Fibroblast activation markers and TGFBI in therapy

Abstract

The present invention relates to a method of detecting fibroblast activation markers in a patient by a sandwich immunoassay for identifying a patient, in particular a cancer patient that benefits from anti-TGF [beta] I therapy. The fibroblast activation marker may be selected from the group consisting of an N-terminal propeptide of type III collagen (Pro-C3), an N-terminal propeptide of type VI collagen (Pro-C6), an N-terminal propeptide of type I collagen (Pro-C1), and / or an internal sequence (P3NP) in the N-terminal region of type 3 collagen. The invention also provides anti-TGF [beta] I therapies for the treatment of cancer and / or fibrosis.

Description

Technical Field

[0001] This invention relates to a method for detecting fibroblast activation markers in patients via immunoassay for the identification of patients, particularly cancer patients who will benefit from anti-TGFβI therapy. The fibroblast activation markers may be selected from the N-terminal propeptide (Pro-C3) of type III collagen, the N-terminal propeptide (Pro-C6) of type VI collagen, the N-terminal propeptide (Pro-C1) of type I collagen, and / or an internal sequence (P3NP) in the N-terminal region of type 3 collagen. This invention also provides anti-TGFβI therapy for the treatment of cancer and / or fibrosis. Background Technology

[0002] Numerous studies have been conducted to understand the changes that occur in extracellular matrix (ECM) dynamics associated with physiological impairment and decreased lung function in diseases such as idiopathic pulmonary fibrosis (IPF) [1-4]. In a healthy state, the ECM forms a thin basement membrane layer in the small airways, separating capillaries from the alveolar space and allowing unimpeded gas diffusion. During fibrosis, the ECM expands, restricting this diffusion. Fibrotic ECM in the lungs contains high levels of type I, III, and VI collagen. Very simply, in pulmonary fibrosis, the specialized basement membrane, which is normally composed of an open structure of type IV collagen backbone and laminin and allows diffusion, is replaced by a dense interstitial ECM composed of a completely different set of collagens and therefore has different functions [5].

[0003] Type III collagen, along with type I collagen, constitutes the major structural proteins in the human body. Type III collagen is crucial for the formation of type I collagen fibrils, except in bone, which is almost entirely composed of type I collagen [6,7]. During fiber assembly, the N-terminal propeptide of type III procollagen is cleaved by a specific N-protease before mature type III collagen is incorporated into the extracellular matrix (ECM), resulting in the release of the N-terminal propeptide into the ECM and into circulation. This N-terminal propeptide molecule, also known as "PIIINP," consists of three identical α-chains with a total molecular weight of 42 kDa. Removal of PIIINP is sometimes incomplete, leaving the propeptide attached to the collagen molecule, resulting in fine fibrils with abnormal cross-linking and thus readily metabolized [8,9]. Therefore, this propeptide can serve as a marker for the formation and degradation of type III collagen.

[0004] WO2014 / 170312A1 discloses a novel C-terminal epitope (also referred to herein as “Pro-C3”) of a PIIINP contained in the C-terminal amino acid sequence CPTGXQNYSP-COOH, where X is Gly or Pro (SEQ ID NO: 1). It also discloses a competitive immunoassay (also referred herein as the “Pro-C3 assay”) that detects the level of the novel C-terminal epitope of PIIINP in a biological sample by contacting the monoclonal antibody with the antibody and measuring the amount of antibody binding.

[0005] P3NP is a general biomarker for fibroblast activation, containing an internal sequence in the N-terminal region of type 3 collagen, including the following internal sequence: 114 PGIPGRNGDP 123 (Uniprot P02461) (SEQ ID NO:2).

[0006] Type VI collagen is a unique extracellular collagen that forms an independent network of microfibrils within the cell's basement membrane. It can interact with other matrix proteins, including collagen, disaccharide proteoglycans, and other proteoglycans. In muscle, type VI collagen is part of the myofibril membrane and participates in anchoring myofibrils to the intracellular extracellular matrix, thus contributing to force transmission. Furthermore, mutations in type VI collagen can cause Bethlemmyopathy and Ullrich's congenital muscular dystrophy. It has been reported that, upon secretion, the C-terminal amino acid sequence of the α3 chain of type VI collagen cleaves from mature type VI microfibrils. However, type VI collagen is not only involved in muscle and muscle loss.

[0007] Type VI collagen, a microfilamentous interstitial collagen composed of triple-helical molecules consisting of chains α1(VI), α2(VI), and α3(VI), is expressed in most connective tissues, and primarily in adipose tissue, where it anchors cells through its interlinking with other ECM proteins. During microfilament formation, the triple-helical core of type VI collagen is released from the propeptide via proteolysis, and the cleavage of the C-terminal propeptide of the α3(VI) chain yields an endogenous protein, an adipokine.

[0008] PRO-C6 is a biomarker for the formation of type VI collagen and the release of endotrophic proteins. It comprises a C-terminal epitope of the C5 domain of the α3 chain of type VI collagen, which is cleaved when new type VI collagen molecules are assembled in the extracellular matrix. This C-terminal epitope is also the C-terminal epitope of the bioactive fragment of endotrophic proteins. The PRO-C6 biomarker and PRO-C6 assay (specifically, the PRO-C6 ELISA) are described in WO2016 / 156526. This assay utilizes a monoclonal antibody that specifically binds to the C-terminal 10 amino acid sequence of the C5 domain of the α3 chain of type VI collagen. Endotrophic proteins have been observed to act as pro-fibrotic, pro-inflammatory, and pro-tumorigenic molecules in preclinical models of breast cancer and liver fibrosis. 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 biomarker for response to glucose-lowering therapy in diabetic patients.

[0009] 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 for type I collagen formation, containing the N-terminal propeptide of type I collagen, and has a sequence... 96 PDGSESPTDQETTGV 110 (uniprotP 02452) (SEQ ID NO: 3).

[0010] Tumor fibrosis and associated extracellular matrix (ECM) and collagen synthesis emerge as key components in defining the vast matrix responses in cancer and in determining prognosis and response to anticancer therapies. Cancer-associated fibroblasts (CAFs) are the primary drivers of this so-called connective tissue-building response and collagen synthesis. Identifying the drivers of collagen synthesis in CAFs is necessary to optimally develop novel and ambitious therapies targeting the excessive deposition of ECM and collagen in the tumor microenvironment.

[0011] While there is significant interest in drugs to alleviate fibrosis (e.g., idiopathic pulmonary fibrosis (IPF), nonalcoholic steatohepatitis (NASH), chronic kidney disease (CKD), and systemic sclerosis (SSC)), the number of Phase II and III failures is increasing. This may be due to both a lack of optimal clinical trial design and robust endpoints, as well as a lack of understanding of the disease drivers. Somewhat concerningly, while the number of novel drug targets and positive animal studies for liver and lung fibrosis is increasing, few or none translate into feasibility for human clinical settings. Understanding the pathways driving fibrosis in humans is essential. In a human clinical setting, we need to understand the commonalities in the indications for central fibrosis and the different pathological pathways of fibrosis to benefit patients.

[0012] The protein TGFβI (transforming growth factor β-inducible protein), also known as BigH3, has been shown to “reprogram” the tumor microenvironment in pancreatic cancer space and is associated with survival outcomes, suggesting that TGFβI may be a promising new target for the development of anticancer drugs in the future

[10]

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

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

[0013] Traditionally, genome-wide association studies (GWAS) have been conducted to analyze these parameters in relation to clinical parameters

[13] . Clinical parameters can be considered as endpoints of disease, while biochemical markers are more relevant to acute disease activity. In direct comparison, PRO-C3, as a measure of type III collagen formation, is an alternative measure of fibrosis formation

[14] . PRO-C3 is a quantitative measure of the propeptide (also known as PNIIIP) of type III collagen chains. When fibroblasts produce type III collagen, the propeptide of collagen is released before the collagen is incorporated into the matrix. Therefore, PRO-C3 can be considered an alternative biomarker for the rate of fibrosis formation. However, type I collagen is the most abundant protein in the body and in the fibrotic extracellular matrix (ECM), while type III collagen is less abundant and more specific to the fibrotic ECM

[15] .

[0014] PRO-C3 levels have previously been associated with prognostic outcomes of liver fibrosis progression

[16] , diagnosis of liver fibrosis

[15] , and pharmacodynamic response to interventions that show reduction in fibrosis

[17] ,

[18] .

[0015] The inventors have identified the relationship between organ fibrosis, PRO-C3, PRO-C1, PRO-C6, and P3NP and pathway genes that lead to higher levels of fibrosis. Summary of the Invention

[0016] To investigate the potential relationship between the biomarker PRO-C3 and genes within pathways leading to high levels of fibrosis, a GWAS analysis was performed based on pro-C3 levels measured using a Pro-C3 assay. A strong association was identified between elevated levels of Pro-C3 and the TGFβI gene. No such association was found between the TGFβI gene and other biomarkers of chronic liver disease.

[0017] Given that high levels of PRO-C3 (the precursor of type III collagen) (a measure of fibrotic activity) are associated with the TGFβI risk allele rs2073511, the inventors investigated whether TGFβI directly affects fibroblast collagen synthesis by stimulating pancreatic cancer-associated fibroblasts (CAFs) in vitro. A paralog of TGFβI is periosteal protein, which is known to play a key and multifaceted role in tumorigenesis

[19] . Both stromal cell proteins contain a single elastin microfibril interface protein (emilin (EMI)) module and four bundle protein-1 (FAS) modules (20). Interestingly, the presence of an EMI domain on another protein (EMILIN-2) is essential for the protein's biological effects (wnt signaling / wnt1 interaction), as deletion mutants lacking this domain have no effect. The EMI domains on TGFβI and periosteal protein are closely associated and share large sequence homology and are generally shorter than other EMI domains ( Figure 1 )[twenty one].

[0018] The inventors have demonstrated that TGFβI leads to a dose-dependent increase in the levels of fibroblast activation markers Pro-C3, Pro-C1, Pro-C6, and P3NP. Furthermore, administration of the EMI domain of periosteal protein also results in an increase in Pro-C3 levels.

[0019] Therefore, in a first aspect, the present invention provides a method for identifying patients more likely to respond to TGFβI therapy, the method using an immunoassay to detect at least one fibroblast activation marker in a patient sample, the method comprising: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody is specifically reactive to the epitopes of the fibroblast activation marker; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

[0020] Fibroblast activation markers can be selected from PRO-C3 (PIIINP), PRO-C6, PRO-C1 and / or P3NP.

[0021] High binding levels indicate elevated levels of fibroblast activation markers. Elevated levels of fibroblast activation markers suggest that patients are more likely to respond to anti-TGFβI therapy, thus making them suitable candidates for anti-TGFβI therapy.

