Metabolic biomarkers that predict response to FGF-18 compounds

Abstract

The present invention relates to biomarkers associated with clinical response to FGF-18 compounds before or during treatment of cartilage disorders. More specifically, the present invention relates to specific proteins present in blood, serum, synovial fluid, or urine that can be used for the diagnosis and treatment of cartilage disorders. The present invention further discloses specific cartilage-related proteins that respond to treatment with FGF-18 compounds, as well as diagnostic tools and kits based on their expression profiles. Thus, the present invention can be used to predict response to treatment with FGF-18 compounds before or during treatment with FGF-18 compounds. The present invention can be used to select / identify subjects to be treated with intra-articular administration of FGF-18 compounds. The use of these biomarkers in diagnosis may lead to increased benefit and reduced risk for subjects. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention generally relates to pharmacogenetics, and more specifically, to biomarkers associated with clinical response to FGF-18 compounds before or during treatment of cartilage disorders. More specifically, the present invention relates to specific proteins present in blood, serum, synovial fluid, or urine that can be used, inter alia, for the diagnosis and treatment of cartilage disorders. The present invention further discloses diagnostic tools and kits based on specific cartilage-associated proteins that respond to FGF-18 compound treatment and their quantity or expression profiles. Thus, the present invention can be used to predict response to FGF-18 compound treatment before or during treatment with FGF-18. The present invention can be used to select / identify subjects to be treated with intra-articular administration of FGF-18 compounds. The use of these biomarkers in diagnosis may lead to increased benefit and reduced risk for subjects. [Background technology]

[0002] Cartilage disorders broadly refer to diseases characterized by abnormal metabolic degeneration in connective tissue, resulting in pain, stiffness, and limited movement in affected body parts. These disorders can be caused by pathology or the result of trauma or injury. Among other things, cartilage disorders include osteoarthritis (OA), cartilage damage (including sports injuries of cartilage and joints, and surgical injuries such as microfractures), and other conditions. Mature cartilage has a limited ability to repair itself, particularly due to the lack of proliferation capacity and lack of blood vessels in mature chondrocytes. Furthermore, cartilage is nutrient-poor and has low oxygen tension. Replacing cartilage damaged by injury or disease, especially articular cartilage, presents significant challenges for physicians, and available surgical procedures are considered to be unpredictable and have limited durations of effectiveness.

[0003] Therefore, most young subjects either do not seek treatment or are advised to postpone it for as long as possible. When treatment is necessary, standard procedures vary depending on age and include total joint replacement, cartilage fragment implantation, or bone marrow stimulation techniques (e.g., microfracture). Microfracture is a common procedure involving penetration of the subchondral bone to stimulate cartilage deposition by bone marrow-derived stem cells. However, this technique has been shown to not adequately repair the cartilage defect, and the new cartilage formed is primarily fibrocartilage, resulting in insufficient or altered function and biomechanics. Indeed, fibrocartilage does not have the same durability and may not properly adhere to the surrounding hyaline cartilage. Therefore, the newly synthesized fibrocartilage may be easily damaged (expected time frame: 5–10 years).

[0004] For subjects with osteoarthritis, non-surgical treatments include, among others, physical therapy, lifestyle modifications (e.g., increased physical activity), supportive devices, oral and injectable medications (e.g., nonsteroidal anti-inflammatory drugs), walking aids, and medical symptom management. Once these treatments fail, surgery, such as joint replacement, becomes the subject's main option. Osteotomy (cutting bone to rebalance worn joints) of the tibia or femur can alleviate symptoms, help maintain an active lifestyle, and delay the need for total joint replacement. Total joint replacement can provide symptomatic relief for advanced osteoarthritis, but generally requires a change in the subject's lifestyle and / or activity level.

[0005] At that time, available drug treatments were primarily directed at pain relief, and there were still no commercially available treatments that reversed or delayed cartilage damage (see Lotz, 2010).

[0006] Fibroblast growth factor 18 (FGF-18) is a member of the FGF family of proteins, closely related to FGF-8 and FGF-17. FGF-18 has been shown to be a proliferation agent for chondrocytes and osteoblasts (Ellsworth et al., 2002; Shimoaka et al., 2002; Gigout et al., 2017). FGF-18 has been proposed alone (WO2008 / 023063) or in combination with hyaluronic acid (WO2004 / 032849) for the treatment of cartilage disorders such as osteoarthritis and cartilage damage.

[0007] Sprifermin is a truncated form of human FGF-18 that is being investigated in clinical trials for the treatment of both osteoarthritis and cartilage damage (for details, see, e.g., NCT01033994, NCT00911469, and NCT01066871). The current dosing regimen for Sprifermin is once a week for three weeks (one treatment cycle), and the drug is administered by intra-articular injection. This treatment cycle can be repeated. This dosing regimen is described in WO2008 / 023063.

[0008] At this point, treatment of OA (osteoarthritis) and cartilage damage with FGF-18 in clinical trials is being administered to subjects without predictive information regarding response, i.e., whether the treatment is likely to be highly effective, moderately effective, only slightly effective, or ineffective (Lohmander et al., 2014; Dahlberg et al., 2016). Currently, after at least one treatment cycle with sprifermin, the majority of the treated population shows a moderate / high response to treatment as measured by MRI technology in cartilage thickness and WOMAC score, but there are also some patients who do not respond to treatment (i.e., no or limited increase in cartilage thickness measured by MRI technology) or who respond but have a higher WOMAC score compared to controls.

[0009] WO2014 / 023703 describes genetic markers (combinations of SNPs, IL-1RN rs9005 and IL-1RN rs315952) associated with the quality of clinical response to treatment of cartilage disorders such as OA, cartilage damage, or microfractures with FGF-18. Such markers are useful for identifying, through pre-treatment genetic screening, subgroups of subjects likely to have a particular response to treatment with FGF-18, such as a very good clinical response to treatment with FGF-18, or conversely, subjects likely to fail the therapy.

[0010] Knowledge of the type of clinical response of a subject to treatment can be used to optimize or select a therapy (such as selecting a treatment using FGF-18 as the first-line therapy), or adapt a dosing regimen.Such information will be clinically useful for the medical management of cartilage disorders, such as OA and / or cartilage damage, in a subject.For example, if an individual with OA or cartilage damage is known to have a high risk of not responding to FGF-18 treatment, a doctor can exclude the subject from FGF-18 treatment, and provide the patient with a risk-free treatment without unnecessary treatment.In addition, such predictive information is also clinically useful for guiding decisions regarding dosing regimens.

[0011] There is a need to identify additional biomarkers that aid in therapy optimization or therapy selection to provide a broader range of solutions for the subjects being treated or for physicians searching for the best therapy for their own patients. Summary of the Invention

[0012] The metabolic biomarkers described herein, such as proC2 or CTX-II, can be used, either alone or in combination, in the detection, diagnosis, and / or treatment of patients with osteoarthritis or cartilage disorders. The expression level (or amount) of at least one of these biomarkers (or a combination thereof) can be used, for example, to detect patients who should be included in or excluded from a particular treatment.

[0013] The present invention provides a method for predicting sensitivity to treatment with an FGF-18 compound in a subject with a cartilage disorder, comprising: a) determining from the sample the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2; b) predicting, from the result of step a), whether the subject is highly, intermediately, poorly or insensitive to treatment with an FGF-18 compound.

[0014] According to the above method, the presence of CTX-II greater than 350±2 SD ng / mmol (>240-260% of the normal mean) and / or ProC2 greater than 4.2±2 SD ng / mL (>120-280% of the normal mean) is indicative of no or a poor response (i.e., poor sensitivity or insensitivity) to treatment with an FGF-18 compound. Conversely, the presence of CTX-II less than 350±2 SD ng / mmol and / or ProC2 less than 4.2±2 SD ng / mL is indicative of a moderate (i.e., moderate sensitivity) or high (i.e., high sensitivity) response to treatment with an FGF-18 compound.

