CA3009445C

CA3009445C - Combined biomarker measurement of fibrosis

Info

Publication number: CA3009445C
Application number: CA3009445A
Authority: CA
Inventors: Federica GENOVESE; Mette Juul NIELSEN; Lisa LARSEN; Diane Julie Oersnes-Leeming; Morten Karsdal
Original assignee: Nordic Bioscience AS
Current assignee: Nordic Bioscience AS
Priority date: 2016-02-03
Filing date: 2017-02-02
Publication date: 2024-03-19
Anticipated expiration: 2037-02-02
Also published as: JP2019510210A; CN108431606A; JP7094219B2; EP3411716A1; KR20180104648A; WO2017134172A1; CA3009445A1; CN108431606B

Abstract

Provided herein is a sandwich immunoassay for detecting cross-linked PIIINP that has at least two strands of PIIINP joined together by inter-strand cross-linking each having a C-terminal neo-epitope of PIIINP that is generated by N-protease cleavage of intact type III procollagen. A biological sample having the cross-linked PIIINP is contacted with a first surface-bound monoclonal antibody and then by a second monoclonal antibody, both specifically reactive with a neoepitope in the C-terminal sequence of PIIINP, and then binding of the second monoclonal antibody is determined. Also provided is a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidases via the immunoassay and a kit containing a solid support binding the first monoclonal antibody and containing the second monoclonal antibody.

Description

2 COMBINED BIOMARKER MEASUREMENT OF FIBROSIS
Technical Field of the Invention The present invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked PIIINP, and its use in evaluating the efficacy of drugs targeting lysyl oxidases (LOXs). The invention also relates to a kit for performing the sandwich immunoassay.
Description of the Related Art Fibrotic diseases (including those listed in Table 1) are a leading cause of morbidity and mortality, e.g. cirrhosis with 800,000 deaths per year worldwide (1).
Table 1. Different fibrotic diseases (2).
Tissue Examples of Causes Liver Viral hepatitis Schistosomiasis Steatohepatitis (Alcoholic or non-alcoholic) Lung Idiopathic pulmonary fibrosis (IPF) Systemic sclerosis (Scleroderma) Kidney Nephrogenic systemic fibrosis (NSF) Diabetes Untreated hypertension Heart Heart attack Hypertension Atherosclerosis Restenosis Eye Macular degeneration, retinal and vitreal retinopathy Skin Systemic sclerosis and scleroderma, keloids, hypertrophic scars, burns, genetic factors N FS
Pancreas Autoimmune/hereditary causes Intestine Crohn's disease/inflammatory bowel disease Brain Alzheimer's disease, AIDS
Bone Cancer, ageing marrow Multi-organ Surgical complications, chemotherapeutic drug-induced fibrosis fibrosis, radiation-induced fibrosis, mechanical injuries A 'fibrotic disease' is any disease giving rise to fibrosis, whether as a main or a secondary symptom. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury.
Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM).
Despite having obvious etiological and clinical distinctions, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, especially collagens and proteoglycans, which progressively remodel and destroy normal tissue architecture (3,4). Despite its enormous impact on human health, there are currently no approved treatments that directly target the mechanisms of fibrosis (5).
Extracellular Matrix (ECM) The ECM is a supramolecular structure with the ability to form aggregates of proteins, thus forming a dynamic scaffold linking cells together in a three dimensional network. This scaffold controls cell-matrix interactions and cell fate through up and down regulation of proteases (6). The ECM consists of collagens, laminins, proteoglycans, and other glycoproteins in various amounts and combinations, thereby providing a variety of biological components which can be modified by proteases to produce scaffolds with specific functions to meet the needs of the individual tissue (7).
Collagen types I and III are the major structural proteins in the human body.
Collagen type III is essential for collagen type I fibrillogenesis in the cardiovascular system and other organs (8,9). During fibrillar assembly the N-terminal propeptide of type III procollagen (which consists of three identical a-chains with a total molecular weight of 42 kDa) is cleaved off by specific N-proteases prior to incorporation of the mature collagen in the ECM. The cleaved propeptides may either be retained in the ECM or released into the circulation. However, the cleavage of the propeptide is sometimes incomplete, leaving the propeptide attached to the molecule. This results in the formation of thin fibrils with abnormal cross-links, which in turn causes the abnormal molecule to be prone to rapid metabolic turnover (10,11). Thus, the level of the N-terminal propeptide of type III collagen (PIIINP) in a suitable sample can be a marker of formation and/or degradation of collagen type III.
Remodeling of the ECM plays an important role in the pathogenesis of various diseases as altered components and non-coded modifications of the ECM leads to tissue stiffness and changes in the signaling potential of the intact ECM and fragments thereof. ECM remodeling is an important prerequisite for tissue function and repair, and

3 is tightly controlled by the enzymes responsible for the synthesis and degradation of the ECM.
During pathological events, such as fibrotic diseases, the balance between the formation and the degradation of the ECM is disturbed, leading to an altered composition of the ECM. Such an alteration results in altered tissue function (12,13). It has been suggested that PIIINP could be used as a biomarker for several fibrotic diseases, such as lung injury (14), viral and non-viral hepatitis (15), systemic sclerosis (16), vascular remodeling (17), and kidney diseases (18).
Limited attention has been given to the ECM remodeling in skeletal muscle tissue. In rat models increased collagen gene expression and biosynthesis have been demonstrated in quadriceps femoris and tibialis anterior muscles after exercise (19,20).
Additionally, increased serum levels of PIIINP have been demonstrated in clinical studies after exercise (21). Therefore, remodeling of the skeletal muscle proteins increases the quantity of PIIINP in the circulation and may serve as a biomarker for detecting early muscle anabolism. Serum levels of PIIINP have previously been suggested as a biomarker of muscular tissue response to testosterone (22), recombinant human growth hormone (23) or the combination thereof (24,25).
In liver fibrosis the fibrillar collagens type I and III are highly up-regulated (26,27).
Type III collagen is dominant in the early stages of fibrosis, while up-regulation of type I
collagen is related to the later stages of fibrosis. Fibrosis occuring in the liver results in the deposition of collagen and release of propeptides, predominantly PIIINP.
Consequently, PIIINP is one of the best studied markers for fibrogenesis (28,29,30).