[0022] This invention provides a method for identifying patients more likely to respond to TGFβI therapy, the method using an immunoassay to detect Pro-C3 in a patient sample, the method comprising: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically reacts with and binds to the epitope of PIIINP; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

[0023] High binding levels indicate elevated Pro-C3 levels. Elevated Pro-C3 levels suggest that the patient is more likely to respond to anti-TGFβI therapy, thus making them a suitable candidate for anti-TGFβI therapy.

[0024] In a preferred embodiment, the PIIINP epitope that specifically reacts with and thus specifically binds to the monoclonal antibody is a novel C-terminal epitope of the PIIINP generated by cleavage of the intact type III procollagen by an N-protein protease. Preferably, the novel C-terminal epitope of the 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 novel C-terminal epitope of the PIIINP is contained in the C-terminal amino acid sequence CPTGPQNYSP-COOH (SEQ ID NO: 4).

[0025] Preferably, the monoclonal antibody nonspecifically recognizes or binds to CPTGXQNYSPQ-COOH, wherein X is an extended form of the C-terminal amino acid sequence of Gly or Pro (SEQ ID NO: 5). Preferably, the ratio between the antibody affinity for the amino acid sequence CPTGXQNYSPQ-COOH (SEQ ID NO: 1) and the antibody affinity 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, more preferably at least 10,000 to 1, more preferably at least 100,000 to 1, and most preferably at least 1,000,000 to 1.

[0026] Preferably, the monoclonal antibody does not specifically recognize or bind to the truncated C-terminal amino acid sequence of CPTGXQNYS-COOH (SEQ ID NO: 6). Preferably, the ratio between the affinity of the monoclonal antibody for the amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 1) and the affinity of the monoclonal antibody for the truncated amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 6) is at least 10:1, preferably at least 100:1, more preferably at least 1,000:1, more preferably at least 10,000:1, more preferably at least 100,000:1, and most preferably at least 1,000,000:1.

[0027] Preferably, the monoclonal antibody is generated against a synthetic peptide comprising the C-terminal amino acid sequence CPTGXQNYSP-COOH, wherein X is Gly or Pro (SEQ ID NO: 1), or a composition thereof. Most preferably, the synthetic peptide has the C-terminal amino acid sequence CPTGPQNYSP-COOH (SEQ ID NO: 4).

[0028] As used herein, the term "new epitope" refers to an epitope resulting from peptide cleavage. "C-terminal new epitope" refers to a new epitope located at the C-terminus of the cleaved peptide (i.e., the C-terminus of the peptide) and should not be interpreted in terms of its general orientation. Similarly, "C-terminal amino acid sequence" refers to the C-terminal peptide sequence located at the end of the peptide, i.e., at the C-terminus of the peptide, and should not be interpreted in terms of its general orientation.

[0029] The terms “peptide” and “polypeptide” are used synonymously in this article.

[0030] As used herein, the term "monoclonal antibody" refers both to an intact antibody and to an antibody fragment that retains the binding specificity of an intact antibody, such as a Fab fragment, an F(ab')2 fragment, a single-chain Fv fragment, or other such fragments known to those skilled in the art. It is well known that intact antibodies typically have a "Y-shaped" structure, consisting of two pairs of identical polypeptide chains, each pair consisting of a "light" chain and a "heavy" chain. The N-terminal regions of the light and heavy chains contain variable regions, while the C-terminal regions of the light and heavy chains constitute constant regions. The variable regions contain three complementarity-determining regions (CDRs), primarily responsible for antigen recognition. The constant regions allow the antibody to recruit cells and molecules of the immune system. An antibody fragment that retains binding specificity contains at least a sufficient portion of the CDRs and the remainder of the variable regions to retain said binding specificity.

[0031] In the method of this invention, monoclonal antibodies comprising any constant region known in the art can be used. Human constant light chains are classified as κ or λ light chains. Constant heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG isotypes have several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4 in humans and IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in mice. Monoclonal antibodies may preferably be IgG isotypes, including any one of IgG1, IgG2, IgG3, or IgG4.

[0032] The CDR of an antibody can be determined using methods known in the art, such as those described by Kabat et al. Antibodies can be generated from B cell clones as described in the examples. The isotype of the antibody can be determined by an ELISA specific to IgM, IgG, or IgA isotypes, or human IgG1, IgG2, IgG3, or IgG4 subclasses, or mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3. The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and cDNA can be generated by reverse transcription using the RNA. The cDNA is then PCR-paired using primers that amplify the antibody heavy and light chains. For example, primers specific to the leader sequence of all VH (variable heavy chain) sequences can be used in conjunction with primers that bind to sequences located in the constant region of the previously determined isotype. The light chain can be amplified using primers that bind to the 3' end of the κ or λ chain and primers annealed to the Vκ or Vλ leader sequence. Full-length heavy and light chains can be generated and sequenced.

[0033] 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 technique known in the art. For example, the monoclonal antibody can be generated against a synthetic peptide containing the amino acid sequence CPTGPQNYSP-COOH (SEQ ID No: 4) or composed thereof, for example by: immunizing rodents (or other suitable mammals) with a synthetic peptide composed of the sequence CPTGPQNYSP-COOH (SEQ ID No: 4), said synthetic peptide optionally being linked to an immunogenic carrier protein (such as keyhole hemocyanin), isolating and cloning individual antibody-producing cells, and measuring the resulting monoclonal antibodies to ensure they have the desired specificity.

[0034] As used herein, the term "specific binding" refers to antibody binding that is selective for antigens and can be distinguished from unwanted or nonspecific interactions. The ability of a monoclonal antibody to bind to a specific epitope or peptide sequence can be measured by the enzyme-linked immunosorbent assay (ELISA) described herein or by other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analysis on BIAcore instruments) and conventional binding assays. The degree to which a monoclonal antibody binds to unrelated proteins is less than about 10% of its binding to an epitope or peptide, a degree that can be measured, for example, by an ELISA method. "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 stated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., the antigen-binding moiety and the antigen). The affinity of a molecule for its partner is typically expressed as a dissociation constant (Kd), which is the dissociation rate constant and the binding rate constant (k, k ... off and k on The ratio of the rate constants to the antigen's binding affinity. Therefore, as long as the ratio of the rate constants remains constant, equivalent affinity can include different rates. Lower Kd values ​​indicate higher binding affinity between the antibody and the antigen, while higher Kd values ​​indicate weaker binding affinity. The dissociation constant represents the antigen concentration at which half of the binding sites on the antibody are occupied. Various methods exist for measuring antibody Kd values, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In some respects, the dissociation constant (KD) of monoclonal antibodies binding to epitopes or peptides is less than 1 pM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10⁸ M or lower, e.g., 10⁸ nM). 8 M to 10 13 M, for example, 10 9 M to 10 13 M).

[0035] Monoclonal antibodies that specifically bind to PRO-C3 may preferably contain 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).

[0036] Preferably, the monoclonal antibody may contain at least 2, 3, 4, 5, or all of the more than 6 listed CDR sequences.

[0037] 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 (SEQ ID. No:11), and CDR-L3: FQGAHDPPA (SEQ ID. No:12).

[0038] Preferably, the monoclonal antibody may have a light chain containing a framework sequence between CDRs, wherein the framework sequence is substantially the same as or substantially similar to the framework sequence between CDRs in the following light chain sequence (wherein CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0039] Preferably, the monoclonal antibody has a heavy chain variable region comprising the following CDR sequence: CDR-H1: NYVIH (SEQ ID. No:7); CDR-H2: YMNPYNDVPKNNAKFRG (SEQ ID. No:8) and CDR-H3: GGFFGPLSY (SEQ ID. No:9).

[0040] Preferably, the monoclonal antibody may have a heavy chain containing a framework sequence between CDRs, wherein the framework sequence is substantially the same as or substantially similar to the framework sequence between CDRs in the following heavy chain sequence (wherein CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0041] As used herein, antibodies are considered substantially identical or substantially similar if the framework amino acid sequence between the CDRs of one antibody has at least 70%, 80%, 90%, or at least 95% similarity or identity with the framework amino acid sequence between the CDRs of another antibody. The similar or identical amino acids may be sequential or discontinuous.

[0042] The framework sequence may contain one or more amino acid substitutions, insertions, and / or deletions. Amino acid substitutions can be conserved, 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 have similar chemical properties. For example, the following groups of amino acids have similar chemical properties, such as size, charge, and polarity: 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.

[0043] Amino acid sequences can be compared using programs such as CLUSTAL. This program compares amino acid sequences and finds the best alignment by inserting appropriate gaps in either sequence. Amino acid identity or similarity (identity plus conservation of amino acid types) can be calculated to obtain the best alignment. Programs like BLASTx align the longest similar sequence fragments and assign a fit value. Therefore, alignments can be performed to find multiple similar regions, each with a different score. This invention covers both types of analysis. It is preferable to calculate identity or similarity over the entire frame sequence length.

[0044] In some preferred embodiments, the monoclonal antibody that specifically binds to PRO-C3 may include a light chain variable region sequence: And / or the heavy chain variable region sequence: (CDR displays in bold and underline, and framework sequences are displayed in italics).

[0045] Immunoassay kits suitable for measuring Pro-C3 levels are available commercially, such as those from Nordic Biosciences (cat# 1700AF06).

[0046] This invention provides a method for identifying patients more likely to respond to TGFβI therapy, the method using an immunoassay to detect Pro-C6 in patient samples, the method comprising: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically reacts with the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen. The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

[0047] High binding levels indicate elevated Pro-C6 levels. Elevated Pro-C6 levels suggest that patients are more likely to respond to anti-TGFβI therapy, thus making them suitable candidates for anti-TGFβI therapy.

[0048] Preferably, the monoclonal antibody that specifically reacts to and thus 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) (also referred to herein as the “PRO-C6 sequence”, or simply “PRO-C6”). Preferably, the monoclonal antibody does not specifically bind to an extended form of the C-terminal amino acid sequence of KPGVISVMGTA (SEQ ID No: 18), or does not specifically bind to a truncated form of the C-terminal amino acid sequence of KPGVISVMG (SEQ ID No: 19).

[0049] Preferably, the ratio of the antibody’s affinity for the C-terminal amino acid sequence KPGVISVMGT (SEQ ID No: 17) to the antibody’s affinity for the extended C-terminal amino acid sequence KPGVISVMGTA (SEQ ID No: 18) and / or for the truncated C-terminal amino acid sequence KPGVISVMGTA (SEQ ID No: 19) is at least 10 to 1, and more preferably at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1.

[0050] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID No: 17) can be produced by any suitable technique known in the art. For example, the monoclonal antibody can be produced against a synthetic peptide containing the amino acid sequence KPGVISVMGT (SEQ ID No: 17) or composed thereof, for example by: immunizing rodents (or other suitable mammals) with a synthetic peptide composed of the sequence KPGVISVMGT (SEQ ID No: 17), said synthetic peptide optionally being linked to an immunogenic carrier protein (such as keyhole hemocyanin), isolating and cloning individual antibody-producing cells, and measuring the resulting monoclonal antibodies to ensure they have the desired specificity.