[0015] Also provided is a method of selecting subjects with cartilage disorders for inclusion or exclusion from treatment or clinical trials with FGF-18 compounds based on their likelihood of sensitivity to said treatment, comprising: a) determining from the sample the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2, wherein the amount of at least one of said proteins is indicative of the subject's risk for being susceptible or insensitive to said treatment; and Also described herein are methods comprising: b) selecting a susceptible subject as suitable for said treatment.

[0016] According to the above method, subjects exhibiting CTX-II greater than 350±2 SD ng / mmol and / or ProC2 greater than 4.2±2 SD ng / mL are excluded from (i.e., not selected for) treatment with FGF-18 compounds. Conversely, subjects exhibiting CTX-II less than 350±2 SD ng / mmol and / or ProC2 less than 4.2±2 SD ng / mL are included in (i.e., selected for) treatment with FGF-18 compounds.

[0017] Also provided is a method for treating a subject with cartilage damage using an FGF-18 compound, comprising: a. determining from the sample the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2, wherein said amount is indicative of the subject's risk for being highly or moderately sensitive to treatment with said FGF-18 compound; b. selecting subjects with CTX-II lower than 350±2SD ng / mmol and / or ProC2 lower than 4.2±2SD ng / mL; c. intra-articularly administering an FGF-18 compound to the subject selected in step b.: Also described is a method comprising:

[0018] Generally, in certain embodiments of the invention, i.e., in any of the methods or uses referred to herein, the FGF-18 compound used as a treatment is sprifermin or a fusion protein comprising an FGF-18 moiety, and the subject has a cartilage disorder selected from the group consisting of osteoarthritis, cartilage damage, a fracture affecting articular cartilage, or a surgical procedure impacting articular cartilage (e.g., a microfracture).

[0019] It should be understood that in any of the methods or uses mentioned herein, before determining the amount of at least one protein, it is necessary to obtain a sample (or test sample) from the subject, for example by collection of blood, serum, synovial fluid, urine, etc. It should further be understood that any of the methods or uses mentioned herein are carried out in vitro and not on an animal or human body.

[0020] (definition) As used herein, the term "FGF-18 compound" or "FGF-18" refers to a protein that maintains at least one biological activity of human FGF-18 protein. FGF-18 may be native, its mature form, or a truncated form. The biological activity of human FGF-18 protein is, inter alia, increasing osteoblast activity (see WO98 / 16644) or increasing chondrogenesis (see WO2008 / 023063). Native, or wild-type, human FGF-18 is a protein produced mostly during skeletal development and is involved in bone and cartilage formation (see Haque et al., 2007). Human FGF-18 was originally designated zFGF-5 and is fully described in WO98 / 16644. SEQ ID NO: 1 corresponds to the amino acid sequence of native human FGF-18, along with a signal peptide consisting of amino acid residues 1 (Met) to 27 (Ala). The mature form of human FGF-18 corresponds to the amino acid sequence from residue 28 (Glu) to residue 207 (Ala) of SEQ ID NO:1 (180 amino acids).

[0021] In the present invention, FGF-18 compounds may be produced by recombinant methods, as described in application WO2006 / 063362. Depending on the expression system and conditions, the FGF-18 of the present invention is expressed in recombinant host cells with a starting methionine (Met) residue or with a signal sequence for selection. When expressed in a prokaryotic host such as E. coli, FGF-18 contains an additional Met residue at the N-terminus of its sequence. For example, the amino acid sequence of human FGF-18, when expressed in E. coli, begins with the N-terminal Met residue (position 1) and continues from residue 28 (Glu) to residue 207 (Ala) of SEQ ID NO: 1.

[0022] The term "FGF-18 compound" also includes native, mature, or truncated variants or mutants of FGF-18, as well as fusion proteins (such as those disclosed in the EP17192467.3 patent family) that contain a bioactive FGF-18 moiety linked to a heterologous protein or chemical compound. In such fusion proteins, the FGF-18 moiety may be native, mature, or truncated FGF-18 protein, or a variant or mutant thereof.

[0023] As used herein, the term "truncated form" of FGF-18 refers to a protein comprising or consisting of residues 28 (Glu) to 196 (Lys) of SEQ ID NO: 1. Preferably, the truncated form of FGF-18 protein is a polypeptide designated "trFGF-18" (170 amino acids), beginning with a Met residue (N-terminus) followed by amino acid residues 28 (Glu) to 196 (Lys) of wild-type human FGF-18. The amino acid sequence of trFGF-18 is shown in SEQ ID NO: 2 (amino acid residues 2 to 170 of SEQ ID NO: 2 correspond to amino acid residues 28 to 196 of SEQ ID NO: 1). trFGF-18 is a recombinant truncated form of human FGF-18 produced in Escherichia coli (see WO 2006 / 063362). The International Nonproprietary Name (INN) of this particular form of FGF-18 is sprifermin. Sprifermin has been shown to exert similar activities to mature human FGF-18, for example, increasing chondrocyte proliferation and cartilage deposition, leading to the repair and reconstruction of various cartilage tissues (see WO2008 / 023063).

[0024] The terms "marker" and "biomarker" are used interchangeably. In the context of the present invention, they are proteins. A "prognostic biomarker" provides information about the condition of a subject, including but not limited to disease progression, disease severity, or disease outcome, regardless of the therapy. A "predictive biomarker" provides information about the effect of a received treatment, including but not limited to efficacy and safety outcomes. Since prognostic and predictive definitions are not mutually exclusive, a biomarker can be either prognostic or predictive. The amount of a biomarker or the expression level of a biomarker is expressed herein as nMol, μMol, mMol, ng, μg, mg, or g of a given protein. The amount or level can be expressed as an absolute value (e.g., 10 ng or 2 μg) or as a concentration (e.g., 10 ng / mL, 2 μg / mL, 10 ng / mmol, or 2 μg / mmol). With respect to these biomarkers, the terms "amount" or "expression level" can be used interchangeably.

[0025] The term "metabolic biomarker" refers to biomarkers such as, but not limited to, CTX-II, ProC2, PIIANP, C2M, ARGS, and AGNx1. More specifically, the term "metabolic biomarker" refers to biomarkers of cartilage metabolism, i.e., collagen and aggrecan turnover and the degradation and / or synthesis of other cartilage matrix components. Various collagen and aggrecan markers have been described (Karsdal et al., 2016).

[0026] The term "CTX-II" or "CTXII" refers to type II collagen cross-linked C-telopeptide, which is a biomarker of type II collagen degradation, which contributes to osteoarthritis (see, e.g., Duclos et al., 2010).

[0027] The term "proC2" refers to a type II collagen neoepitope generated during type II collagen synthesis, with the sequence QDVRQP recognized as an epitope in the proC2 assay. ProC2 is a marker for chondrogenesis (type II collagen formation) assays. ProC2 is associated with repair potential and may be a useful tool for identifying patients with cartilage-related diseases. ProC2 is a biomarker that has been tested in preclinical studies of clinical samples (see, e.g., Gudmann et al., 2016; Munk et al., 2016; Gudmann et al., 2014).

[0028] The term PIIANP refers to the propeptide of type II collagen. Type IIA procollagen contains an N-terminal 69 amino acid cysteine-rich globular domain encoded by exon 2 of the type II collagen gene. Type IIA procollagen has been found to be synthesized by osteoarthritic chondrocytes in diseased cartilage and can serve as a specific arthritis biomarker reflecting the efforts of chondrocytes to repair diseased cartilage (Valdes et al., 2014).