4 Through the years, several radioimmunoassays have been developed for the quantification of PIIINP, with a sensitivity of up to 94% and specificity of up to 81% for the detection of cirrhosis (31,32); however none of the previous assays are neo-epitope specific. Additionally, the current commercially available assays for quantification of PIIINP utilise polyclonal antibodies or monoclonal antibodies targeting internal sequences of the procollagen or the propeptide and do not specifically differentiate between the formation and/or degradation of collagen type III (31,32).
Thus, to differentiate between formation and degradation of collagen type III
we consider that it is necessary to determine and detect a neo-epitopic fragment which is solely produced in the formation process (i.e. a fragment which is produced in the formation of collagen type III but not produced in the degradation of collagen type III).
WO 2014/170312disc1oses a monoclonal antibody which is specific for the C-terminal PIIINP neo-epitope comprised in the terminal amino acids of the C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO:4), wherein X can be Gly or Pro.
Brocks (31) discloses a polyclonal antibody directed to the modified Bovine C-terminal PIIINP sequence IC*QSCPTGGENYSP-COOH (SEQ ID NO: 1) (C* =
acetamido protected Cys; Gin replaced with Glu (E)), however said antibodies are non-specific towards the terminal amino acids of the bovine PIIINP C-terminal sequence ICQSCPTGGQNYSP-COOH (SEQ ID NO: 2) and additionally said antibodies do not recognise human PIIINP.

5 Bayer (33) discloses a sandwich ELISA which utilises a detector monoclonal antibody directed to the sequence H2N-GSPGPPGICQSCPTGPQNYSP-COOH (SEQ
ID NO: 3), however the binding epitope is not defined.
The applicant has now found that a specific sandwich immunoassay which utilises the neo-epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP as disclosed in WO 2014/170312 may be useful in evaluating the efficacy of drugs that target lysyl oxidases (LOXs), particularly LOX antagonist drugs.
Enzymatic collagen crosslinking by LOXs and processing of pro-collagens is key for tissue maturation and stability. In patients with organ fibrosis collagens become highly cross-linked, and thus are less prone to fibrosis resolution. LOXL2, a specific LOX, is a main driver in pathophysiological collagen crosslinking in fibrotic tissue, and novel LOXL2 antagonists are currently undergoing clinical trials. Thus, an assay that could be used to evaluate the efficacy of drugs targeting LOXs, such as LOX antagonists, would clearly be a useful tool for the pharmaceutical industry.
Summary The present invention is directed to a sandwich immunoassay for detecting in a biological sample cross-linked PIIINP where the cross-linked PIIINP comprises at least two strands of PIIINP joined together by inter-strand cross-linking. The method comprises contacting the biological sample comprising the cross-linked PIIINP
with a first monoclonal antibody bound to a surface, where each strand of PIIINP
comprised in

6 the cross-linked PIIINP has a C-terminal neo-epitope of PIIINP generated by N-protease cleavage of intact type Ill procollagen, and adding a second monoclonal antibody. Both monoclonal antibodies are specifically reactive with the C-terminal neo-epitope of PIIINP, and said neo-epitope is comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH, where X is Gly or Pro. The method further comprises determining the amount of binding of the second monoclonal antibody.
The present invention also is directed to a method for evaluating the efficacy of an antagonist drug targeting lysyl oxidases (LOXs). The method comprises using the sandwich immunoassay as described herein to quantify the amount of cross-linked PIIINP in at least two biological samples obtained from a subject at a first time point and at least one subsequent time point during a period of administration of the antagonist drug to the subject. A reduction in the quantity of cross-linked PIIINP from the first time point to the at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.
The present invention is directed further to a kit for use in the sandwich immunoassay as described herein. The kit comprises a solid support to which is bound the first monoclonal antibody as described above and a labelled second monoclonal antibody as described herein.
Figures FIG. 1: Alignment of the targeted PIIINP al chain sequence in human (SEQ ID
NO: 14) and rat (SEQ ID NO: 15) species (highlighted by the box). Position of the

7 corresponding human (¨) and rat ( _______________________________________________ ) sequences within the alpha 1 chain of the N-terminal pro-peptide of type III collagen. The alignment was performed using the NLP
CLUSTALW software.
FIG. 2: Western Blot showing the specific bands of N-terminal propeptide of type III collagen in Amniotic fluid from a) rat and b) human recognized by the monoclonal antibody NB61N62 (lane 1 and 3) and NB61N62 + selection peptide (lane 2+4).
Two bands around 52-60 kDA was observed for the rat, whereas one band was observed for human. Addition of selection peptide resulted in weakness of band intensity for both rat and human.
FIGS. 3A-3D: PRO-C3 ELISA runs showing typical calibration curves and native reactivity against human, rodent, and mouse material. FIG. 3A: Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy human serum, plasma, and amniotic fluid (AF). The calibrator curve was diluted in 2-fold from 76.31 ng/mL, whereas native material was run diluted 1:2 to 1:16 as indicated (--). FIG.
3B:
Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy rat serum, plasma, and AF. The calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1:8 as indicated (--). FIG. 3C:
Calibration curve and inhibition of the competitive PRO-C3 ELISA using healthy mouse serum and plasma. The calibrator curve was diluted in 2-fold from 200 ng/mL, whereas native material was run undiluted to 1:4 as indicated (--). FIG. 3D: Neo-epitope specificity of the PIIINP neo-epitope specific antibody using elongated peptide, i.e. peptide sequence of calibration peptide with one additional amino acid in the C-terminal end.
The

8 calibration curve, elongated peptide, and non-sense peptide were diluted in 2-fold from 76.31 ng/mL. The signal is seen as the optical density at 450 nm, subtracting the background at 650nm, as a function of peptide concentration.
FIG. 4: Results of an in vitro model of lung fibroblasts ("scar-in-a-jar").
FIG. 5: A comparison of Pro-C3X levels in extractions from keloids and extractions from normal skin.
FIGS. 6A-6B: Results of a study of patients with liver fibrosis.
FIG. 7: Pictorial representation of the Pro-C3X assay.
FIG. 8: Results of a study of patients with Alcoholic steatohepatitis.
FIG. 9: Pro-C3X levels in supernatant collected at Day 10 from the Scar-in-a-Jar model. Significance was assessed by one-way ANOVA with Dunnett's multiple comparisons test comparing each condition with TGF-8 alone. Data are shown as mean with SD. *"*p<0.0001. BAPN, (3-aminopropionitrile; TGF-8, transforming growth factor P.
Description of the Invention As used herein the term "neo-epitope" refers to an N- or C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N- or C- terminal end of the of the polypeptide, and is not to be construed as meaning in the general direction thereof.
As used herein the term, the term "competitive ELISA" refers to a competitive enzyme-linked immunosorbent assay and is a technique known to the person skilled in the art.

