COMP cleavage peptide and its use in diagnosis of osteoarthritis

Abstract

There is provided a method of diagnosis comprising providing a sample previously isolated from a subject and analysing the sample for presence of a peptide comprising the amino acid sequence N- terminal-KSSTG (SEQ ID NO 1). The diagnosis may be for example diagnosis of a disease is selected from: osteoarthritis, early stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures and chip fractures of the joint.

Description

[0001] COMP cleavage peptide and its use in diagnosis of osteoarthritis

[0002] FIELD OF THE INVENTION

[0003] This invention relates to a peptide and antibodies against a peptide, and their use in diagnosis, in particular diagnosis of osteoarthritis and its progression.

[0004] BACKGROUND

[0005] Horse sporting events, such as racing or show jumping, draws large crowds to racetracks and other arenas. Occasionally, so-called chip fractures of a leg joint (in particular the carpal joint) of a competing horse result in the horse collapsing on the track due to a so called "catastrophic injury", necessitating to euthanize the horse where it is, in front of the audience. This is extremely tragic and has caused uproar in media. To prevent this type of injuries, horses sometimes undergo radiography in order to detect joint defects before competing. However, radiography of horses is expensive and unpractical.

[0006] Chip fractures of the joints is typically caused by an underlying and undetected osteoarthritis disease.

[0007] In humans, osteoarthritis is a major clinical problem as it affects around 3.8% of the human population. It is estimated that 50% of NSAID pain killer prescriptions are related to OA.

[0008] Osteoarthritis (OA) is a low-grade chronic systemic inflammatory disease that results from breakdown of joint cartilage and underlying bone. The disease progresses from an early phase which is characterized by inflammation and disorganization of matrix proteins in the affected joints, to a more severe phase with damage of the cartilage, synovial membrane and the underlying bone. Hence, it may be considered, at the late stage, as a "whole joint" disease affecting all structures of the joint. The main symptom is joint pain.

[0009] Micro fractures caused by OA may result in chip fractures where a part of the bone and the cartilage detaches. Chip fractures, which are sometimes also referred to as avulsion fractures, are very problematic, in particular if they occur in the carpal joint of a horse. In many cases, the horse cannot recover from such an injury. Additionally, the chip fracture can progress to catastrophic injury and the horse needs to be euthanized. Moreover, a jockey can be severely injured when a gallop racehorse receives a chip fracture during a race.

[0010] The principal reason for having a horse is to be able to use it, and often to use the horses in competitions. OA in horses is a great problem as it causes joint pain and subsequent lameness hence inability to train and compete. OA therefore accounts for the greatest single economic loss in the horse industry.

[0011] Although joint pain often leads a physician (or veterinarian) to suspect OA, it is today difficult to diagnose the early stages of OA. Today diagnosis is often based on identification, using radiology, of structural damages typical of the late stages of OA. X-ray or MRI cannot, however, be used to diagnose early-stage OA ("microscopic osteoarthritis"), because the structural damages are not yet visible using these imaging technologies. Also, X-ray and MRI examination requires expensive equipment, and it is cumbersome to carry out X-ray or MIR on animals.

[0012] Manifestation of OA is similar in horses and humans. In both humans and horses, OA proceeds from an early stage characterized by inflammation over months and years to a later stage with extensive tissue damage (Goldring, M.B. and Otero M. Current Opinion in Rheumatology (2011) 23(5):471). OA disease mechanisms in humans and horses are also very similar on the molecular level (Stenberg J, Ruetschi U, Skibldebrand E, Karrholm J, Lindahl A. Proteome Sci. (2013) Oct 4;11(1):43) (Svala E, Lofgren M, Sihlbom C, Ruetschi U, Lindahl A, Ekman S, Skibldebrand E. Connect Tissue Res.

[0013] (2015);56(4):315-25).

[0014] Fatigue-related changes in the subchondral bone (SCB) microenvironment, is one part of the OA process (Hu et al Bone Res. 2021;9(l):20). This typically happens before onset of damages to the cartilage during OA.

[0015] The articular cartilage of the joint has the following layers from top to bottom: At the surface is the superficial layer. This layer has flattened chondrocytes and an extracellular matrix with low proteoglycan content and abundant collagen fibrils parallel to the surface. Below is the so-called mid layer or transitional zone, comprising more spheroid chondrocytes, thinner collagen fibrils high proteoglycan content. Below the transitional zone of the cartilage is the deep zone, with chondrocytes arranged perpendicular to the joint surface, thick collagen fibrils parallel to the cells and high proteoglycan content. Below the deep zone, limited by the tide mark, is the calcified articular cartilage layer close to the underlying subchondral bone plate.

[0016] Remodelling of the bone and generation of cartilage are two separate and carefully controlled physical phenomena. The two processes are regulated by different signalling mechanisms and are controlled by different cell types. For example, chondrocytes are responsible for remodelling of cartilage whereas osteoblasts and osteoclasts control bone remodelling and modelling. In a healthy joint there is an important crosstalk between the two types of tissues.

[0017] Bone modelling changes the shape and size of bone in response to physiologic and mechanical forces. This is most prominent in the growing animal. Bone remodelling in the adult animal, will replace and renew old bone matrix and maintain its strength and mineral homeostasis. Bone is well- vascularized, and angiogenesis is involved in bone activity.

[0018] Cartilage, on the other hand, is not vascularised and has a very slow turnover with a very low renewal capacity.

[0019] The bone matrix consists of organic components such as collagen type I, versican and inorganic bone salts, mainly hydroxyapatite. In cartilage, collagen type II and aggrecan are the main matrix molecules.

[0020] Cartilage oligomeric matrix protein (COMP), also known as thrombospondin-5, is an extracellular matrix (ECM) protein primarily present in cartilage. In humans it is encoded by the comp gene.

[0021] WO2017216289 discloses a cleavage fragment of COMP with the sequence N-terminal SGPTH that can be used for diagnosis of osteoarthritis, in particular early phase OA.

[0022] WO2022268940 discloses a cleavage fragment of biglycan with the sequence N-terminal-GLGHN that can be used for diagnosis of OA, in particular OA associated with the risk for chip fractures. This peptide is generally associated with damage to the bone part of the joint.

[0023] However, it would be useful if there were further methods of diagnosing and staging OA. In particular, it would be useful to have a marker for various stages of the disease that could be used to follow the progression of the disease from mild OA to later stage or an increased risk for chip fractures.

[0024] Furthermore, it has been recognized that:

[0025] "Despite etiological differences, OA patients continue to be treated as a homogenous population. This 'one-size-fits-all' mentality has not only hampered the evolution of more effective patient care, but has likely contributed to the lack of clinically approved disease-modifying OA drugs" and furthermore that: molecular understanding of OA phenotypes is therefore crucial cand application of this knowledge can facilitate drug development and increase the potential for approval of effective and personalized treatments of OA patients". (Monica T.

[0026] Hannani, Christian S. Thudium, Morten A. Karsdal, Christoph Ladel, Ali Mobasheri, Melanie Uebelhoer, Jonathan Larkin, Jaume Bacardit, Andre Struglics & Anne-Christine Bay-Jensen (2024) From biochemical markers to molecular endotypes of osteoarthritis: a review on validated biomarkers, Expert Review of Molecular Diagnostics, 24:1-2, 23-38, DOI: 10.1080 / 14737159.2024.2315282).

[0027] Molecular typing of OA is an emerging field and it envisaged that that molecular typing of OA will be important for selection of treatment (Im and Moon, Tissue Eng Regen Med (2022) 19(2):321-324).

[0028] Thus, there is a great need for further molecular markers for OA. For example, the peptides described herein may be used as research tools for characterizing OA patients, for example correlating clinical symptoms or development of the disease to the presence of one or more peptides, or predicting treatment response for subgroups of patients.

[0029] Diagnosis is often carried out using blood samples. This may cause discomfort and is not completely without risk for the patients or animal as it involves drawing a blood sample from a vein. It would be useful if such diagnosis could be carried out in a more convenient manner.

[0030] Joint damage may also be caused by excessive running, standing or walking such as certain types of professions or physical exercise. Joint damage is also associated with certain types of footwear and for horses, certain horseshoes. There is a need for preventing this type of joint damage.