[0029] The term C2M refers to a serological type II collagen degradation neoepitope associated with cartilage degradation. C2M is an interhelical fragment of type II collagen generated by MMPs. This biomarker has been described to correlate with drug response, pain measurements, and radiographic severity (Valdes et al., 2014).

[0030] The term ARGS refers to neoepitopes generated during aggrecan degradation. Assays detect aggrecan degradation products in serum and synovial fluid. ARGS levels are associated with the progression of cartilage damage (Struglics et al., 2011; Struglics et al., 2015).

[0031] - The term AGNx1 relates to an assay that detects another neoepitope generated during aggrecan degradation.

[0032] - The terms "marker" or "biomarker" are used interchangeably.

[0033] - The term "SD" means standard deviation and relates to the normal deviation of any validated assay / system.

[0034] As used herein, "cartilage damage" includes damage caused by traumatic injury, cartilage damage, or arthritis. Examples of cartilage damage that can be treated by administering the FGF-18 formulations described herein include, but are not limited to, arthritis such as osteoarthritis, cartilage damage, fractures affecting articular cartilage, or surgical procedures (e.g., microfractures) affecting articular cartilage. This term also includes cartilage or joint degenerative diseases / disorders such as chondrocalcinosis, polychondritis, relapsing polychondritis, ankylosing spondylitis, or costochondritis. The International Cartilage Repair Society has proposed an arthroscopic grading system to assess the severity of cartilage defects. Grade 0: (normal) healthy cartilage; Grade 1: soft spots or blisters in the cartilage; Grade 2: minor cracks visible in the cartilage; Grade 3: deep fissures in the lesion (more than 50% of the cartilage layer); Grade 4: fissures in the cartilage exposing the underlying (subchondral) bone (see, for example, page 13 of http: / / www.cartilage.org / _files / contentmanagement / ICRS_evaluation.pdf).

[0035] The term "osteoarthritis" is used to refer to the most common form of arthritis. The term "osteoarthritis" encompasses both primary and secondary osteoarthritis (see, e.g., The Merck Manual, 17 th(See the American Journal of Clinical Oncology, Vol. 1, No. 1, pp. 449-49.) The most common method of classifying / grading osteoarthritis is using the Kellgren-Lawrence X-ray grading scale (see table below). Osteoarthritis can be caused by the breakdown of cartilage. Pieces of cartilage can break off, causing pain and swelling in the joints between bones. Over time, the cartilage can wear away completely, causing bones to rub against each other. Osteoarthritis can affect any joint, but it usually involves weight-bearing joints such as the hands, hips, knees, feet, and spine. In preferred examples, the osteoarthritis may be knee osteoarthritis or hip osteoarthritis. Osteoarthritis is one of the preferred cartilage disorders that can be treated by administering the FGF-18 compounds of the present invention.

[0036] The Shelgren-Lawrence X-ray grading scale for osteoarthritis is described below:

[0037] [Table 1]

[0038] Grades 1 and 2 can be considered less severe forms of the disease, whereas grades 3 and 4 can be considered more severe forms of the disease.

[0039] The term "cartilage damage" as used herein refers to cartilage damage or cartilage injury, particularly caused by trauma.Cartilage damage may occur especially after traumatic physical damage, especially after accidents or surgery (e.g., microfracture surgery).The term "cartilage damage" also includes cartilage or osteochondral fractures, meniscus injuries, and microfractures.This definition also takes into account sports injuries or sports wear of articular tissues.

[0040] - The "WOMAC total score" or "WOMAC score" ("WOMAC" for "Western Ontario and McMaster Universities Osteoarthritis Index") measures pain (WOMAC pain score), function (WOMAC function score), and contracture (WOMAC contracture score). When applied to the assessment of pain and functional impairment associated with cartilage damage, it consists of a questionnaire containing 24 items divided into three subclasses (5 items related to pain, 2 items related to contracture, and 17 items related to physical function) (see Bellamy et al., 1988; Wolfe, 1999). It is a well-known and widely used instrument, especially in the assessment of OA severity.

[0041] To assess cartilage repair, cartilage volume measurements were performed by magnetic resonance imaging (MRI) measurements, including measurements of lateral cartilage volume (also referred to as LFTC), medial cartilage volume (also referred to as MFTC), total cartilage volume (also referred to as LFTC + MFTC), and new total mean cartilage thickness.

[0042] The term "baseline" means pre-treatment (i.e., at study enrollment). This refers, inter alia, to clinical variables such as, but not limited to, cartilage volume and WOMAC total score of a given subject at study enrollment (i.e., before treatment with an FGF-18 compound or placebo).

[0043] The term "subject" or "patient" refers to both human and non-human animals. The term non-human includes mammals such as rodents (including mice), rabbits, cats, dogs, horses, cows, sheep, or primates.

[0044] "Sensitives" are subjects who respond to treatment of cartilage damage with an FGF-18 compound. Preferably, sensitive subjects (or subjects sensitive / responsive to treatment) show greater increases, i.e., cartilage repair, than placebo-treated subjects, particularly in total cartilage thickness and / or total cartilage volume. Sensitive subjects also show at least equivalent improvements in WOMAC total score compared to placebo. The terms "super-sensitives" (or "high-sensitives" or "highly-sensitives"), "intermediate-sensitives," and "non-sensitives" (including "low-sensitives") refer to different groups of subjects who experience very different degrees of cartilage volume increase following a dosing regimen of an FGF-18 compound. Ultrasensitive subjects show a high response to treatment with FGF-18 compounds (i.e., high cartilage repair), moderately sensitive subjects show a good or moderate response to treatment with FGF-18 compounds (i.e., good or moderate cartilage repair), and non-sensitive subjects show no response or a low response to treatment with FGF-18 compounds. Both ultrasensitive and sensitive subjects have comparable improvements in WOMAC total scores compared to placebo. Conversely, non-responders show significantly smaller improvements in WOMAC total scores than placebo. The terms "ultrasensitive" and "highly sensitive" are used interchangeably. It should be noted that ultrasensitive subjects have been shown to be at a higher risk of AIR events.

[0045] More specifically, "moderately sensitive," "super sensitive," "non-sensitive," "moderate responder," "super responder," and "non-responder" (including poor responders) include, but are not limited to, various subject groups that differ in the degree of increase in cartilage volume and improvement in WOMAC total score after treatment with an FGF-18 compound.

[0046] Suggested criteria for assessing susceptibility / response include (but are not limited to): 1. Positive cartilage gain compared to baseline, 2. A change in cartilage gain that is significantly greater than the change in placebo (e.g., when tested in a linear model adjusted for BMI, KL grade, sex, and age at α=5%), 3. WOMAC score improvement, i.e., reduction, compared to baseline (e.g., reduction of more than 5 points), 4. Change in WOMAC score that is not significantly greater than the change on placebo (e.g., when tested with a linear model adjusted for BMI, KL grade, sex, and age at α=5%).

[0047] A "response" or "sensitivity" to treatment with an FGF-18 compound is understood as 1) an increase in cartilage volume and / or cartilage thickness, e.g., as measured by MRI or X-ray, 2) a decrease in WOMAC total score, or 3) a change in WOMAC total score that does not significantly exceed the change from placebo, at least one year or more preferably two years after the first injection (see also the definition of "sensitives").

[0048] The term "MAD" as used herein means Multiple Ascending Dose. When this term is followed by a number, the number corresponds to the volume of FGF-18 compound injected during treatment. For example, MAD100 refers to a treatment in which the subject receives 100 mcg of FGF-18 compound per injection during the treatment period. The abbreviation "PL" (and "MADPL") refers to placebo.

