EMI active sites.

[0108] In a third aspect, the present invention provides a second-type agent for use in a method of treating cancer and / or fibrosis. Therefore, the present invention provides an agent that binds to the EMI domain of TGFβI or an analog thereof for use in the treatment of cancer. In a preferred embodiment, the present invention relates to a monoclonal antibody that specifically binds to the EMI domain or an analog thereof for use in the treatment of cancer. Cancer may be bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer. Cancer may be stage I, stage II, stage III, or stage IV. Tumor may be a primary tumor or a metastatic tumor. Preferably, the drug is used to treat patients identified as having a relatively high likelihood of responding to anti-TGFbI therapy. In a more preferred embodiment, the patient is identified in the manner of the first embodiment and has elevated levels of fibroblast activity markers described herein. The fibroblast activity markers may be selected from PRO-C3, PRO-C6, PRO-C1, and / or P3NP. In yet another embodiment, the present invention provides a drug for use as a pharmaceutical that binds to the EMI domain of TGFβI or an analog thereof. In a preferred embodiment, the present invention relates to a monoclonal antibody for use as a pharmaceutical that specifically binds to the EMI domain or an analog thereof. [Brief explanation of the drawing]

[0109] figure [Figure 1]Figure 1: (a) Multiple sequence alignments of the EMI domain. Periostin (POSTN_40-94) and TGFBI (TGFBI_40-99) are highlighted in the box. (b) Amino acid sequences of the EMI domain obtained from the following sources: TGFBI (https: / / www.uniprot.org / uniprot / Q15582 amino acid groups 40-94) and periostin (https: / / www.uniprot.org / uniprot / Q15063 amino acid groups 40-99).

[0110] [Figure 2] Figure 2: Manhattan plots of genome-wide association studies using log2(PRO-C3) levels and eight other liver-related biomarkers and scores. Linear additive regression models, adjusted and optimized for baseline age, three leading principal components, serum and blood biochemical parameters identified by elastic nets, and PRO-C3 measurement batches, were performed on 4968 PERF subjects. Linear additive regression models, adjusted for baseline age and the three leading principal components, were also performed for ADAPT, FIB4, APRI, and NFS disease scores, and similarly for log2-conversion levels of the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), and gamma-glutamyl transpeptidase (GGT).

[0111] [Figure 3] Figure 3 shows that biomarkers PRO-C1 (type I collagen propeptide), PRO-C3 (type III collagen propeptide), and PRO-C6 (type VI collagen propeptide) were measured in the CAF supernatant to reflect fibrotic activity and collagen synthesis. The magnification changes between day 3 and day 9 were compared for different doses of TGFBI treatment. [Figure 4]Figure 4 shows that PRO-C3 (type III collagen propeptide) levels were measured in pancreatic fibroblast supernatant to reflect fibrotic activity and collagen synthesis. Biomarker levels were measured on day 12 for different doses of TGF-β, TGFBI, and EMI domains.

[0112] [Figure 5A] Figure 5 shows the effect of increasing BigH3 (TGFBI) levels during the production of markers of fibroblast activity in pancreatic fibroblasts. Figure 5A shows PRO-C1 levels; [Figure 5B] Figure 5B shows the PRO-C3 level; [Figure 5C] Figure 5C shows the PRO-C6 level; [Figure 5D] Figure 5D shows the P3NP level; [Examples]

[0113] Examples The embodiments disclosed herein are described in the following examples. These examples are provided to aid in understanding the disclosure and should not be construed in any way as limiting the scope of the disclosure specified in the claims below. The examples below are provided to a person skilled in the art to provide a complete disclosure and description of how to make and use the described embodiments and are not intended to limit the scope of the disclosure, nor are they intended to mean that the experiments described below are all or only experiments performed. Efforts have been made to ensure accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0114] Materials and methods reagent Unless otherwise specified, all reagents used in the experiments were high-quality chemicals manufactured by Merck (Whitehouse Station, NJ, USA) and Sigma (St. Louis, MO, USA). The synthetic immunogenic protein ovalbumin (OVA)-CGG-CPTGPQNYSP, used in the production of the monoclonal antibody, was purchased from Chinese Peptide Company (Beijing, China).

[0115] Generation of Pro-C3 monoclonal antibodies The production of the Pro-C3 monoclonal antibody was carried out as previously reported

[23] (see also WO2014 / 170312A1). Briefly, the Pro-C3 neo-epitope (the C-terminal neo-epitope of PIIINP generated by N-proteinase) was selected as the target, and the amino acid sequence 144'-CPTGPQNYSP-'153 (SEQ ID NO: 4) in the α1 chain of PIIINP was used to produce an antibody specific to this target. The sequence was aligned for homology to rat and mouse using NPS@: Network Protein Sequencing Analysis with the Uniprot / Swiss-Prot database

[24] , and then blast-aligned for uniqueness to other human proteins. The sequence CPTGPQNYSP (SEQ ID NO: 4) was found to be unique to PIIINP. The generation of monoclonal antibodies, characterization of clones, and characterization of the antibodies were carried out as previously described

[22] . The monoclonal antibodies were found to be specific to the target sequence CPTGPQNYSP (SEQ ID NO: 4) and did not recognize or bind to the extended peptide (CPTGPQNYSPQ (SEQ ID NO: 5)) or the nonsense peptide (GSPGKDGVRG (SEQ ID NO: 41))

[22] .