9 As used herein the term "sandwich immunoassay" refers to the use of at least two antibodies for the detection of an antigen in a sample, and is a technique known to the person skilled in the art.
As used herein the term, the monoclonal antibody NB61N-62 refers to a neo-epitope specific antibody directed towards the C-terminal neo-epitope of PIIINP, said neo-epitope comprising the C-terminal sequence CPTGXQNYSP-COOH (SEQ ID NO:
4), wherein X is Gly or Pro.
As used herein the term, the term "PRO-C3" is used to distinguish the herein described PIIINP assay from the PIIINP assays known in the art which are not based on the specific binding of neo-epitopes originating from PIIINP.
As used herein the term "PRO-C3X" assay refers to the herein described sandwich immunoassay for detecting and quantifying cross-linked PIIINP.
A monoclonal antibody suitable for use in the method of the invention was disclosed in WO 2014/170312 and is specifically reactive with a C-terminal neo-epitope of PIIINP, said neo-epitope being comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO:4), wherein X is Gly or Pro, and wherein said monoclonal antibody does not substantially recognise or bind an elongated version of said C-terminal amino acid sequence which is CPTGXQNYSPQZ-COOH (SEQ ID NO:
5), wherein Z is absent or is one or more amino acids of the sequence of collagen type III.
Preferably, the monoclonal antibody is specifically reactive with the neo-epitope C-terminal sequence CPTGPQNYSP-COOH (SEQ ID NO: 6) in human PIIINP, which is formed by the N-protease cleavage of PIIINP from intact procollagen type III
at the Pro-Gin bond between amino acids P153-Q154 in human PIIINP.
Alternatively, the monoclonal antibody may be specifically reactive with the neo-epitope C-terminal sequence CPTGGQNYSP-COOH (SEQ ID NO: 7) in rodent PIIINP, which said neo-epitope is formed by the N-protease cleavage of PIIINP from intact procollagen type III at the Pro-Gin bond between amino acids P154-Q155 in rodent PIIINP.
Preferably, the ratio of the affinity of the monoclonal antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said monoclonal antibody for elongated amino acid sequence CPTGXQNYSPQZ-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.
Preferably, the monoclonal antibody does not recognise or bind a shortened version of a C-terminal neo-epitope of PIIINP, said shortened neo-epitope having the amino acid sequence CPTGXQNYS (SEQ ID NO: 8).
Preferably, the ratio of the affinity of the monoclonal antibody for amino acid sequence CPTGXQNYSP-COOH (SEQ ID NO: 4) to the affinity of said monoclonal antibody for shortened amino acid sequence CPTGXQNYS-COOH (SEQ ID NO: 8) 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.

The present invention relates to a sandwich immunoassay for detecting in a biological sample cross-linked PIIINP, said cross-linked PIIINP comprising at least two strands of PIIINP joined together by inter-strand cross-linking, said method comprising:
contacting said biological sample comprising said cross-linked PIIINP with a first monoclonal antibody bound to a surface, wherein each strand of PIIINP
comprised in the cross-linked PIIINP comprises a C-terminal neo-epitope of PIIINP generated by N-protease cleavage of intact type III procollagen;
adding a second monoclonal antibody; and determining the amount of binding of said second monoclonal antibody;
wherein both said first monoclonal antibody and said second monoclonal antibody are specifically reactive with said C-terminal neo-epitope of PIIINP, said neo-epitope being comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH, wherein X is Gly or Pro.
Preferably, the monoclonal antibody does not substantially recognise or bind an elongated version of said C-terminal amino acid sequence which is CPTGXQNYSPQZ-COOH, wherein Z is absent or is one or more amino acids of the sequence of collagen type III.
The herein described sandwich immunoassay uses the same antibody as both catcher and detector antibody, therefore a double strand peptide (i.e. cross-linked) can be recognized by the assay.

Preferably, the sandwich immunoassay is used to quantify the amount of cross-linked PIIINP in a biofluid, wherein said biofluid may be, but is not limited to, serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.
The sandwich immunoassay may be, but is not limited to, a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay.
In a preferred embodiment, the second monoclonal antibody may be labeled in order to determine the amount of binding of said second monoclonal antibody.
Preferably, the second monoclonal antibody may be an enzyme-linked antibody.
The enzyme may be, but is not limited to, horseradish peroxidase (HRP).
Preferably, the second monoclonal antibody may be radiolabeled or linked to a fluorophore.
Although these are preferred labels to be used with the invention, it is envisaged that any suitable labeling system may be employed, such as, but not limited to, DNA
reporters or electrochemiluminescent tags.
Alternatively, a further labeled antibody which recognises the second monoclonal antibody may be used to determine the amount of binding of said second monoclonal antibody. The further labeled antibody may be labeled using a label as described above.
In a preferred embodiment of the invention, the sandwich immunoassay may further comprise correlating the quantity of cross-linked PIIINP determined by said method with standard fibrotic disease samples of known disease severity to evaluate the severity of a fibrotic disease. Such a fibrotic disease may be, but is not limited to, liver disease.