[0049] As used herein, the term "storage device" is intended to include any suitable computing or processing device or other device configured or adapted for the storage of data or information. Examples of electronic devices suitable for use with the present invention include stand-alone computing devices, local area networks (LANs), wide area networks (WANs), data telecommunications networks including the Internet, intranets, and extranets, and local and distributed computer processing systems. Storage devices also include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media, magnetic tape, optical storage media such as CD-ROMs, DVDs, electronic storage media such as RAM, ROM, EPROMs, EEPROMs, conventional hard disks, and hybrids of these categories, such as magnetic / optical storage media.

[0050] As used herein, the term "stored" refers to the process of encoding information on a storage device. Those skilled in the art can readily employ any of the currently known methods for recording information on known media to create manufactures containing expression level information.

[0051] (Detailed Description of the Invention) There is a need to predict the clinical effectiveness of FGF-18 compound treatment (especially with respect to cartilage thinning and / or cartilage repair) for the treatment of subjects with cartilage disorders, such as osteoarthritis, cartilage damage, fractures affecting articular cartilage, or surgical procedures that impact articular cartilage (e.g., microfractures). To optimize the treatment of such subjects, it is important to identify biomarkers that can be used as predictors of a given subject's response to FGF-18 compound treatment, especially with respect to cartilage repair. Such predictive biomarkers may be used to identify high-risk groups that are either insensitive or, conversely, sensitive or highly sensitive to treatment. For example, if a subject with osteoarthritis is known to be at high risk of not responding (or being insensitive) to treatment, a physician may decide not to recommend an FGF-18 compound, such as sprifermin or a fusion protein containing an FGF-18 moiety, to the subject. Conversely, if a subject with osteoarthritis is known to be at high risk of being highly sensitive to treatment, a physician may decide to adjust the administration regimen to reduce the dosage of the FGF-18 compound administered to the subject. Indeed, hypersensitivity to FGF-18 compounds may lead to unwanted side effects such as AIR (acute inflammatory response). Such predictive information would be clinically useful to guide medical decisions, particularly regarding the dosing regimen to be applied to patients, or, if necessary (e.g., when FGF-18 compound treatment is not recommended), the timing of joint replacement surgery.

[0052] The surprising findings of the present invention are based on a study aimed at identifying biomarkers potentially associated with the administration of FGF-18 compounds (e.g., sprifermin). In this study, numerous protein markers were used as biomarkers (see Table 1). The relationship between the protein markers and clinical response variables was evaluated. The rationale behind this type of analysis was to identify proteins useful as biomarkers predicting clinical outcomes (particularly with regard to cartilage repair) for subjects treated with FGF-18 compounds, such as sprifermin or fusion proteins containing an FGF-18 moiety. These proteins can be used to stratify and target specific subject populations.

[0053] Various biomarkers indicated changes in response to FGF-18 therapy or indicated changes in outcomes (i.e., cartilage thickness or volume, WOMAC score) by applying criteria of high and low values. Examples include C1M, C3M, and hs-CRP. The inventors surprisingly discovered a relationship between certain metabolic proteins and outcomes (e.g., cartilage repair). CTX-II and ProC2 proteins are of particular interest. Although only CTX-II and / or ProC2 are specifically described herein, it should be noted that other metabolic biomarkers, such as PIIANP, C2M, ARGS, or AGNx1, can also be used. Therefore, it is a routine matter for those skilled in the art to find threshold values ​​for each of these biomarkers based on the teachings of the present invention.

[0054] These proteins have been described in the literature as possibly relevant to osteoarthritis, for example, CTX-II and ProC2 are considered metabolic biomarkers (Bay-Jensen, 2016).

[0055] Surprisingly, the inventors have discovered that the lower the amount of metabolic biomarkers, such as CTX-II and / or ProC2, the better the response to treatment with an FGF-18 compound, such as sprifermin or a fusion protein containing an FGF-18 moiety, in subjects with cartilage damage. These subjects are referred to as sensitive, and this group includes both moderately sensitive and highly sensitive subjects. Conversely, and also surprisingly, the inventors have discovered that the higher the amount of CTX-II and / or ProC2, the better the response or poor response to treatment with an FGF-18 compound, such as sprifermin or a fusion protein containing an FGF-18 moiety, in subjects with cartilage damage (i.e., poor or insensitive to treatment with an FGF-18 compound). These subjects are referred to as non-sensitive subjects, and this group includes both poorly sensitive and insensitive subjects. Even more surprisingly, it has been discovered that each of these biomarkers, when used alone, can provide an effective prediction of response to an FGF-18 compound.

[0056] Thus, the present invention has discovered that the biomarkers CTX-II and / or ProC can be used, either alone or in combination, as predictive biomarkers of a subject's responsiveness to treatment with an FGF-18 compound, such as sprifermin or a fusion protein containing an FGF-18 moiety. Preferably, the subject has a cartilage disorder, such as osteoarthritis, cartilage damage, a fracture affecting articular cartilage, or a surgical procedure impacting articular cartilage (e.g., microfracture). In certain embodiments, a subject is predicted to be insensitive (or less sensitive) to treatment with an FGF-18 compound if the amount of CTX-II is greater than 350±2 SD ng / mmol (>240-260% of the normal mean) and / or the amount of ProC2 is greater than 4.2±2 SD ng / mL (>120-280% of the normal mean). Conversely, a subject is predicted to be sensitive (or a responder or good responder) to treatment with an FGF-18 compound if the CTX-II level is lower than 350±2SD ng / mmol and / or the ProC2 level is lower than 4.2±2SD ng / mL.

[0057] Accordingly, the present invention provides a method for predicting susceptibility to treatment with an FGF-18 compound in a subject with a cartilage disorder, comprising: a) determining the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2 from a biological sample of the subject; b) predicting, from the results of step a), whether the subject will be highly, moderately or insensitive to treatment with an FGF-18 compound.

[0058] Before determining the amount of at least one biomarker, it is necessary to obtain a sample (or biological sample or test sample) from the subject, for example by collecting blood, serum, synovial fluid or urine. The present invention therefore provides a method for predicting sensitivity to treatment with an FGF-18 compound in a subject with cartilage damage, comprising: a) obtaining a sample from said subject; b) determining from the sample the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2; and c) predicting, from the result of step a), whether the subject will be highly, moderately or insensitive to treatment with an FGF-18 compound.

[0059] According to the above method, the presence of CTX-II greater than 350±2 SD ng / mmol (>240-260% of the normal mean) and / or ProC2 greater than 4.2±2 SD ng / mL (>120-280% of the normal mean) is indicative of no response or a low response (e.g., non-sensitive or low-sensitive) to treatment with an FGF-18 compound. Thus, the subject is predicted to be non-sensitive. Conversely, the presence of CTX-II less than 350±2 SD ng / mmol and / or ProC2 less than 4.2±2 SD ng / mL is indicative of a moderate (e.g., moderately sensitive) or high (e.g., highly sensitive) response to treatment with an FGF-18 compound. Thus, the subject is predicted to be sensitive (or responsive) to treatment with an FGF-18 compound (i.e., a responder). From this prediction, a physician can easily select only subjects predicted to be sensitive, including both moderately and highly sensitive, to treatment with an FGF-18 compound.

[0060] The present invention also provides a method for selecting subjects with cartilage disorders for inclusion or exclusion from treatment or clinical trials with FGF-18 compounds based on their likelihood of sensitivity to said treatment, comprising: a) determining from the subject's biological sample the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2, wherein the amount of at least one of these biomarkers is indicative of the subject's risk for being susceptible or insensitive to the treatment; and b) selecting a susceptible subject as suitable for said treatment.