[0051] Monoclonal antibodies that specifically bind to the PRO-C6 sequence may preferably contain one or more complementarity-determining regions (CDRs) selected from the following: CDR-L1: RSSQRIVHSNGITFLE (SEQ ID No: 20) CDR-L2: RVSNRFS (SEQ ID No: 21) CDR-L3: FQGSHVPLT (SEQ ID No: 22) CDR-H1: DFNMN (SEQ ID No: 23) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID No: 24) CDR-H3: WGNGKNS (SEQ ID No: 25).

[0052] Preferably, the antibody contains at least 2, 3, 4, 5 or 6 of the listed CDR sequences.

[0053] Preferably, the variable region of the monoclonal antibody light chain contains a CDR sequence.

[0054] CDR-L1: RSSQRIVHSNGITFLE (SEQ ID No: 20) CDR-L2: RVSNRFS (SEQ ID No: 21) and CDR-L3: FQGSHVPLT (SEQ ID No: 22).

[0055] Preferably, the monoclonal antibody light chain contains a framework sequence between CDRs, wherein the framework sequence is substantially the same as or substantially similar to the framework sequence between CDRs in the following light chain sequence (wherein CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0056] Preferably, the variable region of the monoclonal antibody heavy chain contains the following CDR sequence: CDR-H1: DFNMN (SEQ ID No: 23) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID No: 24) and CDR-H3: WGNGKNS (SEQ ID No: 25).

[0057] Preferably, the monoclonal antibody heavy chain contains a framework sequence between CDRs, wherein the framework sequence is substantially the same as or substantially similar to the framework sequence between CDRs in the following heavy chain sequence (wherein CDRs are shown in bold and underline, and framework sequences are shown in italics).

[0058] In some preferred embodiments, the monoclonal antibody that specifically binds to the PRO-C6 sequence may include a light chain variable region sequence: And / or the heavy chain variable region sequence: (CDR displays in bold and underline, and framework sequences are displayed in italics).

[0059] Immunoassay kits suitable for measuring Pro-C6 levels are available commercially, such as the kit from Nordic Biosciences (cat# 4000AF02).

[0060] This invention provides a method for identifying patients more likely to respond to TGFβI therapy, the method using an immunoassay to detect Pro-C1 in patient samples, the method comprising: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically reacts with the N-terminal epitope of the propeptide of type I collagen; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

[0061] High binding levels indicate elevated Pro-C1 levels. Elevated Pro-C1 levels suggest that patients are more likely to respond to anti-TGFβI therapy, thus making them suitable candidates for anti-TGFβI therapy.

[0062] Preferably, the monoclonal antibody that specifically reacts to and thus specifically binds to the N-terminal epitope of type I collagen propeptide specifically binds to the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID No: 3) (also referred to herein as the “PRO-C1 sequence”, or simply “PRO-C1”). Preferably, the monoclonal antibody does not specifically bind to an extended form of the N-terminal amino acid sequence of PDGSESPTDQETTGVE (SEQ ID No: 30), or a truncated form of the N-terminal amino acid sequence of PDGSESPTDQETTGV (SEQ ID No: 31).

[0063] Preferably, the ratio of the antibody’s affinity for the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID No: 3) to the antibody’s affinity for the extended N-terminal amino acid sequence PDGSESPTDQETTGVE (SEQ ID No: 30) and / or for the truncated N-terminal amino acid sequence DGSESPTDQETTGV (SEQ ID No: 31) is at least 10 to 1, and more preferably at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1.

[0064] As used herein, the term “N-terminus” refers to the N-terminal peptide sequence at the end of a polypeptide, i.e., at the N-terminus of the polypeptide, and should not be interpreted as having any meaning in its general direction.

[0065] Monoclonal antibodies that specifically bind to the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID No: 3) can be produced by any suitable technique known in the art. For example, the monoclonal antibody can be produced against a synthetic peptide containing the amino acid sequence PDGSESPTDQETTGV (SEQ ID No: 3) or composed thereof, for example by: immunizing rodents (or other suitable mammals) with a synthetic peptide composed of the sequence PDGSESPTDQETTGV (SEQ ID No: 3), said synthetic peptide optionally being linked to an immunogenic carrier protein (such as keyhole hemocyanin), isolating and cloning individual antibody-producing cells, and measuring the resulting monoclonal antibodies to ensure they have the desired specificity.

[0066] Immunoassay kits suitable for measuring Pro-C1 levels are available commercially, such as those from Nordic Biosciences (cat# 2800A0E51).

[0067] This invention provides a method for identifying patients more likely to respond to TGFβI therapy, the method using an immunoassay to detect P3NP in a patient sample, the method comprising: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically reacts with an internal sequence in the N-terminal region of type 3 collagen; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

[0068] High binding levels indicate elevated P3NP levels. Elevated P3NP levels suggest that patients are more likely to respond to anti-TGFβI therapy, thus making them suitable candidates for anti-TGFβI therapy.

[0069] Preferably, the monoclonal antibody that specifically reacts to and thus 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) (also referred to herein as the “P3NP sequence”, or simply “P3NP”). Preferably, the monoclonal antibody does not specifically bind to an extended form of the amino acid sequence PPGIPGRNGDP (SEQ ID No: 32) or PGIPGRNGDPG (SEQ ID No: 33), or a truncated form of the N-terminal amino acid sequence GIPGRNGDP (SEQ ID No: 34) or PGIPGRNGDPG (SEQ ID No: 35).

[0070] Preferably, the ratio of the antibody’s affinity for PGIPGRNGDP (SEQ ID No: 2) to the antibody’s affinity for the extended amino acid sequence PPGIPGRNGDP (SEQ ID No: 32) or PGIPGRNGDPG (SEQ ID No: 33) and / or for the truncated amino acid sequence GIPGRNGDP (SEQ ID No: 34) or PGIPGRNGD (SEQ ID No: 35) is at least 10 to 1, and more preferably at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1.

[0071] Monoclonal antibodies that specifically bind to the amino acid sequence PGIPGRNGDP (SEQ ID No: 2) can be produced by any suitable technique known in the art. For example, monoclonal antibodies can be produced against synthetic peptides containing the amino acid sequence PGIPGRNGDP (SEQ ID No: 2) or composed thereof, for example by: immunizing rodents (or other suitable mammals) with a synthetic peptide composed of the sequence PGIPGRNGDP (SEQ ID No: 2), said synthetic peptide optionally being linked to an immunogenic carrier protein (such as keyhole hemocyanin), isolating and cloning individual antibody-producing cells, and measuring the resulting monoclonal antibodies to ensure they have the desired specificity.

[0072] Immunoassay kits suitable for measuring P3NP levels are available commercially, such as those from Nordic Biosciences (cat# 1090BD01).

[0073] The method can measure the levels of one, two, three, or four fibroblast activation markers. The method can measure the level of Pro-C3, and optionally measure the levels of one, two, or three other fibroblast activation markers. The method can measure the levels of Pro-C3 and Pro-C6, Pro-C3 and Pro-C1, or Pro-C3 and P3NP. The method can measure the levels of Pro-C3, Pro-C6, and Pro-C1, or Pro-C3, Pro-C6, and P3NP.

[0074] The method can measure the level of Pro-C6, and optionally the levels of one, two, or three other fibroblast activation markers. The method can measure the levels of Pro-C6 and Pro-C1, or Pro-C6 and P3NP. The method can measure the levels of Pro-C6, Pro-C1, and P3NP.

[0075] The method can measure the level of Pro-C1, and optionally one, two, or three other fibroblast activation markers. The method can measure the levels of Pro-C1 and P3NP.

[0076] The method can measure the level of P3NP, as well as the levels of one, two, or three other fibroblast activation markers.

[0077] The method can measure the levels of Pro-C3, Pro-C6, Pro-C1, and P3NP.

[0078] In a preferred embodiment, the patient sample is a biological fluid. The biological fluid can be, but is not limited to, serum, plasma, urine, amniotic fluid, tissue supernatant, or cell supernatant. Preferably, the biological fluid is blood, serum, or plasma.

[0079] Immunoassays can be, but are not limited to, competitive or sandwich assays. For example, an immunoassay can be a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques well known to those skilled in the art.

[0080] In a preferred embodiment, the immunoassay is a competitive immunoassay. In a preferred embodiment, the competitive immunoassay is a radioimmunoassay, a fluorescence immunoassay, or an enzyme-linked immunosorbent assay (ELISA). Competitive immunoassays, including those performed using radioimmunoassays, fluorescence immunoassays, or ELISA, are methods and techniques well known to those skilled in the art.

[0081] 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). Sandwich immunoassays, including those performed by radioimmunoassay, fluorescence immunoassay, or ELISA, are methods and techniques well known to those skilled in the art. It is well known that in sandwich immunoassays, at least two antibodies are used to detect antigens in a sample, wherein at least one antibody acts as a capture antibody and is typically bound to a solid support, and at least one other antibody acts as a detection antibody. In the case of this invention, a monoclonal antibody can act as either a capture antibody or a detection antibody.

[0082] As described above, monoclonal antibodies can be bound to a solid support. Any suitable form of solid carrier and binding method known in the art can be used. For example, in a particular exemplary embodiment, the monoclonal antibody can be biotinylated and bound to a streptavidin-coated solid support.

[0083] In an alternative implementation, the monoclonal antibody may be labeled to allow detection of the monoclonal antibody and determination of the amount of binding between the monoclonal antibody and a fibroblast activation marker, such as PRO-C3, PRO-C6, PRO-C1, or P3NP, which binds to a first other monoclonal antibody on a solid support.

[0084] For example, in a preferred embodiment, the monoclonal antibody may be an enzyme-linked antibody. The enzyme may be, but is not limited to, horseradish peroxidase (HRP).

[0085] In another preferred embodiment, the monoclonal antibody may be radiolabeled or linked to a fluorophore.

[0086] Although these are the preferred markers used in this invention, it is conceivable that any suitable marker system can be used, such as, but not limited to, DNA reporter genes or electrochemiluminescent tags.

[0087] Alternatively, a labeled second antibody that recognizes the monoclonal antibody can be used to detect the monoclonal antibody, and the binding amount between the second monoclonal antibody and a fibroblast activation marker bound to a first monoclonal antibody on a solid support can be determined. The second labeled antibody can be labeled using the markers described above.

[0088] As used herein, the term "binding amount" refers to the quantification of the binding between a monoclonal antibody and a fibroblast activation marker (e.g., PRO-C3 (PIIINP), PRO-C6, PRO-C1, or P3NP) from a sample. This quantification can be determined, for example, by comparing a measurement of the binding of a fibroblast activation marker from a patient sample with a calibration curve generated using measurements of the binding of a fibroblast activation marker from a standard sample comprising the supernatant from a "Scar-in-a-jar" (SiaJ) model in which healthy primary human lung fibroblasts are cultured in the presence of sucrose and transforming growth factor-β (TGF-β), thereby determining the quantification of the fibroblast activation marker in the patient sample. The fibroblast activation marker may be selected from PRO-C3 (PIIINP), PRO-C6, PRO-C1, and / or P3NP. In the examples described below, an ELISA method is used, wherein spectrophotometric analysis is used to measure the binding amount from the patient sample and when the calibration curve is generated. However, any suitable analytical method may be used.