[0116] The supernatant was collected from the antibody-producing hybridoma cells, and the monoclonal antibodies were purified using a HiTrap protein-G column (GE Healthcare Life Sciences, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions. The antibodies were then labeled using either the Lightning-Link® High-Speed ​​Biotin Binding Kit (Type B) (Innova BioScience) or the Lightning-Link® HRP Binding Kit (Innova BioScience).

[0117] The sequence of the generated antibody was determined, and the CDR (Cellular Discrimination Rate) was then determined. The chain arrangement is as follows (CDR is underlined and bold, constant region is italicized) Heavy chain: amino acid sequence (467aa)

[0118] [ka]

[0119] Light chain: Amino acid sequence (238aa)

[0120] [ka]

[0121] Pro-C3 assay The Pro-C3 assay (Pro-C3 competitive ELISA) was performed as previously reported

[23] (see also WO2014 / 170312A1 incorporated herein by reference). Briefly, the procedure for the Pro-C3 competitive ELISA was as follows: A 96-well streptavidin-coated ELISA plate (cat. 11940279) obtained from Roche was coated with a lysis of the biotinylated peptide Biotin-CGGPTGPQNYSP (SEQ ID NO: 4) dissolved in coater buffer (50 mM PBS-BTE + 10% sorbitol, pH 7.4), incubated at 20°C in the dark for 30 minutes, and then washed with wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Subsequently, 20 μl of peptide calibrator or sample was added to the appropriate wells, followed by 100 μl of a solution of HRP-conjugated Pro-C3 monoclonal antibody (monoclonal antibody NB61N-62) dissolved in incubation buffer (50 mM PBS-BTB + 10% Liquid II (Roche), pH 7.4). The plate was incubated at 4°C for 20 hours and then washed. Finally, 100 μl of tetramethylbenzidine (TMB) (Chem-En-Tech, cat. 438OH) was added, the plate was incubated in the dark at 20°C for 15 minutes, 100 μl of stop solution (1% H2SO4) was added to stop the reaction, and the plate was analyzed using an ELISA reader at 450 nm with 650 nm as the reference (Molecular Device, SpectraMax M, CA, USA). Calibration curves were plotted using a 4-parameter mathematically fitted model.

[0122] Pro-C6 antibody development Pro-C6-specific monoclonal antibodies, as immunogenic peptides, are the last 10 amino acids (i.e., the C-terminal sequence) of the α3 chain of type VI collagen. 3168’ KPGVISVMGT ’3177The development was carried out using (SEQ ID NO: 10) as described in WO2016 / 156526 (Nordic Bioscience, incorporated herein by reference). Briefly, 4-6 week old Balb / C mice were immunized by subcutaneous injection of 200 μL of emulsified antigen containing 60 μg of immunogenic peptide. Sequential immunization was performed every two weeks with Freund's incomplete adjuvant until a stable serum titer level was reached, and blood was collected from the second immunization onward. Serum antibody titers were detected at each blood collection, and mice with the highest anti-serum titer and best innate reactivity were selected for cell fusion. The selected mice were kept in a resting state for one month, and then further immunized by intravenous administration of 100 μL of 0.9% sodium chloride solution containing 50 μg of immunogenic peptide three days before spleen removal for cell fusion.

[0123] Mouse splenocytes were fused with mouse SP2 / 0 myeloma fusion partner cells. The fused cells were generated in a 96-well plate and incubated in a CO2 incubator. Monoclonal proliferation was promoted using the standard limiting dilution method. Cell lines that were specific to the selected peptide and did not cross-react with either the elongated peptide (KPGVISVMGTA (SEQ ID NO: 18), Chinese Peptide Company, China) or the truncated peptide (KPGVISVMG (SEQ ID NO: 19), American Peptide Company, USA) were selected and subcloned. Finally, antibodies were purified using an IgG column.

[0124] The sequence of the generated antibody was determined, and the CDR (Cellular Discrimination Rate) was then determined. The chain arrangement is as follows (CDR is underlined and bold): Heavy chain sequence (mouse IgG1 isotype)

[0125] [ka]

[0126] CDR-H1: DFNMN (Sequence ID 23) CDR-H2: AINPHNGATSYNQKFSG (Sequence ID 24) CDR-H3: WGNGKNS (Sequence ID 25) Light chain sequence (mouse kappa isotype)

[0127] [ka]

[0128] CDR-L1: RSSQRIVHSNGITFLE (Sequence ID 20) CDR-L2: RVSNRFS (Sequence ID 21) CDR-L3: FQGSHVPLT (Sequence ID 22)