[0061] In the assays disclosed above, before determining the amount of at least one protein, it is necessary to obtain a subject sample (or test sample or biological sample), for example, by collecting blood, serum, synovial fluid or urine. The present invention therefore provides a method for selecting subjects with cartilage disorders for inclusion or exclusion from treatment or clinical trials with FGF-18 compounds based on their likelihood of sensitivity to said treatment, comprising: a. obtaining a biological sample from said subject; b. determining from the sample the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2, wherein the amount of at least one of these biomarkers is indicative of the subject's risk for being susceptible or insensitive to said treatment; and c. selecting a susceptible subject as suitable for said treatment.

[0062] According to the above method, subjects exhibiting CTX-II greater than 350±2 SD ng / mmol and / or ProC2 greater than 4.2±2 SD ng / mL are excluded from (i.e., not selected for) treatment with FGF-18 compounds. Conversely, subjects exhibiting CTX-II less than 350±2 SD ng / mmol and / or ProC2 less than 4.2±2 SD ng / mL are included in (i.e., selected for) treatment with FGF-18 compounds.

[0063] Alternatively, a method of selecting subjects with cartilage disorders for inclusion or exclusion from treatment or clinical trials using FGF-18 compounds based on their likelihood of sensitivity to FGF-18 compounds, comprising: (a) subjecting a test sample from a human subject diagnosed with a cartilage disorder to at least one assay adapted to determine the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2; (b) determining from the assay the likelihood of the subject being susceptible or insusceptible to the treatment; and (c) selecting a subject such that if the assay detects CTX-II lower than 350±2SDng / mmol and / or ProC2 lower than 4.2±2SDng / mL, the subject is included in a treatment or clinical trial using an FGF-18 compound, and if the assay detects CTX-II higher than 350±2SDng / mmol and / or ProC2 higher than 4.2±2SDng / mL, the subject is excluded from a treatment or clinical trial using an FGF-18 compound.

[0064] Alternatively, a method of selecting human subjects for clinical trials to test FGF-18 compounds comprises the steps of: a) assaying a biological sample from a human subject diagnosed with a cartilage disorder for the amount of at least one of CTX-II and / or ProC2; b) determining the amount of at least one of CTX-II and / or ProC2; and c) selecting human subjects exhibiting CTX-II greater than 350±2SD ng / mmol and / or ProC2 greater than 4.2±2SD ng / mL for clinical trials.

[0065] The present invention also provides a method for excluding a human subject from a clinical trial testing an FGF-18 compound, comprising: (a) assaying a biological sample from a human subject diagnosed with a cartilage disorder for the amount of at least one of CTX-II and / or ProC2; (b) determining from the assay the likelihood that the subject is susceptible or non-susceptible to the treatment; and (c) excluding from the clinical trial any human subject exhibiting a CTX-II level greater than 350±2 SD ng / mmol and / or a ProC2 level greater than 4.2±2 SD ng / mL.

[0066] The amount (or expression level) of one or more biomarkers in a sample can, if necessary, be compared to a reference amount (or reference expression level) from a reference sample, which can be obtained from a healthy subject or from the same patient diagnosed or treated before or during said treatment.

[0067] FGF-18 compounds are typically administered intra-articularly at a dose of 100 mcg per injection once weekly for three weeks per treatment cycle. An alternative suggested dosing regimen for subjects predicted to be highly sensitive is to administer the FGF-18 compound intra-articularly at a dose of 30 mcg per injection once weekly for three weeks per treatment cycle. A preferred dose is 100 mcg per injection, possibly reduced to 30 mcg per injection in highly sensitive cases, although it should be understood that the present invention is not limited to this dose. Thus, FGF-18 compounds can be administered intra-articularly at a dose of 50-300 mcg per injection, preferably 60-25 mcg, and more preferably 100-200 mcg per injection. For hypersensitive subjects, the dose may be reduced, for example, by half or about half, or one-third, or about one-third. For example, if the usual dose is 50 mcg per injection, the dose may be reduced to between 16-25 mcg per injection.

[0068] The FGF-18 compound is usually intra-articularly administered at a dose of 100 mcg per injection once a week for 3 weeks per cycle.The FGF-18 compound is usually administered in at least one treatment cycle.Preferably, the treatment cycle is repeated at least once, for example, 6 months (or about 26 weeks) after the start of the first treatment cycle.A maximum of 4 treatment cycles within 2 years shows promising results (see Figure 1).

[0069] The present invention further encompasses FGF-18 compounds for use in treating subjects with cartilage disorders characterized in that the subjects have CTX-II below 350 ng / mmol and / or ProC2 below 4.2 ng / mL. Thus, subjects not meeting these criteria are preferably excluded from FGF-18 compound treatment.

[0070] The present invention also relates to an assay for determining sensitivity to treatment with an FGF-18 compound or for determining a treatment regime using an FGF-18 compound, comprising the steps of: (a) subjecting a test sample from a human subject diagnosed with a cartilage disorder to at least one assay for determining the amount of at least one of CTX-II and / or ProC2; (b) determining the amount of at least one of CTX-II and / or ProC2; and (c) determining the sensitivity or insensitivity of the subject to treatment with an FGF-18 compound based on the results of step (b). In this assay, the presence of CTX-II greater than 350 ng / mmol and / or ProC2 greater than 4.2 ng / mL indicates insensitivity to treatment with an FGF-18 compound. Conversely, the presence of CTX-II less than 350 ng / mmol and / or ProC2 less than 4.2 ng / mL indicates sensitivity. From the results of this assay, a physician can easily select only those subjects who are predicted to be sensitive, including both moderately sensitive and highly sensitive, to treatment with an FGF-18 compound. In the assays disclosed above, before determining the amount of at least one biomarker, it is necessary to obtain a biomarker (or test) sample from the subject, for example, by collecting blood, serum, synovial fluid, or urine.

[0071] The present invention also relates to an assay for selecting a treatment regimen for a human subject with cartilage damage, comprising the steps of: (a) subjecting a test sample from a human subject diagnosed with cartilage damage to at least one assay for determining the amount of at least one of CTX-II and / or ProC2, (b) determining the likelihood that the subject is moderately or highly sensitive to FGF-18 treatment, and (c) determining a suitable treatment regimen for the subject from the results of step b). If the subject has CTX-II lower than 350 ng / mmol and / or ProC2 lower than 4.2 ng / mL, the subject can be selected and treated for treatment with the suitable treatment regimen, based on the recognition that this amount is related to response to the compound; and if the subject has CTX-II higher than 350±2SD ng / mmol and / or ProC2 higher than 4.2±2SD ng / mL, the subject can be excluded from treatment with an FGF-18 compound, based on the recognition that this amount is related to response to the compound.

[0072] Also provided is a method for treating a subject with cartilage damage using an FGF-18 compound, comprising: a. determining from the sample the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2, wherein said amount is indicative of the subject's risk for being highly or moderately sensitive to treatment with said FGF-18 compound; b. selecting subjects with CTX-II lower than 350±2SD ng / mmol and / or ProC2 lower than 4.2±2SD ng / mL; c. intra-articularly administering an FGF-18 compound to the subject selected in step b.: Also described is a method comprising:

[0073] Further provided is a method of treating a human subject for a cartilage disorder, comprising: (a) assaying a biological sample from a subject diagnosed with a cartilage disorder for the amount of at least one biomarker selected from the group consisting of CTX-II and / or ProC2; and (b) if the subject has CTX-II below 350±2SD ng / mmol and / or ProC2 below 4.2±2SD ng / mL, administering to the subject a dosage regimen comprising a composition comprising an effective amount of an FGF-18 compound.