[0031] The ground material for riding arenas (so called "footings") are very important for the health of the horse. Regular exercise on hard footings, for example, may lead to OA. New footings have been developed recently. For example, a mixture of sand and polymer fibers have been introduced in recent years. There is some uncertainty as to the effect of new footings on the health of the horse. There is a need for better methods to investigate such footings.

[0032] This invention solves these and other problems.

[0033] SUMMARY OF THE INVENTION

[0034] The inventors have surprisingly found that a cleavage fragment of COMP (COMP644) (N-terminal- KSSTG (NH2-KSSTG) (COMP neoepitope) (SEQ ID NO 1)) is prominent in OA. This cleavage fragment of COMP is present in the deep zone of the articular cartilage. The peptide N- terminal-KSSTG fragment is, surprisingly, present in OA subjects that have low levels of the COMP fragment N-terminal SGPTH, which is a marker for early phase OA. Hence the peptide KSSTG may be used to identify patients with OA and related disease, as the KSSTG peptide may be used to identify OA patients that are negative for the SGPTH peptide. Hence the KSSTG peptide provides independent diagnostic information. The use of the KSSTG peptide provides an improved diagnostic tool for OA.

[0035] The different peptides (SGPTH and KSSTG) will provide precision diagnosis such for the clinician and subsequent crucial guide for intervention, which is lacking today.

[0036] In a first aspect of the invention there is provided a method of diagnosis comprising providing a sample previously isolated from a subject and analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

[0037] The diagnosis may be diagnosis of a disease is selected from: osteoarthritis, early stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures and chip fractures of the joint. In various embodiments, the disease is associated with damage to the deep zone of the cartilage of the joint.

[0038] In various embodiments, in addition, a sample previously isolated from the patient is analysed for the presence of a peptide comprising the amino acid sequence N-terminal-SGPTGH (SEQ ID NO 25) or N- terminal-GLGHN (SEQ ID NO 26), or both.

[0039] In various embodiments, sample may be sample of synovial fluid, serum, blood, blood plasma, urine or saliva.

[0040] In various embodiments, the method may be carried out by repeatedly obtaining samples from an individual and monitoring the progression of the disease. In various embodiments, the method is carried out by repeatedly obtaining samples from an individual and determine the presence of the peptide in the sample, and thereby monitoring the progression of the disease.

[0041] In a second aspect of the invention there is provided a method for preventing osteoarthritis, early- stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures and chip fractures of the joint in a subject to comprising the steps of: a) repeatedly isolating samples from the subject and analysing the presence of a peptide comprising the amino acid sequence N-terminal- KSSTG (SEQ ID NO 1) in the samples, b) if the level of peptide in the subject in a sample is above a predetermined level, determining that the subject should be treated.

[0042] In various embodiments, the samples are, in addition, analysed for the presence of a peptide comprising the amino acid sequence N-terminal-SGPTGH (SEQ ID NO 25) or N-terminal-GLGHN (SEQ ID NO 26), or both.

[0043] The treatment may comprise resting the subject. In various embodiments the treatment may comprise a pharmacological treatment, for example administration of sildenafil, glucose and a anesthetic.

[0044] In a third aspect of the invention there is provided an antibody that specifically binds to a peptide comprising the amino acid sequence N-terminal- KSSTG (SEQ ID NO 1) where the antibody binds to the sequence N-terminal KSSTG.

[0045] In various embodiments, the antibody can be used for the detection of a proteolytic fragment of COMP that exposes the N-terminal.

[0046] The antibody may be for use in diagnosis of a disease selected from: osteoarthritis, early stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures and chip fractures of the joint. The antibody may be used for detecting the amount of a peptide comprising the amino acid sequence N-terminal-KSSTG in a sample from a subject.

[0047] In various embodiments, the sample may be a sample of synovial fluid, serum, blood, blood plasma, urine or saliva.

[0048] In a fourth aspect of the invention there is provided a kit comprising an antibody according to the third aspect of the invention. In various embodiments, the antibody is comprised in a diagnostic device for single use, where the kit additionally comprises a saliva, blood, urine or synovial fluid sampling device. In various embodiments, the kit further comprises an antibody that specifically binds to a peptide comprising the amino acid sequence N-terminal-SGPTGH (SEQ ID NO 25) where the antibody binds to the sequence N-terminal-SGPTH, or an antibody that specifically binds to the sequence N-terminal-GLGHN (SEQ ID NO 26), where the antibody binds to the sequence N-terminal GLGHN. In a fifth aspect of the invention there is provided a peptide comprising the amino acid sequence N- terminal-KSSTG (SEQ ID NO 1).

[0049] In a sixth aspect of the invention there is provided the use of a peptide comprising an amino acid sequence N-terminal-KSSTG (SEQ ID NO 1) for the production of an antibody.

[0050] In a seventh aspect of the invention there is provided a method of treatment of osteoarthritis, early- stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures or chip fractures of the joint comprising, carrying out diagnosis according to anyone claims 1 to 5 and then administering to the subject a phosphodiesterase type 5 (PDE5) inhibitor.

[0051] In various embodiments, a first sample is isolated from the subject at the latest on the same day where the PDE5 inhibitor is administered to the subject and a second sample is isolated from the subject after the PDE5 inhibitor is administered to the subject and where both samples are analysed for presence of the peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

[0052] In an eight aspect of the invention, there is provided a PDE5 inhibitor for use for the treatment of osteoarthritis, early-stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures and chip fractures of the joint in a group of patients where the level of peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1) in samples from the patients is above a predetermined reference level. The patients may have a level of N-terminal SGPTH below a predetermined reference level.

[0053] In ninth aspect of the invention there is provided a method for determining the risk for joint damage comprising obtaining a sample from a subject and analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1) in the sample.

[0054] In various embodiments, the risk relates to a shoe for a human, indoor or outdoor flooring, working conditions for humans, a sports activity for a human, a horseshoe, or footing for a horse, or an exercise program for a subject.

[0055] In various embodiments of the ninth aspect of the invention a sample from the subject is analysed for the presence of a peptide comprising the amino acid sequence N-terminal SGPTGH (SEQ ID NO 25) or N-terminal- GLGHN (SEQ ID NO 26), or both. In tenth aspect of the invention there is provided a method for evaluating a floor or a shoe comprising a) allowing a test subject to use the floor or the shoe, b) isolating a sample from the test subject, c) analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

[0056] In an eleventh aspect of the invention there is provided a method for evaluating a footing, a horseshoe or a method of attaching a horseshoe, or comprising a) allowing a horse to use the footing or the horseshoe or the method of attaching the horseshoe, b) isolating a sample from the horse, c) analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

[0057] In a twelfth aspect of the invention there is provided a method of selecting a horseshoe or a method of attaching a horseshoe for an individual horse comprising a) allowing a horse to use the footing or the horseshoe or the method of attaching the horseshoe, b) isolating a sample from the horse, c) analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

[0058] In various embodiments of the eleventh or twelfth aspect of the invention, in addition, a sample from the subject is analysed for the presence of a peptide comprising the amino acid sequence N- terminal-SGPTGH (SEQ ID NO 25) or N-terminal-GLGHN (SEQ ID NO 26), or both. BRIEF DESCRIPTION OF DRAWINGS

[0059] Fig. 1 is a diagram that shows ELISA data, showing the specificity of an antibody.

[0060] Fig. 2 is a diagram that shows ELISA data from horse synovial fluid.

[0061] Fig. 3 is a diagram that shows ELISA data from horse serum.

[0062] Fig. 4 is a diagram that shows the presence of COMP neo-epitope in saliva from horses.

[0063] Fig. 5 are diagrams that shows the presence of COMP neo-epitope in saliva from healthy horses that undergo training on two different footings.

[0064] Fig. 6 is a graph that shows the presence of the COMP neo-epitope in synovial fluid before and after pharmacological treatment.

[0065] Fig. 7 shows the presence of a prior art fragment (N-terminal SGPTH) in the joints from horses with OA.

[0066] Fig. 8. Shows the presence of the COMP neo epitope N-terminal-KSSTG in the joints from horses with OA.