[0089] As used herein, the term "predetermined cutoff value" means a binding amount that is statistically determined to indicate a high probability of a patient responding to anti-TGFβI therapy, because the measured value of the target peptide in the patient sample is equal to or higher than the statistical cutoff value, corresponding 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%.

[0090] As used herein, the term "correlation value with normal healthy subjects" refers to the standardized binding amount of samples from subjects considered healthy, i.e., without disease, measured by the methods described above, such as those without cancer or a specific cancer of interest; the term "correlation value with known responders of anti-TGFβI therapy" refers to the standardized binding amount of samples from patients known to have responded positively to anti-TGFβI therapy (i.e., patients who have shown significant improvement after treatment with anti-TGFβI drugs), measured by the methods described above. "Significant improvement" can refer to symptom relief or disappearance, tumor regression, improved prognosis, or changes in biomarkers indicating disease severity, thus indicating a reduction in disease severity.

[0091] Therefore, in these embodiments, the method may further include administering anti-TGFβI therapy to a patient identified as potentially responsive to anti-TGFβI therapy. Suitable therapies include administering a neutralizing antibody targeting TGFβI.

[0092] Patients are preferably subjects with cancer and / or fibrosis. 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, II, III, or IV. The tumor may be a primary tumor or a metastatic tumor.

[0093] In a second aspect, the present invention provides a pharmaceutical agent that binds to the EMI domain of TGFβI or periosteum protein or an analogue thereof.

[0094] As used herein, "EMI domain" refers to the EMI domain of periosteum protein or TGFβI, or its analogues. Specifically, "EMI domain" refers to the 56-amino acid EMI domain (bases 40-94 of the protein sequence) from periosteum protein or the 56-amino acid EMI domain (bases 45-99 of the protein sequence) from TGFβI. Preferably, the EMI domain from periosteum protein comprises or is substantially composed of the following sequence: GPNVCALQQILGTKKKYFSTCKNWYKKSICGQKTTVLYECCPGYMRMEGMKGCPA (SEQ ID No: 36).

[0095] Preferably, the EMI domain from TGFβI contains or is substantially composed of the following sequence: GPNVCAVQKVIGTNRKYFTNCKQWYQRKICGKSTVISYECCPGYEKVPGEKGCPA (SEQ ID No: 37).

[0096] Analogs of the EMI domain used in this invention may include proteins comprising sequences similar to the amino acid sequences defined in SEQ ID No: 36 or SEQ ID No: 37, and having the same biological effects, namely, markers that increase fibroblast activation levels, such as increased levels of Pro-C3, Pro-C6, Pro-C1, and / or P3NP. For example, an EMI domain analog may have at least 70%, 80%, 90%, or at least 95% similarity to a sequence or fragment of a sequence defined in SEQ ID No: 36 or SEQ ID No: 37. Alternatively, an EMI domain analog may have at least 70%, 80%, 90%, or at least 95% identity to a sequence or fragment of a sequence defined in SEQ ID No: 36 or SEQ ID No: 37. For example, EMI domain analogs may contain the same amino acid sequence as 50, 51, 52, 53, 54, 55, 56, or 57 amino acids as defined in SEQ ID No: 36 or SEQ ID No: 37. The similar or identical amino acids may be sequential or discontinuous.

[0097] Amino acid sequences can be compared using programs such as CLUSTAL. This program compares amino acid sequences and finds the best alignment by inserting appropriate gaps in either sequence. Amino acid identity or similarity (identity plus conservation of amino acid types) can be calculated to obtain the best alignment. Programs like BLASTx align the longest similar sequence fragments and assign a fit value. Therefore, alignments can be performed to find multiple similar regions, each with a different score. This invention covers both types of analysis. It is preferred to calculate identity or similarity over the full length of SEQ ID No: 36 or SEQ ID No: 37.

[0098] Analogs of the EMI domain may contain one or more amino acid substitutions, insertions, and / or deletions.

[0099] Amino acid substitution refers to replacing an amino acid residue at the same position with another amino acid residue. Amino acid substitution can be conserved, meaning that the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art can understand which amino acids have similar chemical properties. For example, the following groups of amino acids have similar chemical properties, such as size, charge, and polarity: 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.

[0100] The inserted amino acid residues can be inserted at any position, and some or all of the inserted amino acid residues can be adjacent to each other, or none of the inserted amino acid residues can be adjacent to other inserted amino acid residues. For example, EMI domain analogs may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at the N-terminus and / or C-terminus of the amino acid sequence defined in SEQ ID No: 36 or SEQ ID No: 37.

[0101] 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 any position in SEQ ID No: 36 or SEQ ID No: 37.

[0102] The inserted and substituted amino acids can be naturally occurring or non-naturally occurring, for example, they may contain non-natural side chains and / or be linked together by non-natural peptide bonds. Such modified peptide ligands are known in the art. If multiple amino acid residues are substituted and / or inserted, the substituted / inserted amino acid residues can be the same as or different from each other. Each substituted amino acid can have a different side chain than the substituted amino acid.

[0103] Analogs of the EMI domain may contain one or more modified bases, where the amino acid residues may be chemically modified. Examples of chemical modifications include post-translational modifications such as phosphorylation, acetylation, and deamidation. Chemical modifications may differ from those present in vivo. For example, the N-terminus or C-terminus of an EMI domain peptide may be modified to improve peptide stability, bioavailability, and / or affinity. Other examples of non-natural modifications include incorporation of non-coding α-amino acids, photoreactive cross-linking of amino acids, N-methylated and β-amino acids, backbone reduction, reversal using d-amino acids, N-terminal methylation, C-terminal amidation, and polyethylene glycol modification.

[0104] Preferably, the agent 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.

[0105] The present invention relates to a method for generating a monoclonal antibody that specifically binds to an amino acid sequence containing an EMI domain, comprising generating a monoclonal antibody against an amino acid sequence comprising or composed of a synthetic peptide containing an EMI domain.

[0106] The synthetic peptide may contain 5-15 consecutive amino acids of the EMI domain. The synthetic peptide may contain or consist of an amino acid sequence common to the periosteum protein and the TGFβI EMI domain. The synthetic peptide may contain or consist of an amino acid sequence forming the periosteum protein or the TGFβI EMI domain, wherein the amino acid sequence forming the periosteum protein or the TGFβI EMI domain originates from a region with a high percentage of homology between the two sequences, i.e., a region where the amino acid sequences of the periosteum protein and the TGFβI EMI domain are highly similar or identical. Preferably, the synthetic peptide has at least 70%, 80%, 90%, or at least 95% similarity or identity to the periosteum protein and / or the TGFβI EMI domain. The similar or identical amino acids may be continuous or discontinuous. Preferably, the synthetic peptide has 100% similarity or identity to the periosteum protein and / or the TGFβI EMI domain.

[0107] Preferably, the synthetic peptide contains or is composed of an EMI domain active site.

[0108] Preferably, the synthetic peptide comprises GPNVCAX1Q (SEQ ID NO: 38), wherein X1 is V or L, or YECCPGY (SEQ ID NO: 39), or GX2KGCPA (SEQ ID NO: 40), wherein X2 is E or M.

[0109] For example, the method may include immunizing a non-human mammal with an amino acid sequence containing an EMI domain or a synthetic peptide composed thereof; isolating spleen cells from the immunized mammal that produce antibodies that specifically bind to the amino acid sequence of the EMI domain; fusing the isolated spleen cells with hybridoma cells; and culturing the resulting hybridoma cells to ensure monoclonal growth.

[0110] Preferably, the synthetic peptide (via any suitable type of linker) is linked to the immunogenic carrier protein.

[0111] The present invention also relates to a method for screening antibodies that specifically bind to amino acid sequences of EMI domains, comprising contacting a sample containing one or more antibodies with a synthetic peptide containing or composed of EMI domains, and detecting the binding between the antibody and the peptide.

[0112] In a preferred embodiment, the synthetic peptide is an amino acid sequence comprising an EMI domain or a peptide composed of an amino acid sequence comprising an EMI domain, which is linked (through any suitable type of connector) to a solid support and / or linked to a molecule (e.g., biotin) that allows attachment to the solid support. In a particularly preferred embodiment, the synthetic peptide is a peptide composed of an amino acid sequence comprising an EMI domain, which is linked (through any suitable type of connector) to a solid support and / or linked to a molecule (e.g., biotin) that allows attachment to the solid support.

[0113] In a preferred embodiment, the binding between the antibody and the peptide is detected by enzyme-linked immunosorbent assay (ELISA).

[0114] In a preferred embodiment, hybridoma cells that generate antibodies that specifically bind to the amino acid sequence of the EMI domain are generated by the following method: immunizing a non-human mammal with a synthetic peptide containing the amino acid sequence of the EMI domain or derived therefrom; isolating spleen cells from the immunized mammal that generates antibodies that specifically bind to the amino acid sequence of the EMI domain; fusing the isolated spleen cells with hybridoma cells; and culturing the resulting hybridoma cells to ensure monoclonal growth.

[0115] This invention relates to a monoclonal antibody that specifically binds to an 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 generated against a synthetic peptide containing or composed of an amino acid sequence of the EMI domain.

[0116] This invention relates to a method for generating a monoclonal antibody that specifically binds to the amino acid sequence 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), the method comprising generating a monoclonal antibody against a synthetic peptide comprising the amino acid GPNVCAX1Q (SEQ ID NO: 38), YECCPGY (SEQ ID NO: 39), or GX2KGCPA (SEQ ID NO: 40) or composed thereof.

[0117] For example, the method may include immunizing a non-human mammal with a synthetic peptide comprising the amino acid sequence GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40) or composed thereof; isolating from the immunized mammal spleen 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); fusing the isolated spleen cells with hybridoma cells; and culturing the resulting hybridoma cells to ensure monoclonal growth.

[0118] Preferably, the synthetic peptide is a peptide comprising the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38) and / or YECCPGY (SEQ ID NO: 39) and / or GX2KGCPA (SEQ ID NO: 40) or composed thereof, said peptide being linked to an immunogenic carrier protein (through any suitable type of adapter).

[0119] 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 aspect and detecting the binding between the antibody and the peptide.