[0074] Overall, in the context of the present invention, CTX-II and / or ProC2 have been shown to be useful predictive biomarkers of response to FGF-18 compounds and are therefore considered predictive biomarkers.

[0075] In the context of the present invention generally, assaying or otherwise determining the amount of at least one biomarker of the invention may be performed before or during treatment, and indeed, during treatment, dosing regimens may need to be adapted to the context of new biomarkers.

[0076] In general, in the context of the present invention, for patients whose CTX-II levels are within the range of 350±2 SD ng / mmol or ProC2 levels of 4.2±2 SD ng / mL, it is recommended that a diagnostic or biomarker test be completed using another metabolic biomarker. For example, if the CTX-II level is within the range of 350±2 SD ng / mmol, then the ProC2 level can be considered. In rare cases where a patient exhibits a CTX-II level of 350±2 SD ng / mmol and a ProC2 level of 4.2±2 SD ng / mL, it is recommended that a diagnostic or biomarker test be completed using another type of biomarker, such as an inflammatory biomarker or a SNP biomarker, such as those disclosed in WO2014023703.

[0077] In another embodiment of the present invention, a system for obtaining data (and a computer-readable medium for forming the system) is also provided. The data can be used, inter alia, to evaluate the suitability of treatment with an FGF-18 compound in a subject, or to monitor the therapeutic effectiveness of an FGF-18 compound for a given subject, or simply to monitor disease progression. The system can be used when treatment with an FGF-18 compound is envisioned during clinical trials, or when treatment with the compound is already underway.

[0078] Accordingly, an embodiment of the present invention includes a computer system for obtaining data from at least one test sample obtained from at least one subject with cartilage damage, the system comprising: (a) at least one determination module configured to receive at least one said test sample and perform at least one analysis on said at least one test sample to determine the amount of at least one biomarker of the present invention; (b) at least one storage device configured to store data output from the decision module; and (c) at least one display module for displaying content based in part on data output from said decision module, wherein said content includes a signal indicative of the presence of at least one of these conditions, and optionally the absence of any one of these conditions.

[0079] The computer-readable medium may have computer-readable instructions recorded thereon to define software modules for performing a method on a computer. In such a case, the computer-readable storage medium may include: (a) instructions for comparing data stored on a storage device with reference data and providing a comparison result, wherein the comparison is based on the amount of at least one biomarker in accordance with the present invention; and (b) instructions for displaying content based in part on data output from the decision module, wherein the content includes a signal indicative of the presence of at least one condition, and optionally the absence of one or more conditions.

[0080] Computer-readable storage media may be any available tangible media that can be accessed by a computer. Computer-readable storage media includes volatile and non-volatile, removable and non-removable tangible media implemented in any method or technology for storage of information, such as computer-readable instructions, data structures, program modules, or other data. Computer-readable storage media includes, but is not limited to, RAM (random access memory), ROM (read-only memory), EPROM (erasable programmable read-only memory), EEPROM (electrically erasable programmable read-only memory), flash memory or other memory technology, CD-ROM (compact disc read-only memory), DVD (digital versatile disc) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non-volatile memory, and any other tangible medium that can be used to store the desired information and that can be accessed by a computer, including suitable combinations of the foregoing.

[0081] Computer-readable data embodied on one or more computer-readable media may define instructions, for example, as part of one or more programs that, when executed by a computer, instruct a computer to perform one or more functions described herein and / or various embodiments, variations, and combinations thereof. Such instructions may be written in any one of several programming languages, such as, for example, Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or various combinations thereof. The computer-readable media on which such instructions are embodied may reside on any one or more components of a system, or the computer-readable storage media described herein may be distributed across one or more such components.

[0082] The computer-readable medium may be transportable such that the instructions stored on the medium may be stored on any computer resource for carrying out aspects of the present invention discussed herein.

[0083] The information determined in the determination module can be read by a storage device adapted or configured to have expression level or protein level information recorded thereon. Such information may be provided in a digital form that can be electronically transmitted and read, for example, via the Internet, on a diskette, via USB (Universal Serial Bus), or via any other suitable communication mode.

[0084] In the context of the present invention generally, for example in the context of any one of the methods, uses, assays or kits of the present invention, a preferred FGF-18 compound is a truncated FGF-18, such as sprifermin or a fusion protein comprising an FGF-18 moiety, and a preferred cartilage disorder is selected from the group consisting of osteoarthritis, cartilage damage, fractures affecting articular cartilage, or surgical procedures impacting articular cartilage, such as microfractures.

[0085] In the context of the present invention in general, e.g., in the context of any one of the methods, uses, assays, computer systems or kits of the present invention, it should be understood that before determining the amount of at least one biomarker of the present invention (e.g., CTX-II and / or ProC2), it is necessary to obtain a subject sample (or biological sample or test sample), e.g., by collection of blood, serum, synovial fluid or urine, and which may also be obtained from, but is not limited to, cells, tissue, cartilage or synovial fluid.

[0086] Individuals suffering from a cartilage disorder and being tested, examined and / or treated according to any of the methods, uses, assays, kits and other computer systems described herein are human subjects who are candidates for treatment with an FGF-18 compound, such as sprifermin or a fusion protein containing an FGF-18 moiety. In a preferred embodiment, the individual has been diagnosed with or exhibits symptoms of a cartilage disorder.

[0087] In further embodiments, the present invention encompasses kits comprising means for carrying out the above-described methods and instructions for use. Preferably, the kits comprise means for detecting the presence of at least one of the biomarkers of the present invention (e.g., CTX-II and / or ProC2) and means for quantifying it. The kits may also comprise means for detecting the presence of at least two of the biomarkers of the present invention and means for quantifying them.

[0088] The methods and kits of the present invention are useful in clinical diagnostic applications, although the term "diagnostic" as used herein is not limited to clinical or medical applications, and the diagnostic methods and kits of the present invention claimed herein are also useful in any research application or clinical trial in which it is desirable to test a subject for the presence or absence of any of the markers described herein.

[0089] In the context of the present invention, the presence of at least one biomarker of the present invention (e.g., CTX-II and / or ProC2) and their quantification may be detected by any technique known per se to the skilled artisan, including, for example, ELISA.

[0090] Other embodiments of the invention within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention disclosed herein. The description herein, together with the examples herein, is intended to be exemplary only, with the scope and spirit of the invention being defined by the claims herein. [Brief explanation of the drawings]

[0091] DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Scheme of the dosing regimen used for sprifermin in the forward study. [Figure 2] Figure 2: Mean and 95% CI for absolute change from baseline over weeks by treatment in cartilage thickness (mm) of the total femoro-tibial joint - mITT analysis set. [Figure 3] Figure 3: Mean and 95% CI for absolute change from baseline over weeks by treatment in total femoro-tibial joint cartilage volume (μL) - mITT analysis set. [Figure 4] Figure 4: Mean and 95% CI in absolute change from baseline over weeks with treatment for cartilage thickness (mm) at the common femoro-tibial joint by biomarker, by cartilage metabolism subgroup - ITT analysis set - ProC2 metabolic biomarker. [Figure 5]Figure 5: Mean and 95% CI in absolute change from baseline over weeks with treatment for cartilage thickness (mm) at the common femoro-tibial joint by biomarker, by cartilage metabolism subgroup - ITT analysis set - CTX-II metabolic biomarkers. [Figure 6] Figure 6: Mean and 95% CI for absolute change from baseline over weeks with treatment for WOMAC total score in target knee by cartilage metabolism subgroup - ITT analysis set - ProC2 metabolic biomarker. [Figure 7] Figure 7: Mean and 95% CI for absolute change from baseline over weeks with treatment for WOMAC total score in target knee by cartilage metabolism subgroup - ITT analysis set - CTX-II metabolic biomarkers.