[0129] Pro-C6 immunoassay Pro-C6 was measured using an enzyme-linked immunosorbent assay (ELISA) developed by Nordic Bioscience as described in WO2016 / 156526 (incorporated herein by reference) and as detailed in other publications. Briefly, these procedures are as follows: The ELISA plates used for assay development were streptavidin-coated plates obtained from Roche (cat. 11940279). All ELISA plates were analyzed using an ELISA reader (supplied from Molecular Devices, SpectraMax M, CA, USA). We labeled the selected monoclonal antibodies with horseradish peroxidase (HRP) using the Lightning-Link HRP labeling kit (InnovabioScience, Babraham, Cambridge, UK) according to the manufacturer's instructions. A 96-well streptavidin plate was coated with a solution prepared by dissolving the biotinylated synthetic peptide biotin-KPGVISVMGT (SEQ ID NO: 38) (Chinese Peptide Company, China) in coater buffer (40 mM Na2HPO4, 7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 1% BSA, pH 7.4), and incubated at 20°C for 30 minutes. 20 μL of a dilution prepared by diluting a standard peptide or sample in incubation buffer (40 mM Na2HPO4, 7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 1% BSA, 5% Liquid II, pH 7.4) was added to the appropriate wells, followed by the addition of 100 μL of HRP-conjugated monoclonal antibody 10A3, and incubated at 4°C for 21 hours. Finally, 100 μL of tetramethylbenzidine (TMB) (Chem-En-Tech, cat.438OH) was added, and the plate was incubated in the dark at 20°C for 15 minutes. All incubation steps were performed with shaking at 300 rpm. After each incubation step, the plate was washed five times with washing buffer (20 mM Tris, 50 mM NaCl). The TMB reaction was stopped by adding 100 μL of stop solution (1% H2SO4), and measurements were taken at 450 nm with 650 nm as the reference.

[0130] Pro-C1 immunoassay The immunoassay for detecting Pro-C1 was obtained from Nordic BioScience (cat# 2800A0E51) and performed according to the manufacturer's instructions. P3NP immunoassay The immunoassay for detecting P3NP was obtained from Nordic Bioscience (cat# 1090BD01) and performed according to the manufacturer's instructions.

[0131] Research plan The prospective epidemiological risk factor (PERF) study was a follow-up study of Danish postmenopausal women aimed at identifying risk factors associated with age-related diseases.

[25] Participants in PERF were either previously enrolled in a clinical randomized placebo-controlled trial or were screened without randomization for prior studies at the Center for Clinical and Basic Research (CCBR). The participant aggregation procedure was the same as in

[26] . A total of 4,968 subjects were aggregated along with PRO-C3 measurements, serum biochemistry measurements, medical history, and genotype.

[0132] Approval of standard protocols, registration, and patient consent. The study was conducted in accordance with the International Conference on Harmonization – Guidelines for Good Clinical Practice (ICH-GCP), and the study protocol was approved by the local ethics committee. All participants signed informed consent authorizing future analysis.

[0133] Measurement and data collection at reference time At the baseline, participants had completed interviews with a physician or nurse responsible for questioning about their physical health, demographics, lifestyle, and medical history. Fasting serum and DNA samples were collected from subjects who submitted informed consent for the specificity analysis of this study. PRO-C3 levels were measured in blinded serum by enzyme-linked immunosorbent assay (ELISA) at a CAP-certified laboratory, as previously described. Lymphocyte and neutrophil counts were determined using an automated blood cell analyzer (Sysmex). The complete hospital medical history of subjects during the study period (1974-2014) was obtained by linking each individual's personal identification number (CPR number) to the Danish patient registry as of December 31, 2014, the end date of the study. Study participants were kept anonymous, and CPR numbers were not used in any aspect of the study. Patient registry information was available for 5602 patients. Genotyping was performed using a custom Illumina global screening assay (693143 probe) developed in collaboration with deCODE Genetics (Iceland). SNP imputation was performed using the Michigan Imputation Server

[27] and the HRC r1.1.2016,EUR reference panel. Fading was performed using ShapeIt2, and imputation was performed using Minimac3. Positions are reported in the same way as the GRCh37 reference.

[0134] Definition of disease phenotype Eighteen disease phenotypes were defined as the incidence of events over the entire period, based on data available from numerous sources, including: levels of biochemical markers, physiological measurements, hospital records of overall incidence, mortality records, and questionnaires obtained between baseline and follow-up visits. A detailed list of the included phenotypes and their inclusion criteria is provided in Supplementary Table 2.

[0135] Preprocessing of genotype data Standard probe-level filtering was performed using a minimum probe call rate of 97%, a minor allele frequency of ≥1%, and a Hardy-Weinberg equilibrium p-value cutoff of ≥1e-6. Filtering for multi-allelic SNPs was not performed. To investigate the potential for underlying correlations between subjects, the homogeneity coefficient (IBD) was calculated using the Plink--genomics function. The inbreeding coefficient (IBC) was calculated using the Plink--ibc function. Subjects were excluded from one side of the pair's base using a minimum PI_HAT cutoff value of 0.1875, and cutoffs of less than -0.1 or greater than 0.1 were applied to the Fhat2 coefficient.

[0136] Principal component analysis Population-based genetic variation in the dataset was captured using EIGENSTRAT Smartpca 7.2.0[28, 229], and repeated principal component analysis (PCA) of the study population was performed on the non-imputationally filtered variants using default parameters, utilizing the available genotypes.

[0137] Selection of robust elastic network covariates A robust and optimized variable selection procedure was used to select the variables for the GWAS. Fifty randomizations and a 5-fold cross-validated elastic network were performed to select variables related to the log2 level of PRO-C3. Evaluation using coefficient distribution visualization was performed using standard geom_boxplots.

[0138] Linear regression Linear additive regression was performed on the GWAS study population (n=4968) and the genetic association with log2-converted serum PRO-C3 levels was revealed using Plink v1.90p

[30] (Chang et al., 2015) adjusted for baseline age and three leading principal components. The conservative significance thresholds based on a large number of screened variants were determined to be 6.5e-9, i.e., 0.05 / N, and 1.3e-7, i.e., 1 / N, N=7,672,338 for genome-wide association analysis, both of which were insightful values. Manhattan plot visualization was performed using the R package qqman

[31] .