[0120] In a preferred embodiment, the synthetic peptide is a peptide comprising or composed of the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38), and / or YECCPGY (SEQ ID NO: 39), and / or GX2KGCPA (SEQ ID NO: 40), said peptide being linked (through any suitable type of connector) to a solid support and / or linked to a molecule (e.g., biotin) that allows attachment to the solid support. In a particularly preferred embodiment, the synthetic peptide is a peptide comprising the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38), and / or YECCPGY (SEQ ID NO: 39), and / or GX2KGCPA (SEQ ID NO: 40), said peptide being linked (through any suitable type of connector) to a solid support and / or linked to a molecule (e.g., biotin) that allows attachment to the solid support.

[0121] In a preferred embodiment, the binding between the antibody and the peptide is detected by enzyme-linked immunosorbent assay (ELISA).

[0122] In a preferred embodiment, hybridoma cells that generate 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 generated by a method comprising the following steps: immunizing a non-human mammal with a synthetic peptide containing the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38), and / or YECCPGY (SEQ ID NO: 39), and / or GX2KGCPA (SEQ ID NO: 40) or composed thereof; isolating from the immunized mammal spleen cells that generate 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 culturing the resulting hybridoma cells to ensure monoclonal growth.

[0123] This invention relates to monoclonal 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). Preferably, the monoclonal antibody is a monoclonal antibody generated against a synthetic peptide comprising the amino acid sequences GPNVCAX1Q (SEQ ID NO: 38), and / or YECCPGY (SEQ ID NO: 39), and / or GX2KGCPA (SEQ ID NO: 40) or composed thereof.

[0124] Monoclonal antibodies can be produced by generating monoclonal antibodies that target a synthetic peptide containing or composed of an EMI domain active site. Monoclonal antibodies can be produced by suitable techniques known to those skilled in the art, such as, but not limited to, immunizing mice or other non-human mammals, isolating spleen cells (B cells) from the immunized mammal and fusing them with hybridoma cells, and then culturing the resulting hybridoma cells to ensure monoclonal growth.

[0125] For example, monoclonal antibodies can be generated from the B cell clones described in the examples. Preferably, the monoclonal antibody is a humanized antibody containing human frame region and / or constant region sequences. Such antibodies can be generated, for example, from mice (or other non-human mammals) with transgenic human immunoglobulin genes, or by generating chimeric antibodies, thereby first generating mouse (or other non-human mammal) monoclonal antibodies, then determining the antigen-binding sequence (containing at least a CDR) of said antibody, and then generating a recombinant antibody containing said antibody-binding sequence grafted onto human frame region and / or constant region sequences.

[0126] This article also provides a method for screening antibodies that specifically bind to the active site of the EMI domain.

[0127] Methods for screening antibodies that specifically bind to the active site of the EMI domain may include contacting a sample containing one or more antibodies with a synthetic peptide containing or composed of the active site of the EMI domain, and detecting the binding between the antibody and the peptide.

[0128] Binding can be detected by enzyme-linked immunosorbent assay (ELISA). Various suitable forms of ELISA are known in the art and can be used appropriately. For example, the ELISA technique described in the examples can be used, thereby contacting a fluid sample containing one or more antibodies with a solid support that binds to a peptide containing or composed of an EMI domain active site, thereby allowing binding between the peptide and any antibody specific to the EMI domain active site. After removing any unbound antibodies, an enzyme-linked antibody can then be added, which will bind any antibody that binds to the solid support via the EMI domain active site or the peptide composed of it. The activity of the enzyme can then be assessed by incubation with a substrate that produces a measurable product, thereby allowing quantitative detection of any antibody bound to the EMI domain active site or the peptide composed of it that binds to the solid support.

[0129] The aforementioned monoclonal antibodies targeting the EMI domain 2 active site can inhibit the fibrotic effect of TGFβI by binding to and blocking its entry into the EMI active site (thereby eliminating TGFβI-induced fibrosis). In addition to this direct neutralization effect, monoclonal antibodies with an Fc region can also eliminate fibrosis through Fc-mediated antibody binding and clearance of EMI / TGFβI. Therefore, these antibodies can be used to treat fibrotic diseases and pathological fibrosis.

[0130] However, monoclonal antibodies targeting other parts of EMI can also be used to eliminate the effects of TGFβI. In particular, even if such antibodies may not block entry into the EMI active site, they can be used to influence Fc-mediated TGFβI clearance.

[0131] In a third aspect, the present invention provides a pharmaceutical agent for a method of treating cancer and / or fibrosis, representing a second aspect of the invention. Therefore, the present invention provides a pharmaceutical agent that binds to the EMI domain of TGFβI or an analogue thereof for 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 analogue thereof for the treatment of cancer.

[0132] 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, II, III, or IV. The tumor may be a primary tumor or a metastatic tumor.

[0133] Preferably, the agent is used to treat subjects who have been identified as more likely to respond to TGFbI treatment. In a more preferred embodiment, the subject has elevated levels of fibroblast activation markers, which are identified by the method according to the first aspect and described herein. Fibroblast activation markers may be selected from Pro-C3, Pro-C6, Pro-C1, and / or P3NP.

[0134] In another aspect, the present invention provides a pharmaceutical agent that binds to the EMI domain of TGFβI or an analogue thereof for use in medicine. In a preferred embodiment, the present invention relates to a monoclonal antibody that specifically binds to the EMI domain or an analogue thereof for use in medicine. Attached Figure Description

[0135] Figure 1 (a) Shows multiple sequence alignments of the EMI domain. The highlighted parts in the boxes are periosteal protein (POSTN_40-94) and TGFBI (TGFBI_40-99).

[0136] (b) The amino acid sequence of the EMI domain, which is derived from: TGFBI ((https: / / www.uniprot.org / uniprot / Q15582 bases 40-94) and Periosteal protein (https: / / www.uniprot.org / uniprot / Q15063 bases 40-99).

[0137] Figure 2Manhattan plot showing the association between genome-wide associations and log2 (PRO-C3) levels and eight other liver-related biomarkers and scores. An optimized linear additive regression model was used for 4968 PERF participants, corrected for baseline age, three principal components, serum and blood biochemical parameters identified by elasticity net, and PRO-C3 measurement batches. Using a linear additive regression model, corrected for baseline age and three principal components, log2 conversion levels of ADAPT, FIB4, APRI, and NFS disease scores, as well as liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), and gamma-glutamyl transferase (GGT) were analyzed. The red line represents the Bonferroni-corrected genome-wide significance threshold of 6.5. e-9 The blue line represents the suggestive association threshold, which is 1.3. e-7 .

[0138] Figure 3 The assay showed that biomarkers PRO-C1 (type I collagen propeptide), PRO-C3 (type III collagen propeptide), and PRO-C6 (type VI collagen propeptide) were measured in CAF supernatant to reflect fibrotic activity and collagen synthesis. The fold change between day 3 and day 9 was compared among different doses of TGFBI treatment.

[0139] Figure 4 The levels of the biomarker PRO-C3 (type III collagen precursor) were measured in pancreatic fibroblast supernatant to reflect fibrotic activity and collagen synthesis. Biomarker levels were measured on day 12 after treatment with different doses of TGF-β, TGFBI, and EMI domains.

[0140] Figure 5 shows the effect of elevated BigH3 (TGFBI) levels on the production of fibroblast activation markers in pancreatic fibroblasts. Figure 5A The Pro-C1 level was displayed; Figure 5B The Pro-C3 level was displayed; Figure 5C The Pro-C6 level was displayed; Figure 5D The P3NP level is displayed.

[0141] Example The currently disclosed embodiments are described in the following examples. These examples are provided to aid in understanding this disclosure and should not be construed as limiting the scope of this disclosure in any way, which is defined by the following claims. The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of this disclosure, nor to represent that the experiments described below are all or only those experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise stated, portions refer to parts by weight, molecular weight refers to weight-average molecular weight, temperature refers to degrees Celsius, and pressure refers to atmospheric pressure or near atmospheric pressure.

[0142] Materials and methods reagents Unless otherwise stated, all reagents used in the experiments were standard chemicals from Merck (Whitehouse Station, NJ, USA) or Sigma Aldrich (St. Louis, MO, USA). The synthetic immunogenic peptide ovalbumin (OVA)-CGG-CPTGPQNYSP used for monoclonal antibody production was purchased from Chinese Peptide Company (Beijing, China).

[0143] Production of Pro-C3 monoclonal antibodies Pro-C3 monoclonal antibody production was performed as previously reported

[23] (see also WO2014 / 170312A1). In short, a novel Pro-C3 epitope (as described above, a novel C-terminal epitope of PIIINP) was selected as the target, and an antibody specific to the target was generated using the amino acid sequence 144'-CPTGPQNYSP-'153 (SEQ ID NO: 4) in the α1 chain of PIIINP. The sequence was compared for homology with rat and mouse and for uniqueness with other human proteins using NPS@: network protein sequence analysis and the Uniprot / Swiss-Prot database

[24] . The sequence CPTGPQNYSP (SEQ ID NO: 4) was found to be unique to PIIINP.

[0144] As described above, the monoclonal antibody was generated, cloned and characterized. It was found that the monoclonal antibody was specific to the target sequence CPTGPQNYSP (SEQ ID No: 4) and did not recognize or bind the extended (CPTGPQNYSPQ) (SEQ ID No: 5) or nonsense peptide (GSPGKDGVRG (SEQ ID NO: 41))

[22] .

[0145] The supernatant from antibody-producing hybridoma cells was collected, and the monoclonal antibodies were purified using a HiTrap protein-G column (GE healthcareLife Sciences, Little Chalfont, Buckinghamshire, UK) and then purified using Lightning-Link according to the manufacturer's instructions. TM Rapid Biotin Conjugation Kit (Type B) (InnovaBiosciences) or Lightning-Link TM HRP Conjugation Kit (Innova Biosciences) label.

[0146] The generated antibodies were sequenced and CDRs were determined.

[0147] The sequence of chains is as follows (underlined and bold text indicates CDR, italics indicate constant regions): Heavy chain: amino acid sequence (467aa) Light chain: amino acid sequence (238aa) Pro-C3 Measurement As previously reported, a Pro-C3 assay (pro-C3 competitive ELISA) was performed

[23] (see also WO2014 / 170312A1, which is incorporated herein by reference). In short, the PRO-C3 competitive ELISA procedure is as follows: 96-well streptavidin-coated ELISA plates (from Roche, catalog number 11940279) were coated with biotinylated peptide biotin-CGGPTGPQNYSP (SEQ ID No: 4) dissolved in coating buffer (50 mM PBS-BTE + 10% sorbitol, pH 7.4), incubated in the dark at 20°C for 30 min, and then washed in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Subsequently, 20 μl of peptide calibrator or sample was added to the appropriate well, followed by 100 μl of HRP-conjugated Pro-C3 monoclonal antibody (monoclonal antibody NB61N-62) dissolved in incubation buffer (50 mM PBS-BTB + 10% Liquid (Roche), pH 7.4). The plate was incubated at 4°C for 20 hours and washed. Finally, 100 μl of tetramethylaniline (TMB) (Kem-En-Tec catalog number: 438OH) was added, and the plate was incubated at 20°C in the dark for 15 minutes. 100 μl of stop solution (1% H2SO4) was added to terminate the reaction, and the plate was analyzed at 450 nm using an ELISA reader, with 650 nm as a reference (MolecularDevices, SpectraMax M, CA, USA). Calibration curves were plotted using a 4-parameter mathematical fitting model.