[0067] DETAILED DESCRIPTION

[0068] This invention relates to a peptide comprising a cleavage fragment of COMP, in particular the sequence N-terminal-KSSTG (SEQ ID NO 1) ("COMP neo-epitope"), an antibody against this peptide and the use of such an antibody in diagnosis, prognosis and prevention of OA.

[0069] The peptide may have a length of from 5 to 135 amino acids in particular in horses, or from 5 to 117 residues in particular in humans, or preferably from 5 to 30, more preferably from 5 to 20 amino acids, more preferably from 5 to 9 amino acids, as long as the N-terminal has the sequence KSSTG. The lysine residue of this sequence of the peptide thus has the NHz group of the peptide. The peptide may have a length of at least 5 amino acid residues, more preferably at least 6 amino acid residues, more preferably at least 7 amino acid residues and most preferably at least 8 amino acid residues.

[0070] In one embodiment the peptide has a length such that it can be used for immunization.

[0071] The peptide may be an isolated peptide. The peptide may be isolated from for example synovial fluid, blood, plasma or serum, saliva, urine and cerebrospinal fluid. The peptide may also be synthesized, using methods known in the art, for example R. B. Merrifield (1963). "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide". J. Am. Chem. Soc. 85 (14): 2149-2154 and Schnolzer, M. A., P.;

[0072] Jones, A.; Alewood, D.; Kent, S.B.H. (2007). "In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis". Int. J. Peptide Res. Therap. 13 (1-2): 31-44.

[0073] The peptide can be used for the production and isolation of an antibody. The antibody specifically binds to a peptide comprising or consists of the amino acid sequence N-terminal- KSSTG. The term "antibody" also includes Fab, Fab', F(ab')2, Fv, scFv and single-chain antibodies and similar types of proteins that binds to an epitope with a high specificity, in particular proteins that are derived from or comprises a fragment from an antibody, in particular proteins that comprises a variable chain from an antibody that binds to N-terminal KSSTG. Methods for producing antibodies against a peptide are well known. The peptide can be used for screening for antibodies that bind to the peptide and also for purifying the antibody.

[0074] Preferably the antibody has a high affinity for the peptide. Affinity can be expressed using the dissociation constant (Kd). Preferred binding affinities include those with a dissociation constant (Kd) less than 10“6M, more preferably 5xl0“7M, more preferably 10“7M, more preferably 5xl0“8M, more preferably 10“8M, more preferably 5xl0“9M, more preferably 10“9M, more preferably 5xlO“10M, more preferably 1O“10M, more preferably 5xio-11M, more preferably 10-11M, more preferably 5xl0“12M, more preferably 10“12M, even more preferably 5xl0“13M, or and most preferably less than 10“13M. Preferably the antibody is an isolated antibody. The antibody may be a purified antibody. However, a high binding affinity may not be necessary for the uses described herein.

[0075] Preferably the antibody binds specifically to a peptide comprising or consisting of the sequence N- terminal-KSSTG (SEQ ID NO 1), such as N-terminal-KSSTGP (SEQ ID NO 2), or N-terminal-KSSTGPG (SEQ ID NO 3) or N-terminal-KSSTGPGE (SEQ ID NO 4), or N-terminal-KSSTGPGEQ (SEQ ID NO 5), or N- terminal-KSSTGPGEQL (SEQ ID NO 6), or N-terminal-KSSTGPGEQLR (SEQ ID NO 7), or N-terminal- KSSTGPGEQLRN (SEQ ID NO 8), or N-terminal-KSSTGPGEQLRNA (SEQ ID NO 9), or N-terminal- KSSTGPGEQLRNAL (SEQ ID NO 10) or N-terminal -KSSTGPGEQLRNALW (SEQ ID NO 11), or N- terminal-KSSTGPGEQLRNALC, SEQ ID NO 22.

[0076] For generating the antibody, it may be suitable to immunize with a peptide that is longer than N- terminal-KSSTG, for example the immunisation peptide N-terminal KSSTGPGEQLRNALC (SEQ ID NO 22).

[0077] In various embodiments of the invention, the sequence upstream of the COMP cleavage site is used. These peptides and antibodies against these peptides are used in the same manner as the other peptides described herein. In particular, a peptide with the sequence QLKAV-C-terminal (SEQ ID NO 12), or IQLKAV-C-terminal (SEQ ID NO 13), or GIQLKAV-C-terminal (SEQ ID NO 14), or PGIQLKAV-C- terminal (SEQ ID NO 15), or EPGIQLKAV-C-terminal (SEQ ID NO 16), or AEPGIQLKAV-C-terminal (SEQ ID NO 17), or VAEPGIQLKAV-C-terminal (SEQ ID NO 18), or AVAEPGIQLKAV-C-terminal (SEQ ID NO 19) may be detected.

[0078] In various embodiments, in particular when the subject is a horse, the cleavage fragment being detected may have the sequence:

[0079] N-terminal-

[0080] KSSTGPGEQLRNALWHTGDTASQVRLLWKDPRNVGWKDKTSYRWFLQHRPQVGYIRVRFYEGPELVADSNVVL DTTMRGGRLGVFCFSQENIIWANLRYRCNDTIPEDYEIQRLLQAGGKSSGGLGSLMRRHLQGL (SEQ ID NO 20) (horse)

[0081] In various embodiments, in particular when the subject is a human, the cleavage fragment being detected may have the sequence:

[0082] N-terminal-KSSTGPGEQL RNALWHTGDTESQVRLLWKD PRNVGWKDKK SYRWFLQHRP QVGYIRVRFY EGPELVADSN VVLDTTMRGG RLGVFCFSQE NIIWANLRYR CNDTIPEDYE THQLRQA (SEQ ID NO 21) (human).

[0083] The antibody may be any antibody derived from a mammal such as mouse, rat, hamster, rabbit, goat, horse or chicken, and the like, among which mouse and rabbit are preferred. The isotype of the antibodies may be any of IgG, IgM, IgE, IgA, IgY and the like.

[0084] The antibody may be a polyclonal antibody, produced by immunisation of an animal, for example a rabbit, as is known in the art. Preferably the antibody is a monoclonal antibody. Preferably the monoclonal antibody is a mouse or rabbit monoclonal antibody.

[0085] There are well-known methods for producing, purifying and isolating antibodies and determining their binding capacity. There are also well-known methods for using an antibody to determine the presence of an antigen. It is referred to Current Protocols in Immunology and Current Protocols in Molecular Biology for details.

[0086] For example, monoclonal antibodies against the peptide may be generated using the well-known hybridoma technology (Kohler and Milstein, Nature, 256, 495-497, 1975). A single clone can be isolated by limiting dilution analysis, the soft agar assay, a method using a fluorescence activated cell sorter and the like. In the limiting dilution analysis, for example, colonies of the hybridoma are serially diluted to around 1 cell / well in a medium before cultivation to isolate the hybridoma which produces the desired antibody. The antibody may be a chimeric antibody or a humanized antibody.

[0087] Antibody clones may also be generated using other methods, such as, for example, phage display. When the antibody is murine IgG, the antibody can be purified with affinity chromatography using a Protein A-conjugated carrier or an anti-mouse immunoglobulin-conjugated carrier.

[0088] The antibody can be used for diagnosis in different manners. The antibody can be used for measuring the presence, the amount of or concentration of the peptide in a sample from a subject, which may be a human or an animal. The antibody may be contacted with a sample from a subject. The sample may be any type of biological sample, for example synovial fluid, blood, saliva, plasma, serum, spinal fluid (liquor) or urine, ascites, or biological tissues used in histological section. Examples of useful tissues for sections include cartilage, tendon, bone, ligaments and synovial membrane. Preferably the sample is a liquid sample. In a preferred embodiment, the sample is a saliva sample, a serum sample, a blood sample, a plasma sample or a sample of synovial fluid. In one preferred embodiment the sample is a urine sample or a saliva sample, in particular a saliva sample.