In a further aspect, the sandwich immunoassay described herein may be used in a method for evaluating the efficacy of a drug targeting lysyl oxidases (LOXs), such as an antagonist drug targeting LOXs.
Accordingly, the present invention also relates to a method for evaluating the .. efficacy of an antagonist drug targeting lysyl oxidases (LOXs), wherein said method comprises using the sandwich immunoassay described herein to quantify the amount of cross-linked PIIINP in at least two biological samples, said biological samples having been obtained from a subject at a first time point and at least one subsequent time point during a period of administration of the antagonist drug to said subject, and wherein a .. reduction in the quantity of cross-linked PIIINP from said first time point to said at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.
Preferably, the method quantifies the efficaciousness of the antagonist drug.
Preferably, the method evaluates the efficacy of an antagonist drug targeting LOXL2.
In another aspect, the present invention relates to a kit for use in the sandwich immunoassay as described herein, the kit comprising a solid support to which is bound a first monoclonal antibody as described above; and a labelled second monoclonal antibody as described above.

Examples Materials and general considerations All reagents used in the experiments were high-standard chemicals from companies such as Merck (Whitehouse Station, NJ, USA) and Sigma Aldrich (St.
Louis, MO, USA). The synthetic peptides used for monoclonal antibody production and validation were 1) Immunogenic peptide: Ovalbumine (OVA)-CGG-CPTGPQNYSP
(SEQ ID NO: 10), 2) Screening peptide: Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11), and 3) Selection peptide: CPTGPQNYSP (SEQ ID NO 6). All synthetic peptides were purchased from the Chinese Peptide Company, Beijing, China.
Example 1 - Monoclonal antibody NB61-N62 Monoclonal antibody generation The sequence for the N-terminal propeptide of type III collagen was aligned between human, rat and mouse species and selected from homology between the species and uniqueness among other ECM proteins by protein blasting. The amino acid sequence 145'-CPTGPQNYSP-'153 (SEQ ID NO: 6) in the al chain PIIINP is 100%
homologues between human and rat (FIG. 1). Generation of monoclonal antibodies was initiated by subcutaneous immunization of 4-5 week old Balb/C mice with 200 pl emulsified antigen and 50 pg PIIINP neo-epitope C-terminal sequence (OVA-CGG-CPTGPQNYSP (SEQ ID NO: 10)) using Freund's incomplete adjuvant. The immunizations were repeated every 2 weeks until stable serum titer levels were reached. The mouse with the highest serum titer was selected for fusion. The mouse was rested for a month and then boosted intravenously with 50 pg PIIINP neo-epitope C-terminal sequence in 100 pl 0.9% NaCI solution three days before isolation of the spleen. The spleen cells were fused with SP2/0 myeloma cells to produce hybridoma as described by (34), and cloned in culture dishes using the semi-medium method.
The clones were plated into 96-well microtiter plates for further growth employing the limited dilution method to secure monoclonal growth. The supernatants were screened for reactivity against calibrator peptide and native material in an indirect ELISA
using streptavidin-coated plates. Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11) was used as screening peptide, while the free peptide CPTGPQNYSP (SEQ ID NO: 6) was used as calibrator to test for further specificity of clones.
Clone characterization Native reactivity and affinity of the peptide were assessed using different biological materials such as urine, serum, and amniotic fluid (AF) from both humans and rats in a preliminary ELISA using 2 ng/ml biotinylated peptide on streptavidin-coated microtiter plates and the supernatants from growing monoclonal hybridoma cells.
Human AF was obtained from 30 women undergoing elective lower segment Caesarean sections at the Beijing Obstetrics Gynecology Hospital over a 2 month period. 100-200 ml AF was collected directly after incision and the fluid was stored at -C until use. The local ethical board had approved the study and all women provided 20 written consent prior to collection. Rat AF was drawn from the uterus of pregnant Wistar rats two days prior to expected birth. Antibody specificity was tested in a preliminary assay using deselection and elongated peptides (i.e. calibrator peptide with ten amino acid substitutions and calibrator peptide with one additional amino acid at the cleavage site, respectively). The isotype of the monoclonal antibodies was determined using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, AL, USA).
Antibody characterization Prior to Western Blotting, the total protein concentration of human and rat AF
was measured using Bicinchoninic acid (BCA) Protein Assay according to manufacturer's instruction. Briefly, BCA was diluted 2-fold in PBS from 2 mg/ml to produce a standard row for calculation of the samples. Samples were diluted 1:4 in lx phosphate-buffered saline (PBS) and 25 pl sample was added to a microtiter plate along with 200 pl working reagent (Reagent A and B mixed in the ratio 50:1).
The content was mixed on a plate shaker for 30 seconds followed by incubation for minutes at 37 C. After ended incubation the plate was cooled to room temperature and the absorbance was measured in the ELISA reader at 562 nm (Molecular Devices, SpectraMax M, CA, USA). Hereafter, rat or human AF was mixed with sample buffer (x2) and reducing agent (x10), heated at 70 C for 10 minutes, loaded on a 4-20% tris-glycein sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-page), and run for 1 hour at 180V. Protein bands were blotted onto a nitrocellulose membrane using the Invitrogen i-Blot gel transfer system according to manufacturer's instruction.
The membrane was blocked in blocking buffer (5% skimmed milk in Tris-buffered saline with TweenTm (TBST) overnight at 4 C and incubated with 1 pg/ml horseradish peroxidase (HRP)-conjugated PIIINP neo-epitope specific monoclonal antibody 62 for 2 hours. Specificity of the PIIINP neo-epitope specific monoclonal antibody was Date recue/Date received 2023-04-19 investigated by addition of excess PIIINP neo-epitope calibrator peptide and antibody in the ratio 10:1 and allowed to pre-incubate for 1 hour before it was added to the membrane for overnight incubation. After incubation the membranes was washed 4x10 minutes in TBST, incubated with 4 ml chemiluminescence detection kit (ECL), and developed using Amersham Hyperfilm.
Clone selection and characterization The subtype was determined to be an IgG1 subtype. From the Western Blot analysis it was seen that the PIIINP neo-epitope specific monoclonal antibody 62 recognized two bands with molecular sizes around 52-60 kDa in rat amniotic fluid, while only one band around 52 kDa was detected in human amniotic fluid. In addition, the signal could be partly inhibited by the selection peptide in the rat, and inhibited in human (FIG. 2). Native reactivity was observed using the NB61N-62 antibody in the ELISA. Native reactivity was seen towards human serum, plasma, and AF as well as against rodent serum, plasma, and AF (FIGS. 3A-3C). The signal was slightly less inhibited against mouse serum and plasma. The signal of the competitive ELISA
was inhibited using from 1:2 to 1:16, undiluted to 1:8, or undiluted to 1:4 in human, rodent, and mouse native material, respectively. Dilution of the native material approximately followed the same dilution pattern as the calibrator curve for all three species. Human AF inhibited the signal up to 100%; 80% for rat AF; 70% for human serum and plasma and rat serum; 44% for rat plasma, and 35% for mouse serum and plasma. Zero inhibition was observed using the elongated peptide (CPTGPQNYSPQ (SEQ ID NO:
6)) and non-sense peptide (GSPGKDGVRG (SEQ ID NO: 12)) (FIG. 3D).