[0148] Antibody development for PRO-C6 As described in WO 2016 / 156526 (Nordic Bioscience, which is incorporated herein by reference), the last 10 amino acids (i.e., the C-terminal sequence) of the α3 chain of type VI collagen are used. 3168' KPGVISVMGT '3177 (SEQ ID No: 10) was used as an immunogenic peptide to develop a PRO-C6 specific monoclonal antibody. Briefly, 4-6 week old Balb / C mice were subcutaneously immunized with 200 μl of emulsified antigen and 60 μg of the immunogenic peptide. Continuous immunizations were performed at 2-week intervals in Freund's incomplete adjuvant until stable serum titer levels were reached, and blood was collected from the mice starting with the second immunization. Serum titers were measured at each bleeding event, and mice with the highest antiserum titer and best natural reactivity were selected for fusion. The selected mice were allowed to rest for 1 month, followed by an intravenous booster immunization with 50 μg of the immunogenic peptide in 100 μl of 0.9% sodium chloride solution. The spleen was isolated 3 days later for cell fusion.

[0149] Mouse spleen cells were fused with SP2 / 0 myeloma cells. Fusion cells were cultured in 96-well plates and incubated in a CO2 incubator. Standard limiting dilutions were used to promote monoclonal growth. Cell lines specific to the selected peptide and without cross-reactivity to either the extended peptide (KPGVISVMGTA (SEQ ID No: 18), Zhongtai Biochemical Co., Ltd., China) or the truncated peptide (KPGVISVMG (SEQ ID No: 19), American Peptide Company, USA) were selected and subcloned. Antibodies were finally purified using an IgG column.

[0150] The generated antibodies were sequenced and CDRs were determined.

[0151] The sequence of the chain is as follows (CDR is indicated by underline and bold): Heavy chain sequence (mouse IgG1 isotype) CDR-H1: DFNMN (SEQ ID No: 23) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID No: 24) CDR-H3: WGNGKNS (SEQ ID No: 25) Light chain sequence (mouse Kappa isotype) CDR-L1: RSSQRIVHSNGITFLE (SEQ ID No: 20) CDR-L2: RVSNRFS (SEQ ID No: 21) CDR-L3: FQGSHVPLT (SEQ ID No: 22) Immunoassay for PRO-C6 PRO-C6 was measured using an enzyme-linked immunosorbent assay (ELISA) developed by Nordic Bioscience, as described in WO2016 / 156526 (which is incorporated herein by reference) and detailed in other publications. In short, the procedures are as follows: The ELISA plates used for assay development were coated with streptavidin from Roche (catalog number: 11940279). All ELISA plates were analyzed using an ELISA reader from Molecular Devices, SpectraMax M (CA, USA). Selected monoclonal antibodies were labeled with horseradish peroxidase (HRP) using the Lightning Link HRP Labeling Kit according to the manufacturer's (Innovabioscience, Babraham, Cambridge, UK) instructions. 96-well streptavidin plates were coated with biotinylated synthetic peptide biotin-KPGVISVMGT (SEQ ID No: 38) (Zhongtai Biochemical Co., Ltd.) dissolved in coating 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. 100 μL of a standard peptide or sample diluted 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 100 μL of HRP-conjugated monoclonal antibody 10A3, and incubated at 4 °C for 21 h. Finally, 100 μL of tetramethylaniline (TMB) (Kem-En-Tec catalog number: 438OH) was added, and the plate was incubated at 20 °C in the dark for 15 min. All the above incubation steps included shaking at 300 rpm. After each incubation step, the plate was washed 5 times in wash buffer (20 mM Tris, 50 mM NaCl). The TMB reaction was terminated by adding 100 μL of stop solution (1% H₂SO₄), and measurements were taken at 450 nm with 650 nm as a reference.

[0152] Immunoassay for Pro-C1 The immunoassay used to detect Pro-C1 was obtained from Nordic Bioscience (catalog number 2800A0E51) and performed according to the manufacturer's instructions.

[0153] Immunoassay for P3NP The immunoassay used to detect P3NP was obtained from Nordic Bioscience (catalog number 1090BD01) and performed according to the manufacturer's instructions.

[0154] Research Design The Prospective Epidemiological Risk Factor (PERF) study is a follow-up study of postmenopausal women in Denmark designed to identify risk factors associated with age-related diseases.

[25] Participants in the PERF study had previously been enrolled in randomized, placebo-controlled clinical trials or were screened rather than randomized in previous studies at the Center for Clinical and Basic Research (CCBR). The enrollment process for study participants was performed in the same manner as described in

[26] . A total of 4,968 participants were included, and their PRO-C3 assays, serum biochemical markers, medical history, and genotype were all included.

[0155] Standard protocol approval, registration, and patient consent This study was conducted in accordance with the International Council for Harmonisation of Technical Requirements for Clinical Trials of Drugs (ICH-GCP), and the study protocol has been approved by the local ethics committee. All participants signed informed consent forms, agreeing to participate in subsequent analyses.

[0156] Baseline measurements and data collection At baseline, participants completed interviews with their physicians or nurses, including questions related to physical health, demographics, lifestyle, and medical history. All participants provided written consent for this specific analysis, and fasting serum and DNA samples were collected.

[0157] As previously mentioned, serum PRO-C3 levels were measured in a blinded manner using enzyme-linked immunosorbent assay (ELISA) in a CAP-certified laboratory. Lymphocyte and neutrophil counts were determined using an automated hematology analyzer (Sysmex).

[0158] Complete inpatient histories of participants for the period (1974–2014) were obtained by linking each individual’s unique personal identification number (CPR number) to the Danish patient registry system as of December 31, 2014 (corresponding to the end of the study). Study participants were anonymized, and their CPR numbers were not available at any point in the study. Patient registry information for 5,602 participants was available.

[0159] Genotyping was performed in collaboration with deCODE Genetics, Iceland, using a custom-designed Illumina Global ScreeningArray (693143 probes). SNP imputation was performed using the Michigan Imputation Server

[27] , with reference group HRCr1.1.2016, EUR. Phase determination was performed using ShapeIt2, and imputation was performed using Minimac3. Locations are as reported in reference GRCh37.

[0160] Disease phenotype definition Based on data from multiple sources, we defined 18 disease phenotypes as the full-time incidence rate of an event: biochemical marker levels, physiological measurements, full-time incidence rate hospital records, mortality registries, and questionnaires at baseline and during follow-up. A detailed list of included phenotypes and their inclusion criteria are provided in Supplementary Table 2.

[0161] Genotype data preprocessing Standard probe level filtering was performed using a minimum probe recall rate of 97%, minor allele frequencies were greater than or equal to 1%, and Hardy-Weinberg equilibrium p-value cutoff was greater than or equal to 1e-6. Multi-allele SNPs were not filtered.

[0162] To address potential implicit correlations between subjects, we used the Plink

[18] -genomic function to calculate the identity-by-descent (IBD) coefficient and the plink-ibc function to calculate the inbreeding coefficient (IBC). Using one side of a pair as a baseline, a minimum coefficient of 0.1875 was used. PI_HAT Cutoff value: Remove subjects and apply cutoff values ​​less than -0.1 or greater than 0.1 to the Fhat2 coefficient.

[0163] Principal component analysis The population-based genetic variation in the dataset was captured using EIGENSTRAT Smartpca 7.2.0 [28, 29] to perform iterative principal component analysis (PCA) on the study population with available genotypes for non-input filtering variables using default parameters.

[0164] Robust and Resilient Covariate Selection A robust and optimized variable selection procedure was employed to include variables in the GWAS study. An elastic net with 50 randomizations and 5-fold cross-validation was used to screen variables associated with the log2 level of PRO-C3. The distribution of coefficient estimates was visualized using the standard geom_boxplots tool.

[0165] Linear Regression Linear additive regression was performed on the GWAS study population (n = 4968) using plink v1.90p

[30] (Chang et al. 2015) to identify genetic associations with log2-converted serum PRO-C3 levels, corrected for baseline age and three principal components. Conserved significance thresholds based on the number of selected variants for genome-wide and suggestive values ​​were defined as equal to 6.5e-9 (i.e., 0.05 / N) and 1.3e-7 (i.e., 1 / N, N = 7672338), respectively. The R package was used. qqman

[31] Draw a visualization of the Manhattan diagram.

[0166] Phenotypic Association Analysis Phenotypic association analysis at the log2 (PRO-C3) level was performed using a logistic regression model, adjusted for baseline age and BMI. Statistical significance was corrected using the Benjamini-Hochberg method. forestplot An R library visualizes log2(PRO-C3) as a forest plot, showing the disease-related p-values ​​and beta values.

[0167] Correlation analysis To assess the correlation between PRO-C3 and various liver-related variables and disease scores, Pearson's correlation test was performed. Linearity of biochemical concentrations was considered.

[0168] To assess the correlation between the genetic components associated with PRO-C3 and various liver-related variables and disease scores, genetic correlation analyses were performed

[32] . GWAS analyses were performed for each variable as described above, using a standard model adjusted for baseline age and analyzed using three principal components. Log2 transformations of biochemical measurements were considered in the GWAS analyses. We used LD scores estimated based on PERF genotyping array data.

[0169] Pathway enrichment analysis Pathway enrichment analysis was performed using the VEGAS2

[33] and PARIS2.4

[34] programs with default parameters. The VEGAS2 analysis used Biosystems gene / pathway annotation files provided by the software website. The LOKI knowledge base used by PARIS2.4 was compiled by running a script provided in February 2020. It was reported that there was a significant association with the REACTOME pathway, and p<0.05 with both frameworks.

[0170] Data availability Raw data from prospective epidemiological risk factor studies, along with associated data from various health registries, are currently stored at Nordic Bioscience. Researchers must obtain the appropriate ethical permission and sign the corresponding material transfer agreement before accessing this database.

[0171] Example 1 This study included 4,968 participants from the PERF cohort, whose PRO-C3 levels, serum biomarkers, hospitalization information from the Danish patient registry, and genotypes were measured at baseline. Inclusion criteria have been described in the Methods section and in previous studies

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

[0172] Table 1: Baseline characteristics of participants included in the study

[0173] Disease incidence associated with elevated PRO-C3 levels To elucidate the relationship between disease characteristics and PRO-C3 levels in PERF participants, logistic regression analysis was performed, adjusted for baseline characteristics (age and BMI). The logarithmic level of PRO-C3 was strongly associated with an increased incidence of chronic liver disease (OR = 1.74, 95% CI [1.45–2.09], adjusted p = 1.09e-7), and with chronic kidney disease (OR = 1.26, 95% CI [1.10–1.24], adjusted p = 6.04e-3) and rheumatic diseases (OR = 1.35, 95% CI [1.13–1.63], adjusted p = 6.69). e-3 It is nominally related to ).