[0089] A sample of a suitable volume is collected. As a non-limiting example, when the sample is in liquid form, in particular a saliva sample or a synovial fluid sample, a blood sample, a plasma sample or a serum sample, the sample may for example have a volume of from 50 pl to 2000 pl. However, smaller volumes may be used with more sensitive technologies. A saliva sample is suitable at least 300 ul, more preferably at least 500 ul. When the sample is saliva it may be useful to rest the subject before the sample is taken. The resting period may be for example at least 30 minutes, more preferably at least 1 hour. It may also be useful to compare the peptide level when resting with the peptide level immediately after exercise.

[0090] The sample may be isolated from a subject. The sample may be isolated from the subject before the contacting or binding step is carried out. Hence the method of diagnosis may comprise the step of providing a sample that has previously been isolated from a subject. The diagnosis method may be carried out in vitro.

[0091] A convenient manner to measure the concentration of a peptide in a sample with an antibody is ELISA (enzyme-linked immunosorbent assay) is, for example competitive ELISA. The design and use of ELISA well known in the art of diagnostics.

[0092] The antibody can moreover be used in, for example, immunohistochemistry. For example, thin sections of tissue, for example tissue that is frozen, or fixed and paraffin-treated, may be stained using the antibody as is known in the art of histopathology. The antibody can also be used in western blot or Wes or Simple Western systems. Peptide levels may be determined by quantifying a band from western blot. The antibody can be detected in various manners. A frequently used method is to use a secondary antibody that is conjugated with a substance that can be detected (a marker or label), for example an enzyme (such as HRP), a fluorophore or a radiolabel. For example, if the primary antibody is a mouse antibody, the secondary antibody can be a goat anti-mouse antibody. The presence of the marker can be detected with methods known in the art: an enzyme may be detected with reagents that produces a colour or light, a radiolabel may be detected with a scintillator or photographic film, and a fluorophore may be detected with a fluorescence detector or viewed in a fluorescence microscope.

[0093] Alternatively, the primary antibody (anti-N-terminal-KSST-antibody) may be directly conjugated with a marker or label that can be detected, as is known in the field of proteomics.

[0094] A suitable working concentration of the antibody when it is used in various procedures, such as for example ELISA or immunohistochemistry, depends on the affinity of the antibody and can be determined by testing different concentrations of the antibody in order to find a concentration that gives a good signal to noise ratio. As an example, an antibody stock with a concentration of 1 mg / ml may be diluted at 1 / 100, 1 / 200, 1 / 1000 and 1 / 5000 for testing a suitable working concentration. Working concentrations of the antibody in these procedures is usually in the range of pg / ml, for example from 1 ng to 10 pg / ml. The antibody is suitable diluted in PBS, possibly with the use of an additional protein such as BSA and a preservative, such as sodium azide.

[0095] The antibody can be used for diagnosis of a disease in a subject, in particular in a human or in a horse. However, the subject may also be a, a cow, a dog, a cat, a sheep, a pig, a rat or a mouse or any other mammal. For example, the concentration of the peptide in a sample can be determined using standard methods, for example ELISA. The thus determined concentration can be compared against a reference value ("cut-off value"). A peptide level above the reference value may be indicative of a particular disease, or a stage of the disease.

[0096] In various embodiments, the risk for developing a condition is determined. The subject may be suspected of having a condition. Presence of the peptide in the sample may be indicative of the condition or a risk for the condition.

[0097] The diagnosis may thus be diagnosis of osteoarthritis, early-stage osteoarthritis, moderate osteoarthritis, severe osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint, microfractures of the joints, chip fractures of the joint or risk for chip fractures of the joints. In particular, early stage or moderate stage OA may be diagnosed. In some embodiments the disease or condition is associated with damage to the deep zone of the cartilage of the joint. The method of diagnosis or the antibody can be used for diagnosis of osteoarthritis, in mammals, in particular in humans or in horses. In addition, dogs, cats, cows or pigs can be diagnosed.

[0098] In a preferred embodiment, high concentration of the peptide in a sample may indicate the presence of risk for a disease.

[0099] Low concentration of the peptide may indicate a healthy subject or that the osteoarthritis process has not progressed to disease.

[0100] Presence of the peptide may be used for monitoring the progression from physiological to pathological joint changes. Hence the method of diagnosis may be used for monitoring the progression from a healthy state or an early stage of the disease to any later stage of the disease, where the order of stages is: healthy, early stage osteoarthritis, moderate osteoarthritis, severe osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint, microfractures of the joints, and chip fractures of the joint. The order may also be: from healthy to osteoarthritis. Monitoring may be carried out by a user (physician or veterinarian) recording the peptide levels upon detection. Monitoring may comprise, at each time point, determining if treatment shall be performed, or if the patient shall be deemed to have a different diagnosis. Monitoring may comprise determining if a treatment of the patient has effect. Monitoring may result in changing or adding or initiating treatment.

[0101] The level of the peptide may increase with severity of the disease (from healthy subject to mild osteoarthritis, to moderate osteoarthritis, to chip fractures of the joints).

[0102] The reference value may depend on the method used and may be established using standard experiments and appropriate controls. The reference value may also depend on the type of sample and the type of subject (human or animal). For example, the level of the peptide in a sample of subjects with the disease (where the disease is determined in a different manner than using the peptide) is compared with the level of peptide in a group of healthy subjects. Hence the reference value may be predetermined. The reference value for disease may be for example 150%, more preferably 200 %, and most preferably 300 % of the average basal level for healthy subjects. A reference value for synovial fluid or serum for a disease herein may be for example be at least 1200 ng / ml, at least 1000 ng / ml, at least 900 ng / ml, at least 800 ng / ml or at least 700 ng / ml, or at least 600 ng / ml or at least 500 ng / ml. A reference value for saliva for a disease herein may be for example at least 3000 ng / ml, at least 2500 ng / ml, at least 2200 ng / ml, or at least 2000 ng / ml. However, suitable reference values are to be selected on the condition that is being diagnosed, the species of the subject and the type of sample. In some embodiments, a reference level of the COMP peptide is determined for a single subject (a "personalized" reference value). An increase of the level of COMP peptide from the reference level of the subject with more than 25 % more preferably more than 50 % and most preferably more than 100 % may be indicative of disease in the subject.

[0103] In various embodiments samples are repeatedly collected and analysed with a time interval in order to follow the progress of a disease or risk for disease, or the efficacy of treatment. Any suitable time interval may be used. In various embodiments the time between two sampling occasions may be from interval of from 1 day to 1 year may be used. In various embodiments, the time interval may be at least 3 days, or at least 7 days, or at least 10 days, or at least 14 days, or least 20 days or at least 1 month or at least 3 months. In some embodiments, a first sample is isolated at the latest on the same day where a treatment is provided to the subject and a second sample is isolated from the subject at least 7 days, or at least 10 days, or at least 14 days, or least 20 days or at least 1 month or at least 3 months after treatment has been provided to the subject. The time interval may have an upper limit, for example from 20 days, or 1 month, or 3 months or 1 year.

[0104] In other embodiments several samples may be taken on the same day, for example one sample before exercise and one sample after exercise.

[0105] For example, the peptide can be detected in a sample from a subject subjected to training. In case of increased concentration of peptide in for example a horse, in particular a non-lame horse, one treatment option is to decrease the amount of training. The training can be kept at low intensity for a period until the peptide level in synovial fluid, saliva or serum has gone down again. A predetermined reference value may be used or a reference value for an individual may be determined and used to monitor the individual. In this way, the optimal amount of training without risking the development of osteoarthritis for a subject may be established. The same method may be used for humans, in particular athletes.

[0106] The antibody and the necessary reagents may be included in a kit for detecting the peptide in a sample. The kit may be based on ELISA, for example competitive ELISA. The kit may include a stationary phase (such as a plate with wells), secondary antibodies, peptides, buffers and reagents for detecting the marker or label. The kit may include means for detecting other peptides (see below) for example antibodies against other peptides. Hence the kit may comprise an antibody that specifically binds to a peptide comprising the amino acid sequence N-terminal- SGPTH, where the antibody binds to the sequence N-terminal-KSSTG, or an antibody that specifically binds to a peptide comprising the amino acid sequence N-terminal- GLGHN, where the antibody binds to the peptide, or both. In a preferred embodiment, the kit comprises a sample collection device, in particular a saliva collection device, for example a saliva collection device for a human or a horse or other animals. A saliva collection device for human subjects may comprise a test tube with a funnel for a subject to spit a saliva sample into. A saliva collection device for horses or other animals may comprise a handle for holding the device connected to a swab. The user holds the handle and inserts the swab into the mouth of the subject, which may be a horse. The swab then absorbs a sample of saliva. After collection, the swab can be inserted into a sample tube for later analysis. Examples include the EquiSai saliva collection swab by Austin Davies Biologies Ltd. The sample collection device may comprise a buffer for preserving the sample.