Example 2¨ PRO-C3 ELISA using NB61N-62 Supernatant from the antibody producing hybridoma was collected and the monoclonal antibody was purified using HiTrap affinity columns (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK) and labeled with HRP using Lightning-LinkTM HRP Conjugation Kit (Innova Biosciences, Babraham, Cambridge, UK), according to the manufacturer's instructions.
The PRO-C3 competitive ELISA procedure was as follows: A 96-well streptavidin-coated ELISA plate from Roche, cat.11940279, was coated with the biotinylated peptide Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11) dissolved in coater buffer (50mM PBS-BTE + 10% sorbitol, pH 7.4), incubated for 30 min at 20 C in the dark and subsequently washed in washing buffer (20 mM Tris, 50 mM NaCI, pH
7.2).
Thereafter 20 pl of peptide calibrator or sample were added to appropriate wells, followed by 100 pl of HRP-conjugated monoclonal antibody NB61N-62 dissolved in incubation buffer (50 mM PBS-BTB + 10% LiquidII (Roche), pH 7.4) and the plate was incubated for 20 hours at 4 C and washed. Finally, 100 pl tetramethylbenzinidine (TMB) (Kem-En-Tec cat.: 4380H) was added, the plate was incubated for 15 min at 20 C
in the dark and in order to stop the reaction, 100 pl of stopping solution (1%
H2504) was added and the plate was analyzed in the ELISA reader at 450 nm with 650 nm as the reference (Molecular Devices, SpectraMax M, CA, USA). A calibration curve was plotted using a 4-parametric mathematical fit model.

Technical Evaluation A 2-fold dilution of healthy serum and plasma samples from human and rats were used to determine linearity and calculated as percentage of recovery of the 100%
sample. Antibody specificity was calculated as percentage of recovery of the 100%
calibrator peptide (CPTGPQNYSP (SEQ ID NO: 6)), elongated peptide (CPTGPQNYSPQ (SEQ ID NO: 13)), and non-sense peptide (GSPGKDGVRG (SEQ ID
NO: 12)). Lower limit of detection (LLOD) was calculated as the mean +
3xStandard Deviation (SD) of the blank from 21 determinations of standard K (i.e.
buffer). Upper limit of detection (ULOD) was determined as the mean ¨ 3xSD of 10 measurements of Standard A. Lower limit of quantification (LLOQ) was determined as the lowest concentration reproducibly measured with a precision lower than 30%. The intra-and inter-assay variation was determined by 10 independent runs of 8 QC samples, with each run consisting of double determination of the samples. Accuracy of the samples was measured in healthy human serum samples spiked with standard curve or human amniotic fluid at significant concentrations and calculated as the percentage recovery of the theoretical amount of serum. Interference was measured in healthy human serum spiked with hemoglobin, lipemia, and biotin at significant concentrations and calculated as the percentage recovery of the theoretical amount of serum.
Results The measurement range of the human PRO-C3 ELISA was determined by calculating ULOD and LLOQ providing a range from 0.867-60.1 ng/ml with a LLOD
of 0.606 ng/ml. The technical performance of the PRO-C3 ELISA showed acceptable inter-and intra assay variation of mean 11.03% and 4.11% (Table 1), with acceptance range below 15% and 10%, respectively.
Table 1: Inter- and intra-assay variation for the PRO-C3 assay using human serum quality control samples # 1-8 (HS1- HS8). The variation was calculated as the mean variation between 10 individual determinations of each sample.
Sample Value Intra-assay Inter-assay (ng/mL) variability % variability %
HS1 24.24 2.28 5.94 HS2 11.62 2.90 6.45 HS3 8.40 5.31 11.99 HS4 6.54 4.46 11.31 HS5 6.36 3.88 13.09 HS6 5.23 3.98 12.31 HS7 4.29 3.53 12.94 HS8 2.98 4.66 18.56 Dilution recovery was performed using healthy serum and plasma samples from humans, rat and mouse. The dilution recovery was within the acceptable 100 20%