[0174] Logistic regression analysis was performed again using PRO-C3 levels divided by quartiles. The results showed that the association between liver and rheumatic diseases and the Q4 PRO-C3 was more specific, while the association between chronic kidney disease and the Q3-Q4 PRO-C3 was more specific. Notably, the accuracy of the fit was moderate, although the p-values ​​were high: AUC = 0.60 for liver and rheumatic diseases and AUC = 0.70 for kidney diseases.

[0175] Genome-wide association study A cross-validation elastic network-based optimization variable selection procedure was used to perform GWAS analysis on baseline, log2-transformed PRO-C3 levels. The model was adjusted based on the log2-transformed values ​​of the following baseline serum and blood biochemical indicators: alkaline phosphatase (AP), aspartate aminotransferase (AST), sodium (NA), and median cytohemoglobin concentration (MCHC), as well as the PRO-C3 measurement batch number. Results are presented in the form of Manhattan plots, as shown below. Figure 2 As shown, Log2(PRO-C3) levels are associated with the TGFBI / VTRNA2-1 locus (major SNP site rs2073511) on chromosome 5. Figure 2 The effect size was 0.070, with a 95% CI of [0.050–0.090] log₂ ng / mL and a p-value of 8.42e⁻¹². The significant variation at this locus was located throughout the entire TGFBI gene region and showed strong linkage.

[0176] Based on the log2-transformed PRO-C3 quartiles, the study population was stratified, and contingency tables were created to show the presence of the rs2073511 allele in these groups. The results indicated an excessively high proportion of the TGFBI leader variant in the top quartiles of PRO-C3 (chi-square p-value: 1.177e-07).

[0177] Correlation with other liver-related biomarkers and scores To better elucidate the relationship between PRO-C3 and chronic liver disease, this study investigated the correlation between PRO-C3 levels and standard biochemical markers associated with liver disease, as well as liver-related disease scores. Pearson correlation analysis was performed to further reveal potential confounding effects of PRO-C3 biomarkers.

[0178] Pearson correlation analysis showed that PRO-C3 levels were moderately correlated with serum levels of alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma-glutamyl transferase (GGT), and aspartate aminotransferase (AST) (Pearson correlation coefficients ranged from 0.13 to 0.19). Regarding disease scoring, PRO-C3 was denoted as the ADAPT score (Pearson correlation 0.83), which was expected as it is a component of the score calculation.

[0179] GWAS analysis was performed on liver biomarkers AP, ALT, AST, GGT, and ADAPT, APRI, FIB4, and NFS scores, and adjustments were made based on baseline age and three principal components. Figure 2 ).

[0180] When examining the genetic associations of variants associated with these different biomarkers and scores

[32] , the genetic profiles associated with PRO-C3, except for ADAPT, showed no correlation with any of the other biochemical biomarkers or disease scores, and the correlation patterns differed from those calculated based on Pearson correlations of levels and scores. These results suggest that the associations among TGFBI variants are specific to PRO-C3.

[0181] Example 2 In vitro studies Pancreatic CAFs were grown to confluence using the previously described “Scar-in-a-jar” (SiaJ) model method (35) (36) and seeded into 96-well plates at a density of 10,000 cells / well, each well containing 200 µl of 10% FBS Dulbecco modified Eagle medium (DMEM) + GlutaMax. On day 2 post-seeding, the supernatant was removed, and cells were treated with the following concentrations of TGFBI: 0 (vector control), 1 ng / ml, 10 ng / ml, and 100 ng / ml per well in 200 µl of 0.4% FBS DMEM, in quadruplicate. On days 3 and 6 post-treatment, the supernatant was removed and the cells were stored at -20°C, then fresh TGFBI treatment solution in 0.4% FBS DMEM was added to the cells. On day 9 post-treatment, the supernatant was removed, the cells were stored, and the experiment was terminated. 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 to reflect fibrotic activity and collagen synthesis at different dates in the SIAJ setting. The fold change between day 3 and day 9 of different doses of TGFBI treatment was compared.

[0182] In another setup, normal pancreatic fibroblasts were grown and seeded (similar to CAFs) and then treated with TGF-β (0.08 nM), TGFBI (5 nM and 100 nM), and the 56-amino acid EMI domain of periosteum protein at the following concentrations: 0 (vector control), 1 nM, 10 nM, and 100 nM per well, quadruplicate, in 200 µl of 0.4% FBS DMEM. At 3, 6, and 9 days after the initial treatment, the supernatant was removed and stored at -20°C, and fresh 0.4% DMEM solution was added to the cells. The experiment was terminated at 12 days after the initial treatment by removing the supernatant. PRO-C3 levels in the supernatant were measured, and the levels at different treatment doses were compared. Native human quiescent pancreatic fibroblasts and pancreatic CAFs were purchased from Neuromics (cat#SC00A05 and cat#CAF08, respectively, US).

[0183] Result: As Figure 3As shown, most notably, a significant dose-dependent increase in PRO-C3 levels was observed in CAFs treated with TGFBI. In CAFs treated with 100 ng / mL TGBI, PRO-C3 levels increased approximately 28-fold from day 3 to day 9, compared to only a 6-fold increase in untreated CAFs, indicating that PRO-C3 levels in CAFs treated with 100 ng / mL TGFBI increased by more than 450% compared to untreated CAFs. A smaller (50%) dose-dependent increase in PRO-C6 levels was also observed in the supernatant of TGFBI-treated CAFs compared to untreated CAFs. No change in PRO-C1 levels was observed between TGFBI-treated and untreated CAFs. Figure 4 As shown in figure a, 5 nM TGFBI induced PRO-C3 levels in normal pancreatic fibroblasts to the same extent as TGF-β (35% and 34% higher than the vector control, respectively), while high levels of TGFBI (100 nM) induced even greater levels in normal pancreatic fibroblasts (50% higher than the vector control). Interestingly, 100 nM of the EMI domain peptide also induced PRO-C3 levels in normal pancreatic fibroblasts (30% higher than the vector control).

[0184] Conclusion: TGFBI stimulation of normal pancreatic fibroblasts and CAFs led to a dose-dependent increase in the synthesis of specific collagens, particularly type III collagen (PRO-C3). These results indicate a direct association between TGFBI, CAF activity, collagen synthesis, and PRO-C3 levels, supporting TGFBI as a novel target for antifibrotic / anticancer drugs. Furthermore, stimulation with EMI domain peptides also increased PRO-C3 levels, suggesting a potential role for the EMI domain of TGFBI, which may clarify the targeting potential of TGFBI for antifibrotic / anticancer drugs. Example 3: Fibroblast Culture (Scar in a Jar) Pancreatic fibroblasts (PF) were cultured in VitroPlus III, low-serum medium (Neuromics cat#PC00B1, Edina, MN, USA) at 37°C and 5% CO2 in rat tail type I collagen-coated flasks (cat# P8188, Innoprot, Derio, Bizkaia, Spain) at 5 μg / cm2. When the cells reached 80% confluence, they were seeded at a density of 15,000 cells / well in 96-well plates in Gibco Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (FBS) (cat#F7524, Sigma Aldrich, St. Louis, MO, USA) and 1% penicillin / streptavidin (P / S) (cat#P4333, Sigma Aldrich, USA) + GlutaMAX (DMEM) (cat# 31966047, Thermo Fisher Scientific, Waltham, MA, USA). Before seeding cells, highly binding 96-well plates (cat#3361, Corning, New York, USA) were coated with PBS, 2% BSA, or different concentrations of BigH3 at 100 μl / well (50 nm, 100 nm, 200 nm, and 400 nm) and incubated at 37°C for 1 h. After coating, the plates were 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 min. 24 hours later (Day 0), the culture medium was replaced with Ficoll medium (DMEM containing 0.4% FBS, 1% P / S, 56.25 mg / mL 70 kDa Ficoll (cat# 17-0310-50, GE Healthcare, Chicago, IL, USA), 37.5 mg / mL 400 kDa Ficoll (17-0300-50, GE Healthcare, Chicago, IL, USA), and 0.05 mg / mL L-ascorbic acid (cat# A9256, Sigma Aaldrich, St. Louis, MO, USA)). The supernatant was removed and frozen every three days, and frozen once at the end of the experiment (Day 12).

[0185] Evaluation of soluble biomarkers According to the manufacturer's instructions, BigH3 (Abcam, cat# ab220651) and PRO-C3 (Nordic Bioscience, cat#1700AF06) were blindly tested in serum by enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunosorbent assay (CLIA). Similarly, according to the manufacturer's instructions, PRO-C1 (Nordic Bioscience, cat# 2800A0E51), PRO-C6 (Nordic Bioscience, cat#4000AF02), and P3NP (Nordic Bioscience, cat# 1090BD01) were blindly tested in serum by enzyme-linked immunosorbent assay (ELISA).

[0186] BIGH3 is an inducer of PRO-C3 (and other collagen) in pancreatic fibroblasts. After establishing the correlation between BigH3 and PRO-C3, experiments were conducted to determine whether BigH3 was also an inducer of PRO-C3 in pancreatic fibroblasts cultured in vitro in a Scar-in-a-jar model. As shown in Figure 5, incubation of pancreatic fibroblasts with increasing BigH3 doses (50 nM to 400 nM) led to a dose-dependent increase in PRO-C3. Similar phenomena were observed in the type I collagen synthesis biomarker PRO-C1 and the type VI collagen synthesis biomarker PRO-C6, as well as the classic fibrosis marker P3NP (Figure 5). In summary, this indicates that BigH3 is an inducer of PRO-C3 and other collagens in pancreatic fibroblasts, suggesting that BigH3 may be a driver of fibroblast activation to produce more collagen and fibrosis. Importantly, the measurement of the induction of type III collagen synthesis using PRO-C3 directly links this to the initial findings of the relationship between BigH3 SNPs and circulating PRO-C3 levels. Furthermore, it suggests that BIGH3 may be an important therapeutic target for the regulation of tumor fibrosis and cancer-associated fibroblasts in cancer patients with high PRO-C3.

[0187] In this specification, unless otherwise expressly stated, the word “or” refers to the operator that returns a truth value when any one or both of the stated conditions are met, not the operator “exclusive or” that requires only one of the conditions to be met. The word “contains” is used to mean “includes or consists of”. All prior teachings acknowledged above are incorporated herein by reference. No previously published document herein should be construed as an acknowledgment or representation that its teachings are common knowledge in Australia or elsewhere as of the date of this report.