[0139] Phenotype-Association Analysis Phenotype-association analysis using log2(PRO-C3) levels was performed using logistic regression analysis adjusted for baseline age and BMI. Statistical significance was controlled using the Benjamin-Hockberg method. The p-values ​​and beta distributions of log2(PRO-C3) associated with disease were visualized as forest plots using the forestplot R library.

[0140] Correlation analysis Pearson correlation tests were performed to evaluate the correlation between PRO-C3, different liver-related variables, and disease scores. Biochemical concentrations were considered to be linear values. Genetic correlation analysis

[32] was performed to evaluate the correlation between PRO-C3-related gene components, different liver-related variables, and disease scores. For each variable, GWAS analysis was performed as described above using a standard model adjusted for baseline age, and three leading principal components were used to perform these GWAS analyses. Log-2 transformed values ​​of biochemical measurements were considered in the GWAS analysis. We used LD scores evaluated from PERF genotyping array data.

[0141] Pathway enrichment analysis Pathway enrichment analyses were performed using the VEGAS2

[33] and PARIS 2.4

[34] programs with default parameters. For VEGAS2 analyses, biosystem gene / pathway annotation files provided by the software website were used. The LOKI knowledge base used in PARIS 2.4 was compiled by running the script provided by the program in February 2020. Significant associations between REACTOME pathways and both frameworks with p<0.05 were reported to overlap.

[0142] Possibility of data usage Prospective epidemiological risk factor studies and relevant data obtained from various health status registries are currently stored at Nordic Bioscience. Access to this database may be permitted if researchers have appropriate ethical authorizations and consent forms for the transfer of research materials.

[0143] Example 1 A total of 4,968 subjects from the PERF cohort were included in the study, along with PRO-C3, baseline serum biomarker measurements, hospitalizations obtained from the Danish patient registry, and genotypes. Inclusion criteria are described in the Methods section and in previous studies

[26] . Table 1 summarizes the baseline characteristics of the subjects.

[0144] [Table 1]

[0145] To elucidate the disease profile associated with PRO-C3 levels in PERF subjects, logistic regression analysis adjusted for baseline patient characteristics (age and BMI) was performed. Log levels of PRO-C3 were strongly associated with an increased incidence of chronic liver disease (OR=1.74, 95%CI[1.45-2.09], adjusted p=1.09e-7), chronic kidney disease (OR=1.26, 95%CI[1.10-1.24], adjusted p=6.04e-3), and rheumatic disease (OR=1.35, 95%CI[1.13-1.63], adjusted p=6.69). e-3 ) has a slight connection to this. Logistic regression analysis was performed again using quartile-based PRO-C3 levels, showing a more specific association between liver and rheumatic diseases in PRO-C3 at Q4, and between Q3 and Q4 in chronic kidney disease. Despite strong p-values, the fit accuracy is modest: AUC=0.60 for liver and rheumatic diseases, and AUC=0.70 for kidney diseases.

[0146] Genome-wide association studies GWAS analysis of log2-converted PRO-C3 at baseline was performed using an optimized random selection procedure consisting of a cross-validated elastic network. The model was adjusted for log2-converted values ​​of the following serum and blood biochemical measurements at baseline: alkaline phosphatase (AP), aspartate aminotransferase (AST), sodium (NA), and mean corpuscular hyperpigmentation (MCHC), as well as PRO-C3 measurement batch numbers. The results, summarized as a Manhattan plot, are shown in Figure 2. Log2 (PRO-C3) levels are associated with the TGFBI / VTRNA2-1 locus on chromosome 5 containing the read SNP rs2073511 (Figure 2, effect size 0.070, 95% CI [0.050-0.090] log2 ng / mL, p-value = 8.42e-12). Significant variations within the locus are present throughout the genomic region of the TGFBI gene and are strongly associated. The population study was stratified according to the quartile of log2-converted PRO-C3, and a contingency table of rs2073511 allele development was created within these groups. The results suggest that the TGFBI lead variant is excessively present in subjects belonging to the highest quartile PRO-C3 (chi-squared p-value: 1.177e-07).

[0147] Correlation with other liver-related markers and scores To better elucidate the relationship between PRO-C3 and chronic liver disease, we investigated the correlation between PRO-C3 levels and standard biochemical biomarkers associated with liver disease, and further investigated the correlation with liver-related disease scores. Pearson correlation analysis was performed for this investigation to further decipher potential effects that may lead to confusion with PRO-C3 biomarkers. Pearson correlation analysis shows that PRO-C3 levels are mildly correlated with serum levels of alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma-glutamyl transferase (GGT), and aspartate aminotransferase (AST) (Pearson correlation range [0.13–0.19]). Regarding disease scores, PRO-C3 is associated with the ADAPT score (Pearson correlation 0.83), which is expected given that it is a component of the score calculation. Regarding the liver biomarkers AP, ALP, AST, GGT, and the scores ADAPT, APRI, FIB4, and NFS, A GWAS was conducted, adjusting for age at baseline and three leading principal components (Figure 2). We carefully examined the gene correlations of mutations associated with these different markers and scores

[32] , but with the exception of ADAPT, the landscape of genes associated with PRO-C3 did not correlate with any of the other biochemical biomarkers and disease score-related gene landscapes, and the correlation patterns differed from those calculated based on Pearson correlations of levels and scores. These results suggest that the association of TGFBI mutations is specific to PRO-C3.