recovery (Table 2). Further dilution resulted in measurements below LLOQ.
Table 2: Percentage dilution recovery for the PRO-C3 assay using human-, rat-, and mouse samples. Human serum (HS), Human plasma (HP), Rat serum (RS), Mouse serum (MS), Mouse plasma (MP).
PIIINP HS HP RS MS MP
ng/ml (n=2) (n=3) (n=10) (n=2) (n=2) Undiluted 100% - 100% 100% -Dilution 1:2 98 100% 116 96 100%
Dilution 1:4 103 91 110 118 114 Dilution 1:8 114 87 - - -Dilution 1:16 - 92 - - -Mean 105 90 113 107 114 Spiking of calibrator peptide in serum or plasma resulted in a mean recovery of 56% and 55%, respectively (Table 3).
Table 3: Spiking recovery of calibrator peptide in human serum or plasma, and human AF in human serum or plasma. The recovery was calculated as percent recovery of calculated peptide/AF in serum/plasma compared to pure serum/plasma.
Concentration of calibrator peptide were 38.16 ng/ml (StdB), 19.08 ng/ml (StdC), 9.54 ng/ml (StdD), 4.77 ng/ml (StdE), 2.39 ng/ml (StdF) and 1.19 ng/ml (StdG). AF was added in 2-fold dilution starting from 1:2.
Serum (n=3) Plasma Serum (n=3) Plasma Added Std sRE% (n=3) Added AF sRE% (n=3) sRE% sRE%
StdB 16 15 2x 101 103 StdC 29 25 4x 103 108 StdD 42 38 8x 106 113 StdE 58 54 16x 103 112 StdF 70 69 32x 104 115 StdG 82 83 64x 103 110 Buffer 92 100 Buffer 99 104 Mean sRE% 56 100 Mean sRE% 55 111 However, spiking of human AF in 2-fold dilution starting from 1:2 into healthy human serum or plasma resulted in mean recovery of 100% and 111%, respectively. No interference was observed in serum spiked with different concentrations of hemoglobin, biotin, and lipemia (Table 4).
Table 4: Interference of hemoglobin, lipemia and biotin in human serum added in various concentrations. All data are shown as percent recovery compared to pure serum.
Hemoglobin Lipemia Biotin mmol/L RE% mmol/L RE% ng/L RE%
0.5 68 0.56 101 160,000 134 _ -0.25 74 0.28 103 80,000 113 0.13 81 0.14 99 40,000 96 0.063 81 0.07 104 20,000 97 0.031 82 0.04 101 10,000 94 0.016 86 0.00 100 5,000 87 _ 0.008 95 2,500 100 0.000 100 0 100 , Mean 83 101 103 The stability of the analyte was acceptable up to four freeze/thaw cycles with 100 20% recovery compared to 1 freeze/thaw cycle (Table 5).
Table 5: Analyte stability in three human serum and plasma samples in four freeze/thaw cycles. All data are shown as mean percent recovery compared to 1 freeze/thaw cycle.
Freeze/thaw Serum EDTA plasma Heparin plasma Citrate plasma cycle Mean recovery Mean recovery Mean recovery Mean recovery % %

1 100% 100% 100% 100%

Example 3¨ Determining the ratio of binding affinity To determine the ratio of the binding affinity of the monoclonal antibody for the target sequence to the binding affinity of the monoclonal antibody for the elongated or shortened sequence, each of the sequences are synthesized and used as calibrator peptides in the PRO-C3 ELISA as described in example 2. The resultant calibration curves are used to determine the IC50 values of each sequence/antibody combination.
The ratio of IC5o[target] / IC50[elongated or shortened] defines the ratio of binding affinity.
Example 4 - PRO-C3X Assay As noted above, enzymatic collagen crosslinking by lysyl oxidases (LOXs) and processing of pro-collagens is key for tissue maturation and stability. Thus, monitoring inter-strand cross-linking of pro-collagen type III prior to enzymatic processing may prove useful for monitoring in-vivo activity of LOXs. This can be achieved by detecting and quantifying cross-linked PIIINP (i.e. two or more strands of PIIINP bound together by inter-strand links formed by LOXs in pro-collagen type III prior to enzymatic processing of the pro-collagen). A higher level of cross-linked PIIINP
detected in the circulation would be indicative of greater LOX activity. Accordingly, monitoring the level of cross-linked PIIINP during drug trials for drugs targeting LOX, such as LOX

antagonists, could provide useful efficacy data for said drugs.
ELISA
Streptavidin-coated plates were coated with 100 p1/well of 1 pg/ml biotinylated catcher antibody (biotin-linked NB61-N62) and incubated at 20 C, 300 rpm shaking for 30 minutes. Plates were washed five times in washing buffer (20 nM IRIS, 50 mM

NaCI, pH 7.2). Sample, standard or control (20 pl) was added and followed immediately by addition of 100 pl assay buffer and incubated at 4 C, 300 rpm shaking for 20 hours.
After incubation, plates were washed five times in washing buffer. 100 p1/well of 1 pg/ml HRP-labelled detector antibody (HRP-linked NB61-N62) was added and incubated at C, 300 rpm, shaking for 1 hour. After incubation, plates were washed five times in washing buffer. A volume of 100 pl 3,3',5,5' -Tetramethylbenzidine (TMB) was added and incubated for 15 min at 20 C in the dark. To stop the enzyme reaction of TMB, 100 15 pl 0.1% sulphuric acid was added. The enzyme reaction was then read on an ELISA
reader, using a quadratic curve fit. Each ELISA plate included both kit control and in-house quality control samples to monitor inter-assay variation. All samples were measured within the range of the specific assay. All samples below the level of lower limited of quantification (LLOQ) were assigned the value of LLOQ.
20 The technical characteristics of the assay are:
Parameter Results Biological matrix Serum, plasma, supernatants, extraction fluid.