[0107] The kit may comprise a diagnostic device for single use for example a lateral flow device which incorporates the antibody. Such lateral flow devices are known and typically comprise a solid phase, an antibody that specifically bind to an antigen of interest, and a detection means, for example reagents that generate a colour or a fluorescent signal or other signal, if the antibody binds to the antigen. The presence of a colour or fluorescent signal indicates the presence of antigen in the sample. The lateral flow device typically comprise means for determining the presence of a control substance in the sample (positive control). Examples of suitable lateral flow test include the ones disclosed in US6,485,982 and 1159,034,657 and references cited therein.

[0108] In some embodiments the amount of peptide is quantified by reading a colorimetric or fluorescent signal, for example from a lateral flow device. A reader may be arranged to read a colorimetric or fluorescent signal and quantify the signal. In this way, a level of peptide in sample is determined and compared to a reference value. A suitable reader for a diagnostic device may be the Reusable Reader from Lumos Diagnostics Ltd.

[0109] Diagnosis may also be carried out by detecting or determining the amount of the peptides described herein, such as N-terminal- KSSTG or QLKAV-C-terminal, by other means than an antibody. Suitable methods include sequencing peptides by mass spectrometry or sequencing by Edman degradation.

[0110] In various embodiments, the presence of a second peptide in a sample from the subject is determined or detected. The second peptide may be a biomarker for any of the diseases mentioned herein. The same sample which is analysed for N-terminal-KSSTG may be used, or a different sample from the same subject may be used. The second peptide may be N-terminal SGPTH (SEQ ID NO 25), which is a different cleavage fragment of the COMP protein. The second peptide may be N-terminal- GLGHN (SEQ ID NO 26) which is a cleavage fragment of the protein biglycan. In some embodiments the presence of all three peptides is determined. The use of N-terminal SGPTH is described in in WO2017216289. The detection of N-terminal- GLGHN is described in WO2022268940. The presence of these peptides in samples from subject generally correlates with OA or joint damage. The presence of the SGPTH peptide correlates with OA, in particular early phase OA, or the risk for these conditions. The presence of the GLGHN peptide may correlate with the subchondral bone destruction associated with bone sclerosis, fractures, chip fractures of the joint, avulsion fractures, bone bruise or osteoporosis, or risk for these conditions.

[0111] The inventors have surprisingly found that the OA patient group, in particular subjects with early OA, consist of subgroups that have different COMP cleavage products. Hence some OA patients may have the SGPTH peptide and not the KSSTG peptide, whereas other OA patients may have the KSSTG peptide and not the SGPTH peptide. Hence by detecting both peptides further patients may be diagnosed. In some embodiments, the subject has OA and has a KSSTG-peptide level above a reference value but is negative for the SGPTH peptide, i.e. a SGPTH level below a reference value.

[0112] The inventors have furthermore surprisingly found that the peptide N-terminal KSSTG is a marker for damage in the deep zone of the cartilage of the joint whereas N-terminal SGPTH is a marker for damage in the superficial layer of the cartilage. Hence using each peptide has the advantage of providing additional information about various endotypes of OA patients, by characterizing the disease at the molecular level. The deep zone may be characterised by chondrocytes arranged perpendicular to the joint surface and / or collagen fibrils parallel to the cells.

[0113] The KSSTG peptide may be used to determine that a subject has early or moderate OA rather than OA associated with damage to the bone, which may be associated with the N-terminal-GLGHN peptide.

[0114] Briefly, the same methods of detection as for N-terminal KSSTG may be used for the second and third peptide. The same type of sample may be used. Specific antibodies, for example monoclonal antibodies, to the respective peptides may be used. For example, one sample may be divided into three aliquots, and each aliquot is assayed for presence of one of the peptides. Alternatively, a multiplex technology may be used to detect all three peptide fragments in the same volume of sample. Various multiplex technologies are known in the art. For example, monoclonal antibodies from different species are used for each of the three analytes and secondary antibodies specific for the species are used, and where each secondary antibody is coupled to a different fluorophore. Fluorescence is then detected. In some embodiments, ELISA in a multiplex format is used.

[0115] In general, the methods and properties of the antibodies that are used for N-terminal-KSSTG (apart from specificity of the antibody) also apply to the other two peptides. For example, references values for the SGPTH peptide and the GLGHN peptide may be determined as above for the KSSTG peptide.

[0116] In various non-limiting embodiment, a reference value for the SGPTH peptide in the horse, in synovial fluid concentration, may be 500 ng / ml or more, 600 ng / ml or more, 1 pg / m or more 20 pg / ml or more, 30 pg / ml or more 40 pg / ml or more, or 200 pg / ml or more. These values may be indicative of early lameness and thus early OA. In various non-limiting embodiments, for the GLGHN peptide, a reference value for serum or synovial fluid in the horse may be 300 ng / ml or more, 500 ng / ml or more, more preferably 600 ng / ml or more, preferably 800 ng / ml and most preferably 1000 ng / ml or more. A cut-off value for saliva may be 20 ng / ml or more, more preferably 100 ng / ml or more, and more preferably 500 ng / ml or more, and most preferably 1000 ng / ml or more, or 1500 ng / ml or more or 2000 ng / ml or more.

[0117] However, suitable reference values are to be selected on the condition that is being diagnosed, the species of the subject and the type of sample.

[0118] Treatment

[0119] When the method has been used to determine a diagnosis, the subject may be treated. The condition being treated may be one or more of osteoarthritis, early-stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures or chip fractures of the joint.

[0120] The subject being treated may be any subject mentioned herein, such as a humans, horse, dog or cat. One useful treatment is intra-articular injections with a pharmaceutical or mixture of two or more pharmaceuticals. Pharmacological treatment may for example be treatment with a phosphodiesterase 5 inhibitor (PDE5 inhibitor) described in W02020 / 084113 which is incorporated herein by reference. In general, a pharmacologically effective amount of a PDE5 inhibitor or a sildenafil analogue is provided to a patient in need thereof. The PDE5 inhibitor may be selected from the group consisting of sildenafil, avanafil, lodenafil, mirodenafil, tadalafil, vardenafil and udenafil, hongdenafil, aildenafil, homosildenafil, nitrosoprodenafil, sulfoaildenafil and zaprinast, where sildenafil is preferred. The PDE5 inhibitor may be administered in any suitable manner, including by injection in the affected joint or orally, topically, intravenously, intradermally, or intraarticularly, intramuscular, nasal, or into the cerebrospinal fluid.

[0121] In a preferred embodiment the PDE 5 inhibitor is delivered locally to the affected joint. In an even more preferred embodiment, the formulation is delivered intraarticularly, in particular with one or more intraarticular injections into the affected joint, i.e. injection into the joint. Injection may be carried out once, twice or three times or more. The plurality of injections may be carried out with an interval of 2-60 days, more preferably 5-30 days, even more preferably 8-25 days and most preferably 10-18 days.

[0122] The PDE5 inhibitor may be administered by topical administration by, for example, transdermal administration. The transdermal formulation is specifically advantageous in regard of simplicity and from a patient comfort standpoint. It may, for instance, take the form of a transdermal patch.

[0123] In one embodiment, the PDE5 inhibitor or the sildenafil analogue is delivered so that at least some systemic distribution of the PDE5 inhibitor is achieved. This may be achieved local administration where the drug spreads through the body or with systemic administration, for example oral administration or intravenous administration.