[0148] Example 2 In vitro research Applying the previously described “scar in a jar” (SiaJ) model research methods (35)(36), pancreatic CAFs were cultured until confluent and inoculated into 96-well plates with 200 μl of 10% FBS Dulbecco’s modified Eagle medium (DMEM) + GlutaMax to a cell density of 10,000 cells / well. One day after inoculation, the supernatant was removed and the cells were subjected to TGFBI treatment at the following concentrations: four wells were prepared with 200 μl of 0.4% FBS DMEM at concentrations of 0 (medium control), 1 ng / ml, 10 ng / ml, and 100 ng / ml. Three and six days after the initial treatment, the supernatant was removed and stored at -20°C, and the cells were subjected to a fresh TGFBI treatment using 0.4% FBS DMEM. Nine days after the initial treatment, the supernatant was removed and stored, and the experiment was concluded. Biomarkers PRO-C1 (type I collagen propeptide), PRO-C3 (type III collagen propeptide), and PRO-C6 (type VI collagen propeptide) were measured in the supernatant and reflected in the fibrotic activity and collagen synthesis obtained from the SIAJ experiment on a different day. The magnification changes between day 3 and day 9 for different doses of TGFBI treatment were compared.

[0149] In additional experiments, normal pancreatic fibroblasts were grown and inoculated (similarly to CAFs), then treated with TGF-β (0.08 nM), TGFBI (5 nM and 100 nM), and then with a 56-amino acid EMI-domain derived from periostin at the following concentrations: four wells were prepared using 200 μl of 0.4% FBS DMEM at concentrations of 0 (medium control), 1 nM / ml, 10 nM / ml, and 100 nM / ml. Three, six, and nine days after the initial treatment, the supernatant was removed and stored at -20°C, and the cells were subjected to a new treatment using 0.4% DMEM. The experiment was terminated after removing the supernatant 12 days after the initial treatment. PRO-C3 levels were measured in the supernatant, and the levels obtained from different treatment doses were compared. Natural human resting pancreatic fibroblasts and pancreatic CAFs were purchased from Neuromics (cat#SC00A05 and cat#CAF08, US, respectively).

[0150] result As shown in Figure 3, a clear dose-dependent increase in PRO-C3 was most pronounced in CAFs treated with TGFBI. Compared to an approximately 6-fold increase in untreated CAFs, the increase in PRO-C3 from day 3 to day 9 in CAFs treated with 100 ng / mL TGFBI was approximately 28-fold, which corresponds to an increase of over 450% in PRO-C3 in CAFs treated with 100 ng / mL TGFBI compared to untreated CAFs. A smaller (50%) dose-dependent increase was also observed for PRO-C6 levels in the supernatant from TGFBI-treated CAFs compared to untreated CAFs. There was no change in PRO-C1 levels between TGFBI-treated and untreated CAFs. As shown in Figure 4a, 5 nM TGFBI induced PRO-C3 levels in normal pancreatic fibroblasts to a similar degree as TGF-β (increases of 35% and 34%, respectively, from the medium control), and high levels of TGFBI (100 nM) increased the induction even more significantly in normal pancreatic fibroblasts (a 50% increase from the medium control). Interestingly, 100 nM EMI-domain peptide also induced PRO-C3 level induction in normal pancreatic fibroblasts (a 30% increase from the medium control).

[0151] Conclusion: TGFBI stimulation of normal pancreatic fibroblasts and CAFs induces a dose-dependent increase in specific collagen synthesis, particularly type III collagen (PRO-C3) synthesis. These results suggest a direct correlation between TGFBI, CAF activity, collagen synthesis, and PRO-C3 levels, and support TGFBI as a novel target for anti-fibrotic / anti-cancer drugs. In addition, stimulation with EMI-domain peptides also induced an increase in PRO-C3 levels, indicating a potential role for the EMI-domain of TGFBI overall, thereby potentially allowing for clear identification of TGFBI as a target for anti-fibrotic / anti-cancer drug targeting.

[0152] Example 3: Culture of fibroblasts (scar in a jar) Pancreatic fibroblasts (PFs) were cultured in VitroPlus III, low serum (Neuromics cat# PC00B1, Edina, MN, USA) at 37°C and 5% CO2 in culture flasks coated with rat tail type I collagen (cat# P8188, Innoplot, Derio, Bizkaia, Spain) (5 μg / cm2). When the cells reached 80% confluence, they were inoculated into 96-well plates diluted with Divco-Dulbecco's modified Eagle medium + GlutaMax (DMEM) (cat# 31966047, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine (FBS) (cat# F7524, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin / streptavidin (P / S) (cat# P4333, Sigma-Aldrich, USA) to a cell concentration of 15,000 cells / well. Prior to inoculation, a highly bound 96-well plate (cat#3361, Corning, New York, USA) was coated with 100 μl / well of either PBS, 2% BSA, or BigH3 at different concentrations (50 nm, 100 nm, 200 nm, and 400 nm), and the plate was incubated at 37°C for 1 hour. After coating, the plate was washed once with 200 μl / well of PBS, and then blocked with 200 μl / well of 2% BSA in PBS at 37°C for 20 minutes. After 24 hours (day 0), the culture medium was changed to Focoll medium (DMEM containing 0.4% FBS, 1% P / S, 56.25 mg / mL of 70 kDa Focoll (cat# 17-0310-50, GE Healthcare, Chicago, IL, USA), 37.5 mg / mL of 400 kDa Focoll (17-0300-50, GE Healthcare, Chicago, IL, USA), and 0.05 mg / mL of L-ascorbic acid (cat# A9256, Sigma-Aldrich, St. Louis, MO, USA)). The supernatant was removed and frozen every 3 days and at the end of the study (day 12).