[0092] (Array description) SEQ ID NO: 1: Amino acid sequence of native human FGF-18. SEQ ID NO: 2: Amino acid sequence of recombinant truncated FGF-18 (trFGF-18). SEQ ID NO: 3: Amino acid sequence of the marker CTX-II. SEQ ID NO: 4: Amino acid sequence of marker PROC2. [Example]

[0093] 1. FGF-18 Compound The FGF-18 compound used as treatment in this example is sprifermin, a truncated form of FGF-18, as defined in the "Definitions" section. Two strengths of sprifermin were provided for this study: 30 μg and 100 μg. Sprifermin was supplied as a white, sterile, lyophilized powder in 3 mL glass vials. Each vial contained either 31.5 μg or 105 μg of sprifermin active substance; these amounts were included in 5% excess to allow extraction of 30 μg or 100 μg of sprifermin active substance, respectively, followed by reconstitution with 0.9% w / v sodium chloride injection (referred to herein as "saline"). The excipients for this formulation were sodium phosphate buffer (pH 7.2), sodium hydroxide, O-phosphate, sucrose, and poloxamer 188. The kit for the 30 μg treatment contained one glass vial of sprifermin (strength 30 μg) and one glass ampoule of sterile saline for injection (2 mL / ampule). The kit for the 100 μg treatment contained one glass vial of sprifermin (strength 100 μg) and one glass ampoule of sterile saline for injection (2 mL / ampule). The dosing volume for all treatment groups was 2 mL.

[0094] 2. Method Forward Test This study is based on a forward study (EMR700692-006). Five groups of patients were studied: Group 1 (placebo 4 cycles; hereafter referred to as placebo): 108 subjects. Group 2 (2 cycles of sprifermin 30 μg / injection alternated with 2 cycles of placebo; hereafter referred to as sprifermin / placebo 30 μg): 110 subjects. Group 3 (4 cycles of Sprifermin 30 μg / injection; hereafter referred to as Sprifermin 30 μg): 111 subjects. Group 4 (2 cycles of sprifermin 100 μg / injection alternated with 2 cycles of placebo; hereafter referred to as sprifermin / placebo 100 μg): 110 subjects. Group 5 (4 cycles of Sprifermin 100 μg / injection; hereafter referred to as Sprifermin 100 μg): 110 subjects.

[0095] According to this forward study, patients received four cycles of treatment, each consisting of three intra-articular injections given once a week for three consecutive weeks, at six-month intervals (see Figure 1). All injections were administered intra-articularly (into the joint).

[0096] The primary efficacy outcome was the change from baseline in cartilage thickness in the total femoro-tibial joint as assessed by MRI at 2 years.

[0097] Exploratory endpoints included baseline protein and / or genetic markers associated with response to treatment or disease progression (response assessed by MRI and / or questionnaires).

[0098] Inclusion / Exclusion Criteria The study enrolled adult subjects of either sex with primary tibiofemoral OA according to the American College of Rheumatology (ACR) clinical and radiographic criteria, with a Kjellgren-Lawrence Grade (KLG) of 2 or 3 and a minimum joint space width (JSW) of ≥ 2.5 mm in the medial compartment. Subjects must have pain in the target knee on most days and / or have required symptomatic treatment for knee pain with paracetamol (acetaminophen), systemic nonsteroidal anti-inflammatory drugs (NSAIDs), including COX inhibitors (COXibs), or tramadol on most days in the previous month, and must have both: 1) a history of pain due to OA of the target knee for at least 6 months, and 2) a target knee pain score of 4 to 9 points in response to question 1 of the Western Ontario-McMaster Universities Osteoarthritis Index (WOMAC) pain index ("How much pain have you felt when walking on level ground [over the past 48 hours in the target knee]?") at screening and baseline, after at least 5 half-lives of washout of acetaminophen, topical or oral NSAIDs, COXibs, opioids, and / or tramadol analgesic medication. Women of childbearing potential used a contraceptive method with a failure rate of less than 1% per year throughout the study.

[0099] Major exclusion criteria included >5 degrees of malalignment in the femoro-tibial axis of the target knee, clinical signs of inflammation (i.e., redness) in the target knee, intra-articular administration of corticosteroids or hyaluronic acid to either knee within 6 months prior to screening, planned any knee surgery within the following 2 years (affecting either the target knee or the contralateral knee), coexisting conditions or treatments deemed incompatible with study participation, contraindications to MRI scanning (including inability to fit into the scanner or knee coil), pregnancy or breastfeeding, participation in another clinical trial within the past 30 days, and legal incapacity or limited legal capacity.

[0100] Written informed consent was obtained before each study was conducted.

[0101] statistical methods The treatment effect on the primary endpoint was assessed using repeated measures analysis of variance (ANOVA, using PROC MIXED in SAS) on absolute change from baseline, including baseline value, treatment group, assessment time point, and country as factors, and treatment-by-time point interaction, by dose-ranging. The primary efficacy analysis consisted of testing the linear dose relationship and overall treatment effect at 2 years. The significance level was set at 5% two-sided for both studies. Pairwise comparisons (sprifermin vs. placebo, and between sprifermin dose and regimen groups) were performed within the context of this modeling framework. For each pairwise comparison, the difference between treatments and the corresponding 95% confidence interval (CI) and p-value were presented. The same ANOVA model used for the primary endpoint was used to assess treatment effects on persistent secondary endpoints, including MRI endpoints, WOMAC endpoints (total, pain, function, and contracture scores), and radiographic endpoints at each assessment time point and over time. Logistic regression was used to assess treatment effects on binary efficacy endpoints, such as OMERACT-OARSI response rate. Point estimates and corresponding 95% CIs and p-values ​​for each pairwise comparison are shown.

[0102] Pain and functional assessment The WOMAC is a validated instrument used to assess symptom modification in clinical OA trials. This clinical score was developed in 1981 and is considered a valid instrument by both clinical researchers and regulatory agencies. The WOMAC has been extensively used and validated in clinical trials of hip and knee OA.

[0103] Subjects had to self-complete 24 questions (i.e., 5 items regarding pain, 2 items regarding contracture, and 17 items regarding physical function) regarding the past 48 hours using an 11-box NRS rating scale (categories 0 to 10). Different forms of the questionnaire existed for the right and left knees; to reduce confounding of the WOMAC response by symptoms in the contralateral knee, subjects used the WOMAC questionnaire specific to the target knee. Following this, instructions regarding the WOMAC 3.1 index were given for administration of the questionnaire.

[0104] X-ray evaluation of JSW Change in JSW measured by radiography is an endpoint approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for use in efficacy trials in OA. JSW was measured using standardized techniques.

[0105] qMRI evaluation The primary endpoint for the DBPC treatment phase was the change from baseline in total femoro-tibial joint cartilage thickness assessed by qMRI in the mITT at year 2. Total femoro-tibial joint cartilage thickness was calculated in two ways: 1. Average cartilage thickness (total volume divided by total surface area) 2. Total cartilage thickness (sum of cartilage thicknesses of the medial and lateral compartments).

[0106] Treatment effects on the primary endpoint were assessed across the dose range using a repeated measures analysis of variance (ANOVA) on absolute change from baseline, including treatment group, assessment time point, and (pooled) country as fixed factors and baseline value as covariate, and the interaction between treatment group and assessment time point. Repeated measurements over time demonstrate the use of "unctructured" covariance patterns.