Intra-assay variation 2% (accepted if <10%) Inter-assay variation 6% (accepted if <15%) Measurement range 0.965-17.586 ng/ml Lower limit of detection 0.251 ng/ml Normal range in healthy serum 4.022 ( 2.24) ng/ml Required volume 30 pl serum/plasma; 60 pl supernatant/extraction Spiking recovery Peptide in serum: 94%
Serum in serum: 86%
Results Scar Tissue Preliminary results using an in vitro model of lung fibroblasts ("scar-in-a-jar") strongly suggest that an enzyme of the LOX family is the responsible for the cross-link in PIIINP (TGF-8 is known in the art to increase lysyl oxidase (LOX) enzyme activity).
Briefly, Pro-C3X (i.e. cross-linked PIIINP) was generated by culturing lung fibroblasts for 5 days in crowded conditions and under TGF48 stimulation. Pro-C3X was significantly elevated after 12 days of culturing with TGF-13, with negligible quantities of Pro-C3X
being observed in the absence of TGF-8 (FIG. 4). Similarly, Pro-C3X was elevated in extractions from keloids when compared to extractions from normal skin (FIG.
5).
Liver Fibrosis A study of patients with liver fibrosis was conducted using the "Pro-C3X"
assay and compared to the herein described "Pro-C3" competitive ELISA. It was found that Pro-C3X was significantly elevated in later stages of disease, when fibrosis is more severe, with levels similar to healthy controls in early stages of disease (FIG. 6A). In comparison, Pro-C3 levels differed at all stages of the disease (FIG. 6B).
The difference in selectivity between the Pro-C3X assay and Pro-C3 assay is attributed to the Pro-C3X assay only recognizing cross-linked PIIINP, whereas the Pro-C3 assay recognises both cross-linked and non-cross-linked PIIINP. FIG. 7 shows the Pro-C3X assay and provides a pictorial explanation for the reasoning behind this conclusion:
Pro-C3X assay: if cross linked PIIINP is present then the first antibody will bind to the free epitope on a first strand of PIIINP and subsequently the second antibody will bind to the free epitope on a second strand of PIIINP. However, if non-cross-linked PIIINP is present then the surface-bound antibody will bind to the free epitope of the non-cross linked collagen type III, but the second antibody will fail to bind as the binding epitope is already occupied, therefore addition of the second antibody will fail to produce a signal. Thus, the signal from the Pro-C3X assay is exclusively due to the detection of cross linked PIIINP.
Conversely, substantially all of the antibodies in the Pro-C3 assay will bind to strands of PIIINP comprising a free binding epitope, irrespective of whether the PIIINP is or is not cross-linked. Thus, the signal obtained from the Pro-C3 assay is an aggregate signal of cross-linked and non-cross-linked PIIINP.
Accordingly, it is this selectivity that prompts the use of the Pro-C3X assay for evaluating the efficacy of drugs targeting LOX; monitoring the level of PIIINP
cross-linking which, as noted above, is suggested to be a result of LOX activity during a period of drug administration to a subject could be used to monitor drug activity and thus efficacy of said drug.
Alcoholic steatohepatitis A study of patients with alcoholic steatohepatitis was conducted using the "Pro-C3X" assay and compared to the herein described "Pro-C3" assay. In alcoholic steatohepatitis, both Pro-C3 and Pro-C3X were elevated in later stages of the disease (Metavir 2-4). However, for patients with cirrhosis Pro-C3 did not correlate with MELD
(Model For End-Stage Liver Disease) score (P=0.527), whereas Pro-C3X strongly correlated (corr coefficient 3.34, P<0.001). Moreover, ProC3 only correlated negatively with albumine, while ProC3X correlated with albumine, bilirubine and Gamma-Glutamyl Transpeptidase (GGT) (FIG. 8). The late stage increase in Pro-C3X suggests increase in cross-linking of PIIINP, which would be in accordance with increased scarring of the liver (i.e. increased LOX activity).
Example 5 Assessment of Pro-C3X in the Scar-in-a-Jar model Background: Fibrosis is the accumulation of extracellular matrix (ECM) within affected tissues, which can lead to organ failure and ultimately death. Following stimulation by e.g. transforming growth factor (TGF)-13, fibroblasts are the main cell type responsible for the excessive accumulation of ECM proteins, especially collagens. Here we describe the use of the in vitro model "Scar-in-a-Jar", known to generate ECM and cross-links, in combination with the Pro-C3X ELISA to investigate collagen formation and cross-linking during fibrogenesis. This tool can be used in the investigation of novel anti-fibrotic compounds by assessing the modulation of these fibrotic processes.
Methods: Healthy human lung fibroblasts (L248) were grown to confluence, after which they were seeded at a density of 30,000 cells/well. The cells were grown in DMEM
media containing 0.4% FCS, 225 mg/mL ficoll 70, 150 mg/mL ficoll 400, and 1%
ascorbic acid for 18 days. The cells were stimulated with 1 ng/mL TGF-6 with or without the lysyl oxidase (LOX) inhibitor 6-aminopropionitrile (BAPN; 0.02 or 0.2 mM) to inhibit formation of cross-links. Unstimulated cells or cells grown in media not containing ficoll were used as controls. Media was changed on day 3, 6, 10, and 14. Fibroblast viability was assessed using the AlamarBlue assay. Pro-C3X levels were assessed in the collected supernatant, using the Pro-C3X sandwich ELISA described above.
.. Results: TGF-6 stimulation induced the release of Pro-C3X from day 3 with a peak in Pro-C3X levels at day 10 showing a 14-fold increase as compared with unstimulated cells (p<0.0001; Figure 9). Treatment with 0.02 mM BAPN had no significant effect, but 0.2 mM BAPN induced a significant decrease in Pro-C3X levels as compared with TGF-6 stimulation only (0.59-fold change, p<0.0001; Figure 9).
Conclusions: The pan LOX inhibitor BAPN significantly reduced Pro-C3X levels at a concentration of 0.2 mM, indicating that the Pro-C3X ELISA assesses a cross-linked epitope. Thus. the Pro-C3X ELISA can be used to evaluate fibroblast activity and thus be used to screen potential anti-fibrotic compounds. TGF-13 stimulation induced the release of Pro-C3X, a marker of the cross-linked collagen type III pro-peptide.
In conclusion, the Pro-C3X assay is a second generation assay combining the cleavage neo-epitope of the collagen type III pro-peptide and the presence of cross-linking in the molecule. This assay is therefore providing additional information to the measurement of Pro-C3, because it describes a different process in the fibrosis timeline; that is, the cross-linking of collagen molecules in the scar. The Pro-C3X assay can therefore be used to test the efficacy of drugs targeting LOX, particularly LOX
antagonists/inhibitors, since the use of a LOX inhibitor was shown to reduce the presence of the cross-linked PIIINP biomarker.
In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.
***
In some aspects, embodiments of the present disclosure as described herein include the following items:
Date recue/Date received 2023-04-19 Item 1. A sandwich immunoassay method for detecting in a biological sample cross-linked PIIINP, said cross-linked PIIINP comprising at least two strands of PIIINP
joined together by inter-strand cross-linking, said method comprising:
contacting said biological sample comprising said cross-linked PIIINP with a first monoclonal antibody bound to a surface, wherein each strand of PIIINP
comprised in the cross-linked PIIINP comprises a C-terminal neo-epitope of PIIINP generated by N-protease cleavage of intact type III procollagen;
adding a second monoclonal antibody; and determining the amount of binding of said second monoclonal antibody;
wherein both said first monoclonal antibody and said second monoclonal antibody are specifically reactive with said C-terminal neo-epitope of PIIINP, said neo-epitope being comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH
of SEQ ID NO: 4, wherein X is Gly or Pro, wherein said first monoclonal antibody and said second monoclonal antibody bind to different strands of the crosslinked PNIIIP.
Item 2. The sandwich immunoassay method of item 1, wherein the first monoclonal antibody and/or the second monoclonal antibody does not recognise or bind an elongated version of said C-terminal amino acid sequence which is CPTGXQNYSPQZ-COOH of SEQ ID NO: 5, wherein Z is absent or is one or more amino acids of the sequence of collagen type III.