[0153] Evaluation of soluble biomarkers Serum BigH3 (Abcam, cat# ab220651) and PRO-C3 (Nordic BioScience, cat# 1700AF06) were blinded and measured by enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunosorbent assay (CLIAs) according to the manufacturer's instructions. Similarly, serum PRO-C1 (Nordic BioScience, cat# 2800A0E51), PRO-C6 (Nordic BioScience, cat# 4000AF02), and P3NP (Nordic BioScience, cat# 1090BD01) were blinded and measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.

[0154] BIGH3 is an inducer of PRO-C3 (and other collagens) in pancreatic fibroblasts. After confirming the correlation between BigH3 and PRO-C3, experiments were conducted using the Scar-in-a-Jar model to determine whether BigH3 also acts as an inducer of PRO-C3 in pancreatic fibroblasts cultured in vitro. As shown in Figure 5, when pancreatic fibroblasts were incubated in the presence of BigH3 and the dose of BigH3 was increased (from 50 nM to 400 nM), PRO-C3 increased in a dose-dependent manner. Furthermore, a similar phenomenon was observed for the type I collagen synthesis biomarker PRO-C1 and the type VI collagen synthesis biomarker PRO-C6, and also for P3NP, a classical fibrosis indicator component (Figure 5). Overall, this indicates that BigH3 is an inducer of PRO-C3 and other collagens in pancreatic fibroblasts, suggesting that BigH3 may act as a driving force for fibroblast activity, leading to increased collagen and fibrosis production. Importantly, the induction of type III collagen synthesis was measured using PRO-C3, and therefore directly relates to early findings regarding the relationship between BIGH3 SNPs and PRO-C3 in circulation. Furthermore, it is suggested that BIGH3 may be an important therapeutic target for regulating tumor fibrosis and cancer-associated fibroblasts in cancer patients with high PRO-C3 levels.

[0155] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns true if one or both of the stated conditions are met, in contrast to the "exclusive OR" operator, which requires that only one of several conditions be met. The word "comprising" is used to mean "including, or consisting of." All prior teachings acknowledged above are incorporated into this specification by reference thereto. Any recognition of prior publications in this specification should not be taken as an admission or statement that the teachings in such publications were common knowledge in Australia or other countries at the time of this specification.

[0156] [References] 1. Jenkins RG, et al. Long-term changes in collagen degradation markers in idiopathic pulmonary fibrosis: A prospective, multi-center PROFILE study. Lancet Respir Med. 2015a;3:462-72. 2. Organ, LA, and others. Biomarkers for collagen synthesis predict disease progression in the PROFILE idiopathic pulmonary fibrosis cohort. Respir Res.2019;20:148. 3. Christensen JH, et al. Circulating MMP-7-degrading elastin levels are elevated in lung diseases. Clin Biochem. 2015;48:1083-8. 4. Reaming DJ, and others. Serological studies on the collagen degradation profiles of patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. Biomark Insights. 2012;7:119-26. 5. Karsdal MA, et al. Regarding fibrosis, the role of good or bad collagen in signal transduction and organ function. Adv Drug Deliv Rev.2017;121:43-56.

[0157] 6. Bao X, Zen Y, Wei S, Wang G, Liu C, Sun Y, Chen Q, and Li H. Growth changes in Col3a1 mRNA expression in muscle, and their relationship to intramuscular collagen in pigs. J Genet Genomics 2007; 34(3): 223-228. 7. Jensen LT and Host NB. Collagen: A scaffolding material related to recovery or death. Cardiovasc Res 1997; 33(3): 535-539. 8. Niemera O, Listerie L, Parkinen J, and Listerie J. Purification and characterization of the N-terminal propeptide of human type III procollagen. Biochem J 1985; 232(1): 145-150. 9. One WM, Ge G, Rim MH, Nagase H, and Greenspan DS. TIMP-3 inhibits procollagen N-proteinase ADAMTS-2. Biochem J 2006; 398(3): 515-519. 10. Goelig D, et al. The substrate protein βIG-H3 reprograms the tumor microenvironment in pancreatic cancer [Internet]. Gut 2019;68(4):693-707.