[0124] In general, a pharmacologically effective amount of a PDE5 inhibitor or a sildenafil analogue is provided to a patient in need thereof. The appropriate dose range for the compound can be established in routine experiments. The dose may be lower than the sildenafil dose used to treat erectile dysfunction (which is often about 100 mg). The dose per administration of sildenafil given to a horse may be from 0.001 mg to 1000 mg, in particular 0.01 mg to 10 mg, in particular 0.01 mg to 1 mg, in particular 0.01 mg to 0.1 mg and most preferably 0.01 mg to 0.05 mg or 0.01 mg to 0.03 mg. Accordingly, the dose may be at least 0.001 mg, or at least 0.01 mg, or at least 0.1 mg, or at least 1 mg, or at least 10 mg. The dose given to human is approximately the same, in particular in the case of local administration, for example when intra-articular injection is used. Alternatively, a lower dose, which is approximately a fifth of the dose given to the horse, is given to the human.

[0125] The PDE5 inhibitor may be administered together with a local anaesthetic and glucose. Examples of useful local anaesthetic include bupivacaine, mepivacaine, lidocaine, prilocaine, ropivacaine, chloroprocaine and articaine. The local anaesthetic may be an amino amide type local anaesthetic, such as mepivacaine. The dose of glucose may be from 100 mg to 1000 mg, or from 150 mg to 400 mg. The dose of mepivacaine may be from 10 mg to 200 mg or from 10 mg to 30 mg. A pharmaceutical such an injection fluid, used to treat a condition herein, may comprise a PDE5 inhibitor, for example sildenafil, glucose and mepivacaine as described in W02020 / 084113.

[0126] Another treatment option is to rest the subject. For example, the amount of training is reduced. A suitable resting time may be determined by monitoring the level of the peptide as described herein. A suitable resting time may be at least one week, before commencing training again, but more likely it will be at least one month. Furthermore, when the subject is a human, in particular an athlete, the treatment may be to reduce training or to rest.

[0127] Determination of risk for joint damage

[0128] The presence of the peptide correlates with joint damage. The peptide is secreted into various tissues and biological fluids when the joint is subjected to physical stress. Such stress may arise for example in a human during for example exercise such as running, or in a horse when exercising the horse. The risk for joint damage also depends on the properties of the shoes or the flooring (or horseshoe when the subject is a horse), where a different or softer shoe decreases the risk for damage. However, the softness of the shoe and the flooring may not fully explain the risk for joint damage. The peptide may be used as a research tool when determining joint damage or risk for joint damage.

[0129] The peptide or antibody or method herein may be used for determining a risk related to a shoe for a human such as working shoes, running shoes or other types of sports shoes, or the evaluation of floorings in particular flooring for sports or work, in particular flooring for workplaces where the worker need to stand or walk long periods of time such as for example hospitals or restaurants. Example of flooring materials that can be evaluated include materials for sports tracks or sport arenas, such as football fields or running tracks.

[0130] The antibody, peptide or methods herein may be used for determining the risk for a human subject to damage the joints at the workplace, in particular a workplace where the workers need to stand or walk for long periods of time such as for example hospitals or restaurants.

[0131] The antibody, peptide or methods herein may be used for determining the risk for groups of athletes or activities, in particular athletes in sports that involve running or jumping such as, for example, football, gymnastics, volleyball, basketball, and track and field, or activities such as aerobics, dancing, weightlifting, running or hiking. The conformation of the equine hoof is considered an important factor affecting performance of the horse. Empirical observation, personal experience, and pragmatism have sustained the activities of trimming and shoeing for thousands of years (Mark Caldwell, (The Art and Science of Horse Shoeing, blogpost, April 29, 2022). The selection of the proper horseshoe and its attachment is crucial for the wellbeing of the horse. The peptide or the antibody or the method herein may be used for determining the properties of a horseshoe or method of attaching a horseshoe, or for determining if a certain horseshoe or method of attaching a horseshoe to a hoof is suitable for a certain horse.

[0132] The peptide or antibody may be used for determining the properties of a footing for a horse arena. A softer footing will generate less impact and stress to the joint and will result in a lower amount of the peptide in a sample from the horse.

[0133] Hence, a method for determining risk for joint damage may comprise obtaining a sample from a subject, or a group of subjects that has been exposed to the risk (such as wearing a particular shoe or participating in a certain activity) and analysing the sample for presence of a peptide in the sample. The subjects are preferably rested before being exposed to the risk. The results may be compared to results from a control group that has not been exposed to the risk. Alternatively, the peptide levels before and after being subjected to the risk is determined.

[0134] The peptide, antibody or methods herein may be used for selecting a shoe for a subject such as a shoe for an individual human or a horseshoe (or way of attaching a horseshoe) for an individual horse. For example, the subject is first rested and then allowed to use the shoe. A sample may be isolated from the user after the use, and optionally also before the use. The level of peptide in the sample is determined and used to determine if the shoe (human) or horseshoe. In some embodiments the method is used for selecting a horseshoe or choosing not to have a horse shoe on the horse.

[0135] The reference levels described above may be used. As an example, a reference level in saliva may have a maximum value of for example 3000 ng / ml, 2500 ng / ml 2200 ng / ml, or 2000 ng / ml.

[0136] EXAMPLE 1

[0137] The COMP cleavage site AVAEPGIQLKAV* KSSTGPGEQLRNAL (SEQ ID NO 24) where the * indicates the cleavage site is located between amino acids 644 and 645 from the N-terminal of native horse COMP. The corresponding cleave site in the human sequence is between residues 646 and 647.

[0138] A monoclonal antibody against the amino acid peptide N-terminal KSSTGPGEQLRNALC (SEQ ID NO 22) was produced (GenScript) in rabbits.

[0139] Bioinformatics analysis revealed the amino acid sequence around the cleavage site (AVAEPGIQLKAV* KSSTGPGEQLRNAL) is completely conserved in all analysed mammal species (human, horse, dog, cat, cow, pig, mouse) (Data not shown). Hence it can be expected that the antibody will be useful for detecting the neo-epitope in all mammals.

[0140] EXAMPLE 2

[0141] An inhibitory ELISA was developed and evaluated for detection of the COMP neo-epitope in horse serum using a chemically synthesised peptide KSSTGPGEQLRNALC (SEQ ID NO 22) also referred to as "COMP644peptide", below. A chemically synthesized peptide with the sequence KSSTGPGEQLRNALC (SEQ ID NO 10) was used as a calibration standard. The freeze-dried peptides were reconstituted according to the GenScript peptide solubility guidelines. In short, the peptides were reconstituted in distilled water to a concentration of 2mg / mL and thereafter aliquoted, frozen and stored in -80°C until use.

[0142] The inhibitory ELISA started with coating the plate with COMP644peptide. Using Nunc MaxiSorp™ Clear Flat-Bottom 96-Well Plates (Invitrogen) and the addition of COMP644peptide (100pL / well, GenScript) diluted to lpg / mL in lOOmM carbonate buffer with pH 9.6, the peptide was coated over night at 4°C, denoted as the ELISA-plate.

[0143] The calibration standard peptide was prepared from the stock solution. (2mg / mL). First the highest standard point was set at 2000 ng / mL with a dilution in Sample Extra (Kementech) and thereafter using 9 step-l:2 serial dilution (ImL peptide + ImL Sample Extra (Kementech) the calibration standard was made ranging from 0 (the 9th with no peptide) to 2000 ng / mL.

[0144] Serum samples were prepared by dilution 1:4, in Sample Extra (Kementech). Serum dilution was determined after analyzing the serial dilution of normal serum where the primary antibody found the most peptide at 1:4 dilution. As a serum control we used Equidae serum (lot.2109875, Gibco).

[0145] The monoclonal antibody of Example 1 (0.445mg / mL) was used as primary antibody. The primary antibody was diluted in Sample Extra (Kementech) to a concentration of 30ng / mL. lOOpL of each concentration of calibration standard and samples (in duplicates) was added to Nunc™ 96-well polypropylene Sample processing & Storage Microplates (Life Technologies). The 30ng / mL diluted primary antibody (100pl / well) was added to each standard as well as samples and thereafter preincubated overnight in humid chamber within a rotation incubator (250rpm) with temperature set at 37°C.