Date recue/Date received 2023-04-19 Item 3. The sandwich immunoassay method of item 1 01 2, wherein the sandwich immunoassay method is used to quantify the amount of cross-linked PIIINP in the biological sample.
Item 4. The sandwich immunoassay method of item 3, further comprising correlating the quantity of cross-linked PIIINP determined by said method with standard fibrotic disease samples of known disease severity to evaluate the severity of a fibrotic disease.
Item 5. The sandwich immunoassay method of item 4, wherein the fibrotic disease is liver disease.
Item 6. The sandwich immunoassay method of any one of items 3 to 5, wherein the biological sample is a biofluid.
Item 7. The sandwich immunoassay method of item 6, wherein said biofluid is serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.
Item 8. The sandwich immunoassay method of any one of items 1 to 7, wherein the sandwich immunoassay is a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay.

Date recue/Date received 2023-04-19 Item 9. The sandwich immunoassay method of any one of items 1 to 8, wherein the second monoclonal antibody is labeled.
Item 10. The sandwich immunoassay method of item 9, wherein the second monoclonal antibody is an enzyme-linked antibody.
Item 11. The sandwich immunoassay method of item 10, wherein the enzyme is horseradish peroxidase (HRP).
Item 12. The sandwich immunoassay method of item 9, wherein the second monoclonal antibody is radiolabeled or linked to a fluorophore.
Item 13. The sandwich immunoassay method of any one of items 1 to 8, wherein a further labeled antibody which recognises the second monoclonal antibody is used to determine the amount of binding of said second monoclonal antibody.
Item 14. A method for evaluating the efficacy of an antagonist drug targeting lysyl oxidases (LOXs), wherein said method comprises using the sandwich immunoassay method of item 1 to quantify the amount of cross-linked PIIINP in at least two biological samples, said biological samples having been obtained from a subject at a first time point and at at least one subsequent time point during a period of administration of the antagonist drug to said subject, and wherein a reduction in the quantity of cross-linked Date recue/Date received 2023-04-19 PIIINP from said first time point to said at least one subsequent time point during the period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.
Item 15. The method of item 14, wherein the method evaluates the efficacy of an antagonist drug targeting LOXL2.
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Claims

1. A sandwich immunoassay method for detecting in a biological sample cross-linked PIIINP, said cross-linked PIIINP comprising at least two strands of PIIINP
joined together by inter-strand cross-linking, said method comprising:
contacting said biological sample comprising said cross-linked PIIINP with a first monoclonal antibody bound to a surface, wherein each strand of PIIINP
comprised in the cross-linked PIIINP comprises a C-terminal neo-epitope of PIIINP generated by N-protease cleavage of intact type III procollagen;
adding a second monoclonal antibody; and determining the amount of binding of said second monoclonal antibody;
wherein both said first monoclonal antibody and said second monoclonal antibody are specifically reactive with said C-terminal neo-epitope of PIIINP, said neo-epitope being comprised in a C-terminal amino acid sequence CPTGXQNYSP-COOH of SEQ ID
NO: 4, wherein X is Gly or Pro, wherein said first monoclonal antibody and said second monoclonal antibody bind to different strands of the crosslinked PNIIIP.

2. The sandwich immunoassay method of claim 1, wherein the first monoclonal antibody and/or the second monoclonal antibody does not recognise or bind an elongated version of said C-terminal amino acid sequence which is CPTGXQNYSPQZ-COOH of SEQ ID NO: 5, wherein Z is absent or is one or more amino acids of the sequence of collagen type III.

Date recue/Date received 2023-04-19

3. The sandwich immunoassay method of claim 1 or 2, wherein the sandwich immunoassay method is used to quantify the amount of cross-linked PIIINP in the biological sample.

4. The sandwich immunoassay method of claim 3, further comprising correlating the quantity of cross-linked PIIINP determined by said method with standard fibrotic disease samples of known disease severity to evaluate the severity of a fibrotic disease.

5. The sandwich immunoassay method of claim 4, wherein the fibrotic disease is liver disease.

6. The sandwich immunoassay method of any one of claims 3 to 5, wherein the biological sample is a biofluid.

7. The sandwich immunoassay method of claim 6, wherein said biofluid is serum, plasma, urine, amniotic fluid, tissue supernatant or cell supernatant.

8. The sandwich immunoassay method of any one of claims 1 to 7, wherein the sandwich immunoassay is a radioimmunoassay, fluorescence immunoassay, or an enzyme-linked immunosorbent assay.
Date recue/Date received 2023-04-19

9. The sandwich immunoassay method of any one of claims 1 to 8, wherein the second monoclonal antibody is labeled.

10. The sandwich immunoassay method of claim 9, wherein the second monoclonal antibody is an enzyme-linked antibody.

11. The sandwich immunoassay method of claim 10, wherein the enzyme is horseradish peroxidase (HRP).

12. The sandwich immunoassay method of claim 9, wherein the second monoclonal antibody is radiolabeled or linked to a fluorophore.

13. The sandwich immunoassay method of any one of claims 1 to 8, wherein a further labeled antibody which recognises the second monoclonal antibody is used to determine the amount of binding of said second monoclonal antibody.

14. A method for evaluating the efficacy of an antagonist drug targeting lysyl oxidases (LOXs), wherein said method comprises using the sandwich immunoassay method of claim 1 to quantify the amount of cross-linked PIIINP in at least two biological samples, said biological samples having been obtained from a subject at a first time point and at at least one subsequent time point during a period of administration of the antagonist drug to said subject, and wherein a reduction in the quantity of cross-linked PIIINP from said first time point to said at least one subsequent time point during the Date recue/Date received 2023-04-19 period of administration of the antagonist drug is indicative of an efficacious antagonist drug targeting LOXs.

15. The method of claim 14, wherein the method evaluates the efficacy of an antagonist drug targeting LOXL2.

Date recue/Date received 2023-04-19