[0158] 11. Sato T, et al. Identification and characterization of light conversion growth factor beta-induction in circulating cancer cell sublines derived from pancreatic cancer cell lines [Internet]. Cancer Sci. 2018;109(11):3623-3633. 12. Naku, and others. Macrophages produce TGF-beta-inducible (beta-ig-h3) after ingesting apoptotic cells, regulating MMP14 levels and collagen turnover in fibroblasts [Internet]. J. Immunol. 2008;180(7):5036-5044. 13. Takumajidou I, Hatchikotourasu K, Sausum L, and others. Identification of novel therapeutic targets for osteoarthritis through genome-wide analysis of UK biobank data. Nat Genet 2019;51:230-6. doi:10.1038 / s41588-018-0327-1 14. Nielsen MJ, Weydal SS, Karsdal MA, et al. Plasma Pro-C3 (N-terminal type III collagen propeptide) predicts the progression of fibrosis in patients with chronic hepatitis C. Liver Int 2015;35:429-37. 15. Daniels SJ, Reaming DJ, Eslam M, and others. Algorithms incorporating ADAPT:PRO-C3 accurately identify patients with NAFLD and advanced fibrosis. Hepatology 2019;69. doi:10.1002 / hep.30163

[0159] 16. Nielsen MJ, Nedergaardo AF, Sun S, et al. Neo-epitope-specific PRO-C3 ELISA accurately measures the formation of type III collagen related to liver and muscle parameters. Am J Transl Res 2013;5:303-15. 17. Hirschfield GM, Chazo Willelis, Drens JP, and others. Efficacy of FGF19 analog, NGM282, in primary sclerosing cholecholitis: A multi-site, randomized, double-blind, placebo-controlled phase 2 trial. J Hepatol 2018. 18. Brill F, Reaming DJ, Karsdal MA, et al. Use of plasma fragments of propeptides of type III, V, and VI procollagens for the detection of hepatic fibrosis in type 2 diabetes. Diabetes Care 2019;42. doi:10.2337 / dc18-2578 19. Kai Luang, et al. The multifaceted role of periostin in tumor-forming cells. Mol Life Sci. 2009 Jul;66(14):2219-30 doi: 10.1007 / s00018-009-0013-7 20. Dine F. Moscher, et al. Periostin and TGF-β-inducing protein: Two micronuclei within the pod? Crit Rev Biochem Mol Biol. 2015;50(5):427-39. doi: 10.3109 / 10409238.2015.1069791.

[0160] 21. Stefano Marastoni, et al. EMILIN2 downregulates the Wnt signaling pathway, suppressing the proliferation and migration of breast cancer cells. J Pathol. 2014 Mar;232(4):391-404. doi: 10.1002 / path.4316. 22. Nielsen MJ, Nedergaardo AF, Sun S, et al. Neo-epitope-specific PRO-C3 ELISA accurately measures the formation of type III collagen related to liver and muscle parameters. Am J Transl Res 2013;5(3):303-15. 24. Combet C, Blanchett C, Jowlion C, Direage G. NPS@: Network Protein Sequence Analysis. Trends Biochem Sci. 2000;25(3):147-150. 25. Nirgaard JS, Dragusbayek K, Kehlet SN, et al. Prospective Epidemiological Risk Factor (PERF) Study Int J Epidemiol 2016;:1-10. doi:10.1093 / ije / dyw251

[0161] 26. Tan M-HE, Breyer JPM, Bäger CL, and others. Matrix metalloproteinase-degraded type I collagen is associated with APOE / TOMM40 gene alterations and preclinical dementia. Neurol Genet 2020;6:e508. doi:10.1212 / NXG.0000000000000508 27. DAS S, Forear L, Shoan Hill S, and others. Next-generation genotyping imputation services and methods: HHS Public Access Producer Manuscript Nat Genet 2016;48:1284-7. doi:10.1038 / ng.3656 28. Price AL, Patterson NJ, Prange RM, et al. Principal component analysis corrects for stratification in genome-wide association studies. Nat Genet 2006;38:904-9. doi:10.1038 / ng1847 29. Patterson N, Price AL, Leitch D, et al. Group structure and eigenvalue analysis. PLoS Genet 2006;2:190. doi:10.1371 / journal.pgen.0020190 30. Chan CC, Chou CC, Terrier LC, and others. PLINK 2nd Generation: The beginning of a challenge to larger and richer datasets. 2015;4:7. doi:10.1186 / s13742-015-0047-8

[0162] 31. Turner SD. qqman: An R package for visualizing GWAS results using QQ and Manhattan plots. bioRxiv 2014;:005165. doi:10.1101 / 005165 32. Brix-Sullivan B, Finucin HK, Antila V, and others. An illustration of the overall genetic correlations between human diseases and traits. Nat Genet 2015;47:1236-41. doi:10.1038 / ng.3406 33. Michelin A, McGregor S. VEGAS2: Software for more flexible gene-based analysis. Twin Res Hum Genet 2015;18:86-91. doi:10.1017 / thg.2014.79 34. Butokiwikuz M, Kuuki Bailey JN, Freyse A, et al. Path analysis using randomized inclusion structure - PARIS: Update. Bioinformatics 2016;32:2361-3. doi:10.1093 / bioinformatics / btw130

[0163] 35. Roenno SR, and others. Extended Scar-in-a-Jar: An in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis. Respir. Res. 2020;21(1):108. 36. Nissen NI, Karsdal M, Wilmssen N. Abstract 3958: Establishment of an in vitro model for studying cancer-associated fibroblasts and their direct effects on stromal fibrosis [Internet]. In: Cancer Research. American Association for Cancer Research (AACR); 2020:3958-3958

Claims

1. A method for identifying patients who are relatively likely to respond to anti-TGFβI therapy, comprising the following steps, using an immunoassay to detect at least one marker of fibroblast activity in a patient sample: The patient's sample is brought into contact with a monoclonal antibody that specifically reacts to an epitope of a marker for fibroblast activity; Determining the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals with known responses to anti-TGFβI therapy and / or a predetermined cutoff value.