[0146] Overnight, after 17 hours, the coated ELISA-plate was washed 4 times in the wash buffer (10 mM PBS with 0.05 % Tween, pH 7.4) using Tecan Hydro wash and thereafter blocked with synthetic blocker (Kementec) for 1 hour at 37°C. After the blocking step the pre-incubated standards and samples (lOOOpL / well) were transferred to the ELISA-plate and incubated for 1 hour at room temperature on the ELISA-plate shaker set at 600rpm. After the 1-hour incubation with primary antibody, standard and samples, the ELISA-plate was washed 4 times in wash buffer. The secondary polyclonal goat anti rabbit (IgG) HRP lmg / mL (Abeam) was diluted 1:50 000 in 10 mM PBS with 0.05 % Tween and 0.1% BSA, pH 7.4. Then, lOOpL / well of the secondary antibody was added to the standard and sample wells in the ELISA-plate and incubated in the dark for 30 minutes on the ELISA-shaker set at 600rpm. Thereafter the ELISA-plate was washed 8 times in wash buffer. Next TMB was added, lOOpL / well, and incubated in the dark at RT and stopped after 5 minutes with 0.18M H2SO4. Absorbance was evaluated at 450nm and scanned in SPARK multifunctional plate reader using Magellan software (Tecan).

[0147] To evaluate the specificity of the primary antibody we used an overlapping control peptide (OL) with the sequence QLKAVKSSTGPGEQLRNAL (SEQ ID NO 23) as coating peptide and as antigen in the preincubation were we made series dilution such as the calibration standard.

[0148] The intra-assay precision was investigated for the inhibitory COMP644peptide ELISA. The Equidae control serum were used in 6 replicates. The inter-assay variation was also examined for the Equidae control serum as 6 replicates in a total of 5 assays on different occasions. The lowest and highest detections level was investigated during one ELISA with n=6 replicates of the standard curve points and a CV calculation of the replicates below 20% was granted as detectible values. An assay of spike and recovery was also done using 2 different samples with spiked 125ng / ml. The recovery was thereafter calculated.

[0149] The specificity of the primary antibody against COMP644peptide and the overlap peptide (OL) was tested. Both peptides were serial diluted as calibration standards ranging from 0 - 2000 ng / mL. The monoclonal antibody showed high specificity for COMP644peptide. We verified that the monoclonal antibody could not detect the OL peptide (Fig.l). This shows that the epitope was detectable in serum, and that the N-terminal of the epitope was crucial for antibody binding. EXAMPLE 3

[0150] The concentration of COMP neoepitope N-terminal-KSSTG in synovial fluid from horses was analyzed.

[0151] The following cohorts were sampled:

[0152] Normal joints: joints from 13 healthy horses with macroscopically normal cartilage and bone.

[0153] Mild osteoarthritis ("OA"): joints from 7 horses with mild articular cartilage lesions as defined by arthroscopy.

[0154] Moderate osteoarthritis: joints from 8 horses with moderate articular cartilage lesions as defined by arthroscopy. These horses have a high risk of developing chip fractures.

[0155] Chip fractures: joints from 14 horses with severe articular cartilage lesions (i.e. chip fractures), as defined by arthroscopy.

[0156] Synovial fluid was diluted 4x in Sample Extra (Kementech). The concentration of the neo-epitope in synovial fluid showed a significant increase in joints with chip fractures compared to normal joints (P < 0.0001). Additionally, the concentration of COMP neo-epitope in synovial fluid showed a significant increase in joints with moderate osteoarthritis compared to normal joints and (P < 0.05) and chip fractures compared to mild osteoarthritis (P < 0.05). (Fig 2).

[0157] This shows that detection of the inventive peptide N-terminal-KSSTG is useful for diagnosis of various stages of OA, and that detection of the peptide can be used to monitor the progress of the disease.

[0158] EXAMPLE 4

[0159] The COMP neo epitope N-terminal-KSSTG was detected in serum from healthy horses.

[0160] Material (horses): 19 young Standardbred trotters in training. The horses started their training at the age of 1.5 years. Saliva samples was taken every 6 weeks for up to 9 months month. No difference in the serum concentration of the COMP neoepitope was found in these horses. We conclude that degradation of COMP has not started yet and therefore no increase in neoepitope concentrations could be observed. (Fig. 3). However, this showed that the peptide could be detected in serum and that the level of peptide was stable over time in healthy individuals. EXAMPLE 5

[0161] Presence of the COMP neo-epitope in saliva samples was determened using the ELISA desribed above above.

[0162] Horses with osteoarthritis were recruited from Hallands Djursjukhus, Kungsbacka Horse clinic, Sweden (N =5) and University Animal Hospital (UDS), Uppsala, Sweden (N =4). The recruited horses showed clinical lameness by the reaction to the flexion test and were diagnosed with osteoarthritis by determining visible abnormalities in the joint or by radiology or ultrasound. The reference group samples were saliva collected from Standardbred trotters (N =19) aged 1.5 years, trained by the same professional trainer. The horses were entered into a long-term training program just a month before saliva collection, followed by a training program with a slow trot distance of 2 km, a maximum of four days per week.

[0163] Saliva was collected from horses using Equisal saliva collection kit (Austindavis biologies ltd), inserting the swab through the interdental space on the horse tongue until the volume indicator changed colour. Approximately 600 pl of crude saliva was sampled with the device. The samples were stabilised in 1 ml preservative buffer (lxPBS sodium chloride 137 mM, potassium chloride 2.7 mM, disodium phosphate 11.9 mM), 0.05% tween 20, 0.05% bromonitrodioxane and 0.05% sodium azide), temporarily stored at -20°C before being thawed and centrifuged at 3000g for 5 min. All samples were aliquoted in a minimum volume of 500 pl and stored at -80°C prior to analysis.

[0164] The results are shown in Fig. 4 Horses with radiographic changes associated with osteoarthritis ("OA") showed a significantly increased concentration of COMP neo-epitope in saliva compared to reference horses.

[0165] EXAMPLE 6

[0166] A short-term training study was performed on riding horses (N =7) recruited from Ida farm, Wellington, Florida, USA. The horses were trained on two different footings in a crossover design: a) sand (CapillaryFlow-Wellington, FL, USA) and b) sand-fibre. The Orono biomechanical surface tester (OBST) was used to measure the surface of the tracks in vertical and horizontal directions, for impact firmness, cushioning, responsiveness, grip and uniformity and were graded accordingly (Adepu et al, Osteoarthr Cartil Open 2023 Mar 15;5(2):100354). The mean number of measured test sites for all variables per arena was 15. It was concluded, based on the measurements, that sand-fibre footing was harder than sand footing.

[0167] The saliva collection was carried out according to the following scheme: TP1) In the stable at rest i.e., 1 hour pre-training, TP2) 10 min post warmup (5 min free-walk and 5 min walk, trot and canter), TP3) 20 min post-training (intensive workout with increased collection in all gaits), TP4) 5 min post cooldown (freewalk) and TP5) 1 h post-training respectively. The total intense interval was 20 min. The total warm-up and cool-down times were 15 min.

[0168] According to OBST grading sand footing is graded as ideal for impact firmness, cushioning, responsiveness, grip and uniformity where sand-fiber footing is graded as harder for impact firmness and more compact for cushioning when compared to sand footing. It was was observed that saliva COMP neoepitope levels increased at T3 (intensive exercise interval) during the training and this increase was seen on sand-fiber footing which is more hard and compact compared to sand footing (Fig 5). This indicates that that detecion of the peptide is useful for testing the impact to the load on various footings.

[0169] EXAMPLE 7

[0170] Horses (n=10) diagnosed with mild osteoarthritis were treated with one intra-articular injection of combination of sildenafil, mepivacaine and glucose (visit 1). The injection solution comprised sildenafil: 0.002 mg / ml, mepivacaine: 2 mg / ml, glucose: 20 mg / ml. 10 ml was administered to each horse hence the dose was sildenafil: 0.02 mg, mepivacaine: 20 mg and glucose 200 mg.

[0171] A sample of synovial fluid was obtained from each horse at visit 1- and 14-days post treatment (visit 2). A significant reduction of the COMP neoepitope was found at visit 2 (Fig 6). This shows that presence of the neoepitope can be used to monitor the treatment of osteoarthritis.