[0188] References 1. Jenkins RG, et al. Longitudinal change in collagen degradationbiomarkers in idiopathic pulmonary fibrosis: an analysis from theprospective, multicentre PROFILE study. Lancet Respir Med. 2015a;3:462–72. 2. Organ LA, et al. Biomarkers of collagen synthesis predictprogression in the PROFILE idiopathic pulmonary fibrosis cohort. RespirRes.2019;20:148. 3. Kristensen JH, et al. Levels of circulating MM P-7 degradedelastin are elevated in pulmonary disorders. Clin Biochem. 2015;48:1083–8. 4. Leeming DJ, et al. Serological investigation of the collagendegradation profile of patients with chronic obstructive pulmonary disease oridiopathic pulmonary fibrosis. Biomark Insights. 2012;7:119–26. 5. Karsdal MA, et al. The good and the bad collagens of fibrosis—their role in signalling and organ function. Adv Drug Deliv Rev.2017;121:43–56. 6. Bao X, Zeng Y, Wei S, Wang G, Liu C, Sun Y, Chen Q, and Li H.Developmental changes of Col3a1 mRNA expression in muscle and theirassociation with intramuscular collagen in pigs. J Genet Genomics 2007; 34(3): 223-228. 7. Jensen LT and Host NB. Collagen: scaffold for repair or execution.Cardiovasc Res 1997; 33(3): 535-539. 8. Niemela O, Risteli L, Parkkinen J, and Risteli J. Purification andcharacterization of the N-terminal propeptide of human type III procollagen.Biochem J 1985; 232(1): 145-150. 9. Wang WM, Ge G, Lim NH, Nagase H, and Greenspan DS. TIMP-3 inhibitsthe procollagen N-proteinase ADAMTS-2. Biochem J 2006; 398(3): 515-519. 10. Goehrig D et al. Stromal protein βig-h3 reprogramM es tumourmicroenvironment in pancreatic cancer [Internet]. Gut 2019;68(4):693–707. 11. Sato T et al. Identification and characterization of transforminggrowth factor beta-induced in circulating tumor cell subline from pancreaticcancer cell line [Internet]. Cancer Sci. 2018;109(11):3623–3633. 12. Nacu N et al. Macrophages produce TGF-beta-induced (beta-ig-h3)following ingestion of apoptotic cells and regulate MM P14 levels andcollagen turnover in fibroblasts [Internet]. J. ImM unol. 2008;180(7):5036–5044. 13. Tachmazidou I, Hatzikotoulas K, Southam L, et al. Identificationof new therapeutic targets for osteoarthritis through genome-wide analyses ofUK Biobank data. Nat Genet 2019;51:230–6. doi:10.1038 / s41588-018-0327-1 14. Nielsen MJ, Veidal SS, Karsdal MA, et al. Plasma Pro-C3 (N-terminal type III collagen propeptide) predicts fibrosis progression inpatients with chronic hepatitis C. Liver Int 2015;35:429–37. 15. Daniels SJ, Leeming DJ, Eslam M, et al. ADAPT: An AlgorithmIncorporating PRO-C3 Accurately Identifies Patients With NAFLD and AdvancedFibrosis. Hepatology 2019;69. doi:10.1002 / hep.30163 16. Nielsen MJ, Nedergaard AF, Sun S, et al. The neo-epitope specificPRO-C3 ELISAmeasures true formation of type III collagen associated withliver and muscle parameters. Am J Transl Res 2013;5:303–15. 17. Hirschfield GM, Chazouillères O, Drenth JP, et al. Effect ofNGM282, a FGF19 analogue, in Primary Sclerosing Cholangitis: a Multicentre,Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial. J Hepatol 2018. 18. Bril F, Leeming DJ, Karsdal MA, et al. Use of plasma fragments ofpropeptides of Type III, V, and VI procollagen for the detection of liverfibrosis in type 2 diabetes. Diabetes Care 2019;42. doi:10.2337 / dc18-2578 19. Kai Ruan, et al. The multifaceted role of periostin intumorigenesis Cell Mol Life Sci. 2009 Jul;66(14):2219-30 doi: 10.1007 / s00018-009-0013-7 20. Deane F Mosher et al. Periostin and TGF-β-induced protein: Twopeas in a pod? Crit Rev Biochem Mol Biol. 2015;50(5):427-39. doi: 10.3109 / 10409238.2015.1069791. 21. Stefano Marastoni, et al. EMILIN2 down-modulates the Wntsignalling pathway and suppresses breast cancer cell growth and migration. JPathol. 2014 Mar;232(4):391-404. doi: 10.1002 / path.4316. 22. Nielsen MJ, Nedergaard AF, Sun S, et al. The neo-epitopespecific PRO-C3 ELISAmeasures true formation of type III collagen associatedwith liver and muscle parameters. Am J Transl Res. 2013;5(3):303-315. 24. Combet C, Blanchet C, Geourjon C, Deléage G. NPS@: networkprotein sequence analysis. Trends Biochem Sci. 2000;25(3):147-150. 25. Neergaard JS, Dragsbæk K, Kehlet SN, et al. Cohort Profile: TheProspective Epidemiological Risk Factor (PERF) Study. Int J Epidemiol 2016;:1–10. doi:10.1093 / ije / dyw251 26. Tang M-HE, Blair JPM, Bager CL, et al. Matrix metalloproteinase-degraded type I collagen is associated with APOE / TOMM 40 variants andpreclinical dementia. Neurol Genet 2020;6:e508. doi:10.1212 / NXG.0000000000000508 27. Das S, Forer L, Schönherr S, et al. Next-generation genotypeimputation service and methods HHS Public Access Author manuscript. Nat Genet2016;48:1284–7. doi:10.1038 / ng.3656 28. Price AL, Patterson NJ, Plenge RM, et al. Principal componentsanalysis corrects for stratification in genome-wide association studies. NatGenet 2006;38:904–9. doi:10.1038 / ng1847 29. Patterson N, Price AL, Reich D. Population structure andeigenanalysis. PLoS Genet 2006;2:190. doi:10.1371 / journal.pgen.0020190 30. Chang CC, Chow CC, Tellier LC, et al. Second-generation PLINK:rising to the challenge of larger and richer datasets. 2015;4:7. doi:10.1186 / s13742-015-0047-8 31. Turner SD. qqman: an R package for visualizing GWAS resultsusing Q-Q and manhattan plots. bioRxiv 2014;:005165. doi:10.1101 / 005165 32. Bulik-Sullivan B, Finucane HK, Anttila V, et al. An atlas ofgenetic correlations across human diseases and traits. Nat Genet 2015;47:1236–41. doi:10.1038 / ng.3406 33. Mishra A, Macgregor S. VEGAS2: Software for more flexible gene-based testing. Twin Res Hum Genet 2015;18:86–91. doi:10.1017 / thg.2014.79 34. Butkiewicz M, Cooke Bailey JN, Frase A, et al. Pathway analysisby randomization incorporating structure—PARIS: an update. Bioinformatics2016;32:2361–3. doi:10.1093 / bioinformatics / btw130 35. Rønnow SR et al. Prolonged Scar-in-a-Jar: an in vitro screeningtool for anti-fibrotic therapies using biomarkers of extracellular matrixsynthesis. Respir. Res. 2020;21(1):108. 36. Nissen NI, Karsdal M, Willumsen N. Abstract 3958: Establishmentof an in vitro model to study cancer associated fibroblasts and their directeffect on desmoplasia [Internet]. In: Cancer Research. American Associationfor Cancer Research (AACR); 2020:3958–3958.

Claims

1. A method for identifying patients more likely to respond to TGFβI therapy, the method comprising using an immunoassay to detect at least one fibroblast activation marker in a patient sample, the method comprising: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody is specifically reactive to the epitopes of the fibroblast activation marker; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

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

3. The method according to claim 1 or 2, wherein the method comprises: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically binds to the epitope of PIIINP; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

4. The method of claim 3, wherein the epitope of the PIIINP to which the monoclonal antibody specifically binds is a novel C-terminal epitope of the PIIINP contained in the C-terminal amino acid sequence CPTGXQNYSP-COOH, wherein 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 an extended form of the C-terminal amino acid sequence of CPTGXQNYSPQ-COOH (SEQ ID NO: 5), and / or a truncated form of the C-terminal amino acid sequence of CPTGXQNYS-COOH (SEQ ID NO: 6).

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

7. The method according to any one of the preceding claims, wherein the method comprises: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically binds to the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen. The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

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 said type VI collagen specifically binds to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID No: 17).

9. The method according to claim 7 or claim 8, wherein the monoclonal antibody does not specifically recognize or bind to the extended form of the C-terminal amino acid sequence of KPGVISVMGTA (SEQ ID No: 18) and / or the truncated form of the C-terminal amino acid sequence of KPGVISVMG (SEQ ID No: 19).

10. The method according to any one of claims 7-9, wherein the monoclonal antibody is generated against a synthetic peptide comprising the C-terminal amino acid sequence KPGVISVMGT (SEQ ID No: 17) or composed thereof.

11. The method according to any one of the preceding claims, wherein the method comprises: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically binds to the N-terminal epitope of the propeptide of type I collagen; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

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

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

14. The method according to any one of claims 11-13, wherein the monoclonal antibody is generated against a synthetic peptide comprising the N-terminal amino acid sequence PDGSESPTDQETTGV (SEQ ID No: 3) or composed thereof.

15. The method according to any one of the preceding claims, wherein the method comprises: The patient sample is contacted with a monoclonal antibody, wherein the monoclonal antibody specifically binds to an internal sequence in the N-terminal region of type 3 collagen; The amount of antibody bound is determined; as well as The binding amount is correlated with values ​​in normal healthy subjects, and / or with values ​​in known responders of anti-TGFβI therapy, and / or with predetermined cutoff values.

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

17. The method of claim 15 or claim 16, wherein the monoclonal antibody does not specifically recognize or bind to an extended form of the C-terminal amino acid sequence of PPGIPGRNGDP (SEQ ID No: 32) or PGIPGRNGDPG (SEQ ID No: 33), and / or a truncated form of the C-terminal amino acid sequence of GIPGRNGDP (SEQ ID No: 34) or PGIPGRNGDPG (SEQ ID No: 35).

18. The method according to any one of claims 15-17, wherein the monoclonal antibody is produced against a synthetic peptide comprising the amino acid sequence PGIPGRNGDP (SEQ ID No: 2) or composed thereof.

19. The method according to any one of the preceding claims, wherein the patient sample 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 preceding claims, wherein the immunoassay is a competitive immunoassay.

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

23. The method according to any one of the preceding claims, further comprising administering anti-TGFβI therapy to a patient identified as potentially responsive.

24. An agent that binds to the EMI domain of TGFβI or periosteum protein or an analogue thereof.

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

26. The pharmaceutical preparation according to claim 24 or claim 25, used in medicine.

27. The pharmaceutical agent according to any one of claims 24-26, and its method of treating cancer or fibrosis.

28. A pharmaceutical agent for use according to claim 26 or claim 27, wherein the pharmaceutical agent is used to treat patients identified by the method according to any one of claims 1-22 as more likely to respond to TGFβI therapy.

Images