2. The method according to claim 1, wherein the marker for fibroblast activity is selected from PRO-C3 (PIIIINP), PRO-C6, PRO-C1, and / or P3NP.

3. The method of claim 1 or claim 2, wherein the method includes the following steps: The patient's sample is brought into contact with a monoclonal antibody that specifically binds to the PIIINP epitope; Determining the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals with known responses to anti-TGFβI therapy and / or a predetermined cutoff value.

4. The method of claim 3, wherein the epitope of PIIINP to which the monoclonal antibody specifically binds is the C-terminal neo-epitope of PIIINP contained in the C-terminal amino acid sequence CPTGXQNYSP-COOH (where X is Gly or Pro) (SEQ ID NO: 1).

5. The method of claim 4, wherein the monoclonal antibody does not specifically recognize or bind to CPTGXQNYSPQ-COOH (SEQ ID NO: 5), which is an extension of the C-terminal amino acid sequence, and / or CPTGXQNYS-COOH (SEQ ID NO: 6), which is a truncated version of the C-terminal amino acid sequence.

6. The method according to any one of claims 3 to 5, wherein the monoclonal antibody is produced in opposition to a synthetic peptide containing or consisting of the C-terminal amino acid sequence CPTGXQNYSP-COOH (wherein X is Gly or Pro) (SEQ ID NO: 1).

7. The method of any of the above claims, wherein the method includes the following steps: The patient's sample is brought into contact with a monoclonal antibody that specifically binds to the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen; Determining the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals with known responses to anti-TGFβI therapy and / or a predetermined cutoff value.

8. The method of claim 7, wherein the monoclonal antibody that specifically binds to the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen specifically binds to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO: 17).

9. The method of claim 7 or claim 8, wherein the monoclonal antibody does not specifically recognize or bind to KPGVISVMGTA (SEQ ID NO: 18), which is an extension of the C-terminal amino acid sequence, and / or KPGVISVMG (SEQ ID NO: 19), which is a truncated version of the C-terminal amino acid sequence.

10. The method according to any one of claims 7 to 9, wherein the monoclonal antibody is produced in opposition to a synthetic peptide containing or consisting of the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO: 17).

11. The method of any of the above claims, wherein the method includes the following steps: The patient's sample is brought into contact with a monoclonal antibody that specifically binds to the N-terminal epitope of the propeptide of type I collagen; Determining the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals with known responses to anti-TGFβI therapy and / or a predetermined cutoff value.

12. The method of claim 11, wherein the monoclonal antibody that specifically binds to the N-terminal epitope of the propeptide of type I collagen specifically binds to the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID NO: 3).

13. The method according to claim 11 or claim 12, wherein the monoclonal antibody does not specifically recognize or bind to PDGSESPTDQETTGVE (SEQ ID NO: 15), which is an extension of the C-terminal amino acid sequence, and / or DGSESPTDQETTGV (SEQ ID NO: 17), which is a truncated version of the C-terminal amino acid sequence.

14. The method according to any one of claims 11 to 13, wherein the monoclonal antibody is produced in opposition to a synthetic peptide containing or consisting of the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID NO: 3).

15. The method of any of the above claims, wherein the method includes the following steps: The patient's sample is brought into contact with a monoclonal antibody that specifically binds to the internal sequence of the N-terminal region of type 3 collagen; Determining the amount of the monoclonal antibody bound; and, The binding amount is correlated with a value associated with normal healthy individuals and / or a value associated with individuals with known responses to anti-TGFβI therapy and / or a predetermined cutoff value.

16. The method of claim 15, wherein the monoclonal antibody that specifically binds to the internal sequence of the N-terminal epitope region of type 3 collagen specifically binds to the amino acid sequence PGIPGRNGDP (SEQ ID NO: 2).

17. The method of claim 15 or 16, wherein the monoclonal antibody does not specifically recognize or bind to PPGIPGRNGDP (SEQ ID NO: 32) or PGIPGRNGDPG (SEQ ID NO: 33), which are elongated C-terminal amino acid sequences, and / or GIPGRNGDP (SEQ ID NO: 34) or PGIPGRNGD (SEQ ID NO: 35), which are truncated C-terminal amino acid sequences.

18. The method according to any one of claims 15 to 17, wherein the monoclonal antibody is produced in opposition to a synthetic peptide containing or consisting of the amino acid sequence PGIPGRNGDP (SEQ ID NO: 2).

19. The method according to any one of the claims, wherein the sample of the patient is a biological fluid.

20. The method of claim 19, wherein the biological fluid is serum or plasma.

21. The method according to any one of the claims, wherein the immunoassay is a competitive immunoassay.

22. The method of claim 21, wherein the competing immunoassay is a radioimmunoassay, a fluorescent immunoassay, or an enzyme-linked immunosorbent assay.

23. The method according to any of the claims, further comprising administering anti-TGFβI therapy to patients identified as likely to respond to anti-TGFβI therapy.

24. A drug that binds to the EMI domain of TGFβI, periostin, or their analogues.

25. The drug according to claim 24, wherein the drug is an antibody.

26. A pharmaceutical agent according to claim 24 or claim 25, for use as a medicine.

27. A drug according to any one of claims 24 to 26, for use in a method of treating cancer or fibrosis.

28. The agent according to claim 26 or 27, wherein the agent is used to treat a patient identified as having a relatively high likelihood of responding to anti-TGFβI therapy by any one of claims 1 to 22.

Images