[0172] EXAMPLE 8

[0173] Histopathology on articular cartilage sections from horses with OA was carried out using polyclonal antibodies against the peptide N-terminal-KSSTG and N-terminal SGPTH, respectively.

[0174] Articular cartilage was immersed in 10% buffered formaldehyde, dehydrated, embedded in paraffin and tissue sections were stained with a polyclonal antibody against the COMP156 and COMP644 neo-epitope and native COMP(Ab74524). Briefly, specimens were sectioned and mounted onto slides, deparaffinized, rehydrated and washed in phosphate-buffered saline (PBS; 0.01 mol / L phosphate, 0.15 mol / L NaCI, pH 7.4). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in PBS. Nonspecific binding was blocked with 2% normal goat serum(X0907), prior to incubation with antiserum. After rinsing in PBS, sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (K5007) and visualization (Real EnVision™ Detection Systemk5007 [ready-to-use kit]) was performed using the color developer 3,3- diaminobenzidine. For negative (isotype) controls, the primary antibody was substituted with nonimmune rabbit serum (X0936) at the same dilution used for the primary antibody. The sections were evaluated by light microscopy.

[0175] Fig. 6 shows staining of native COMP in the extracellular matrix of the osteoarthritic joint. Staining of SGPTH peptide is prominent in the cells of the superficial layer of the osteoarthritic joint.

[0176] Fig. 7 shows taining of native COMP in the extracellular matrix of the osteoarthritic joint. Staining of KSSTG peptide is prominent in the cells of the deep zone of the osteoarthritic joint but absent from the matrix and absent from the upper layers.

[0177] Microspy of hematoxhylin / eosin staining was used to verify that the location of the damage to the cartilage correlated with the staining of the respecctive peptide.

[0178] We conclude that the KSSTG peptide is specific for damage to the deep zone of the joint.

[0179] EXAMPLE 9

[0180] The presence of the N-terminal-KSSTG peptide (COMP cleavage fragment 2) as well as the N- terminal-SGPTH peptide (COMP cleaveage fragment 1) and the N-terminal-GLGHN peptide (Biglycan cleavage fragment) was determined using ELISA assays out in synovial fluid samples from horses with early phase OAThe results are seen in Table 1. Table 1. Concentration of respective peptide synovial fluid from the carpal joints of horses. Reference values: SGPTH: 600 ng / ml, KSST: 600 ng / ml, GLGHN: 2000 ng / ml

[0181] Two horses, Hera and Giant Laser (enhanced outlines in Table 1) has KSSTG peptide above the reference values whereas they have SGPTH concentration and GLGHN concentrations below the reference values. We conclude that the COMP2 fragment (N-terminal KSSTG) provide independent diagnostic information.

[0182] These findings highlight the importance of precision diagnosis, which enables targeted, endotype- driven treatment for osteoarthritis.

Claims

CLAIMS1. A method of diagnosis comprising providing a sample previously isolated from a subject and analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal- KSSTG (SEQ ID NO 1).

2. The method of claim 1 where the diagnosis is diagnosis of a disease is selected from: osteoarthritis, early stage osteoarthritis and moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures and chip fractures of the joint.

3. The method of claim 2 where the disease is associated with damage to the deep zone of the cartilage of the joint.

4. The method of anyone of claims 1 to 3 where in addition, a sample previously isolated from the patient is analysed for the presence of a peptide comprising the amino acid sequence N- terminal-SGPTGH (SEQ ID NO 25) or N-terminal-GLGHN (SEQ ID NO 26).

5. The method of claim 5 where the sample is analysed for the presence of N-terminal-SGPTH (SEQ ID NO 25).

6. The method of claim 5 where the sample is analysed for the presence of N-terminal-GLGHN (SEQ ID NO 26).

7. The method of claim 5 where the sample is analysed for the presence of both N-terminal-SGPTH (SEQ ID NO 25) and N-terminal-GLGHN (SEQ ID NO 26).

8. The method of any one of claims 1 to 7 where the sample is a sample of synovial fluid, serum, blood, blood plasma, urine or saliva.

9. The method of any of claims 1 to 8 where the method is carried out by repeatedly obtaining samples from an individual and determine the presence of the peptide in the sample, and thereby monitoring the progression of the disease.2910. A method for preventing osteoarthritis, early-stage osteoarthritis, moderate osteoarthritis, osteoarthritis associated with risk for chip fractures of the joint or risk for avulsion fractures, severe osteoarthritis, microfractures of the joints, avulsion fractures and chip fractures of the joint in a subject comprising the steps of: a) repeatedly isolating samples from the subject and analysing the presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1) in the samples, b) if the level of peptide in the subject in a sample is above a predetermined level, determining that the subject should be treated.

11. The method of claim 10 where the treatment comprises resting the subject.

12. The method of any one of claims 10 or 11 where in addition, samples are analysed for the presence of a peptide comprising the amino acid sequence N-terminal-SGPTGH (SEQ ID NO 25) or N-terminal-GLGHN (SEQ ID NO 26).

13. The method of claim 12 where the sample is analysed for the presence of N-terminal-SGPTH (SEQ ID NO 25).

14. The method of claim 12 where the sample is analysed for the presence of N-terminal-GLGHN (SEQ ID NO 26).

15. The method of claim 12 where the sample is analysed for the presence of both N-terminal- SGPTH (SEQ ID NO 25) and N-terminal-GLGHN (SEQ ID NO 26).

16. An antibody that specifically binds to a peptide comprising the amino acid sequence N-terminal- KSSTG (SEQ ID NO 1) where the antibody binds to the sequence N-terminal KSSTG.

17. A kit comprising an antibody according to claim 16.

18. The kit according to claim 17 further comprising an antibody that specifically binds to a peptide comprising the amino acid sequence N-terminal-SGPTGH (SEQ ID NO 25) where the antibody binds to the sequence N-terminal-SGPTH, or an antibody that specifically binds to the sequenceN-terminal-GLGHN (SEQ ID NO 26), where the antibody binds to the sequence N-terminalGLGHN.

19. A method for determining the risk for joint damage comprising obtaining a sample from a subject and analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

20. The method of claim 19 where in addition, a sample from the subject is analysed for the presence of a peptide comprising the amino acid sequence N-terminal SGPTGH (SEQ ID NO 25) or N-terminal- GLGHN (SEQ ID NO 26).

21. The method of claim 20 where the sample is analysed for the presence of N-terminal SGPTH (SEQ ID NO 25).

22. The method of claim 20 where the sample is analysed for the presence of N-terminal- GLGHN (SEQ ID NO 26).

23. The method of claim 20 where the sample is analysed for the presence of both N-terminal SGPTH (SEQ ID NO 25 and N-terminal GLGHN (SEQ ID NO 26).

24. The method of any one of claims 19 to 23 where the risk relates to a shoe for a human, indoor or outdoor flooring, working conditions for humans, a sports activity for a human, a horseshoe or footing for a horse, or an exercise program for a subject.

25. A method for evaluating a floor or a shoe comprising a) allowing a test subject to use the floor or the shoe, b) isolating a sample from the test subject, c) analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

26. The method of claim 25 where in addition, a sample from the subject is analysed for the presence of a peptide comprising the amino acid sequence N-terminal-SGPTGH (SEQ ID NO 25) or N-terminal-GLGHN (SEQ ID NO 26).

27. A method for evaluating a footing, a horseshoe or a method of attaching a horseshoe, or comprising a) allowing a horse to use the footing or the horseshoe or the method of attaching the horseshoe, b) isolating a sample from the horse, c) analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

28. A method of selecting a horseshoe or a method of attaching a horseshoe for an individual horse comprising a) allowing a horse to use the footing or the horseshoe or the method of attaching the horseshoe, b) isolating a sample from the horse, c) analysing the sample for presence of a peptide comprising the amino acid sequence N-terminal-KSSTG (SEQ ID NO 1).

29. The method of any one of claims 27 or 28 where in addition, a sample from the subject is analysed for the presence of a peptide comprising the amino acid sequence N-terminal- SGPTGH (SEQ ID NO 25) or N-terminal-GLGHN (SEQ ID NO 26).

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