[0107] Pairwise comparisons of absolute change from baseline in cartilage thickness (sprifermin treatment group vs. placebo) were performed within the context of the modeling framework described above. For each pairwise comparison, the difference between treatments and the corresponding 95% confidence interval (CI) and p-value were presented. p-values ​​(corresponding to a type 3 test of fixed effects) are reported for all covariates (i.e., baseline value, treatment, assessment time, treatment group x assessment time interaction, country) in the original "overall" model for all assessment time points combined, and for all assessment time points. Estimated coefficients, p-values, and 95% CIs are presented across time and at each assessment time point for: (i) the dose relationship (linear trend) and (ii) each pairwise comparison between dose level and placebo.

[0108] To assess the robustness of the primary outcome, tests for a linear dose-relationship and tests for overall treatment effect were repeated using the PP analysis set. For the mITT analysis set, a nonparametric analysis was performed on the ordered data of cartilage thickness within the total femoro-tibial joint as an alternative to the primary analysis. Data were ranked by absolute change from baseline over 2 years during the DBPC treatment phase using a rank transformation.

[0109] Biomarker measurements Serological and urinary biochemical markers of bone and joint tissue turnover and synovial inflammation were evaluated. Potential biomarkers of ointment metabolism include, but are not limited to, the neo-epitope propeptide of type II collagen degradation (proC2) and the cross-linked C-telopeptide of type II collagen (CTX-II). Blood and urine samples for systemic biomarker assessment were collected at the following time points: Week 0 (before the first injection of sprifermin), Week 26, Week 54, Week 80, and Week 104. For evaluation time points where injections were also administered, samples were taken before injection. Synovial fluid samples were collected at these time points. These samples were collected as part of the intra-articular injection procedure and immediately before injection, using the same needle as used for injection. For urine collection, a second morning voided urine sample was obtained.

[0110] The following assessments were performed as exploratory endpoints: - Changes from baseline in serum and urinary markers associated with administration of the compound. - Baseline protein and / or genetic markers associated with response to treatment or disease progression (response assessed by MRI and / or questionnaire).

[0111] 3.Results Primary endpoint (allcomer) Total femorotibial joint: At 2 years, a statistically significant treatment effect was observed for the change from baseline in cartilage thickness at the common femoro-tibial joint (see Figure 2). Both the sprifermin / placebo 100 μg group and the sprifermin 100 μg group demonstrated greater improvement at week 104 (mean change from baseline: +0.02 mm and +0.03 mm, respectively) compared with the placebo group (mean change from baseline: -0.02 mm) (p<0.001 for both comparisons). This treatment group difference began at week 78 for the sprifermin / placebo 100 μg group and week 52 for the sprifermin 100 μg group. Statistical significance was maintained throughout week 104 in both groups. The placebo group did not demonstrate improvement from baseline at any visit through week 104. There was a statistically significant difference overall (all weeks) in increased cartilage thickness between the Sprifermin / placebo 100 μg group and the Sprifermin 100 μg group compared to the placebo group (p=0.002 and p<0.001, respectively). The ANCOVA model was statistically significant for treatment (p<0.001), week (p<0.001), treatment*week (p=0.029), and country with pooled data (p=0.009).

[0112] Cartilage volume within the common femoro-tibial jointThere was a statistically significant treatment effect on the change from baseline in cartilage volume within the total femoro-tibial joint (Figure 3; p<0.001). The sprifermin / placebo 100 μg and sprifermin 100 μg groups showed greater improvement compared to the placebo group. Statistical significance for sprifermin 100 μg compared to placebo began at week 78 and persisted through week 104; statistical significance for sprifermin / placebo 100 μg compared to placebo was evident at week 26 and persisted from week 78 to week 104. The mean changes from baseline to week 104 were -28.2 μL, +9.5 μL, +96.6 μL (p<0.001), and +116.5 μL (p<0.001) in the sprifermin / placebo 30 μg, sprifermin 30 μg, sprifermin / placebo 100 μg, and sprifermin 100 μg groups, respectively, and -55.5 μL in the placebo group. There was a statistically significant effect for countries with pooled data (p=0.011).

[0113] Exploratory endpoints (biomarker statistics) The overall objectives of the pharmacodynamic (PD) / biomarker analysis are: - Identification of predictive biomarker(s) for determining patients who maintain positive structural outcomes (based on differences in MRI total cartilage thickness with sprifermin vs. placebo) while improving symptomatic outcomes (WOMAC total score and WOMAC pain index score) versus placebo; -Identification of predictive biomarker(s) for safety parameters such as AIR; - Characterization of functional biomarkers as potential predictive biomarkers and evaluation of potential predictive cut-off values; -Identification of potential prognostic biomarkers (placebo group only), -Evaluation of biochemical biomarkers (e.g., ProC2 and CTX-II) as PD biomarkers.

[0114] Stratification and Cartilage Thickness at the Common Femorotibial Joint: Changes from baseline in cartilage thickness at the common femorotibial joint showed notable differences between biomarker subgroups at week 104. Subjects with lower levels of cartilage metabolism biomarkers (proC2 and CTX-II) at baseline showed improved total cartilage thickness after 104 weeks of treatment with sprifermin (vs. placebo) compared with subjects with higher levels of cartilage metabolism biomarkers. This differential response was primarily due to the differential placebo response (Figures 4 and 5). Surprisingly, the results indicate that highly metabolic chondrocytes (high proC2 and / or high CTX-II) respond inconsistently to anabolic therapies such as sprifermin.

[0115] Stratification and WOMAC total score Changes from baseline in WOMAC total score at week 104 showed notable differences between biomarker subgroups: Subjects with lower levels of markers of cartilage metabolism (proC2 and CTX-II) than baseline demonstrated improved WOMAC total scores after 104 weeks of treatment with sprifermin compared to subjects receiving placebo or subjects with higher levels of biomarkers of cartilage metabolism (Figures 6 and 7). These results surprisingly demonstrate that joints with lower metabolism (low proC2 and / or low CTX-II) not only respond better to anabolic therapies such as sprifermin in terms of cartilage thickness, but also have a positive effect on WOMAC total score.

[0116] References [ka]

[0117] (abbreviation) OA = osteoarthritis CI = confidence interval DBPC = double-blind placebo-controlled CTX-II = type II collagen cross-linked C-telopeptide ICOAP = Intermittent and Persistent Osteoarthritis Pain Measure ITT = intention-to-treat KOOS Symptom Index = Knee Injury and Osteoarthritis Outcome Score Symptom Index KOOS QOL = Knee Injury and Osteoarthritis Outcome Score Quality of Life LOCF = last observation performed forward LFTC = lateral femoro-tibial compartment MFTC = medial femoro-tibial compartment mITT = modified intention-to-treat MOS SF-36 = Medical Outcomes Study Short Form-36 Health Status Survey MRI = magnetic resonance imaging NRS pain score = Numerical Scale Pain Score PGA = patient global assessment PGIC = Patient Global Impression of Change PK = pharmacokinetics PROC2 = type II collagen propeptide neoepitope W=week WOMAC = Western Ontario-McMaster University Osteoarthritis Index.

Claims

1. 1. A method for assisting in predicting sensitivity to treatment with an FGF-18 compound in a subject with a cartilage disorder, comprising: 1) determining the amount of at least one biomarker selected from the group consisting of CTX-II and ProC2 from a sample derived from the subject; and 2) using the determined value obtained from the results of step 1) to aid in predicting the subject's susceptibility to treatment with an FGF-18 compound.

2. 2. The method of claim 1, wherein step 2) comprises using the value determined in step 1) to aid in predicting the subject's sensitivity to treatment with an FGF-18 compound, and wherein if the sample derived from the subject exhibits ProC2 lower than 4.2 ng / mL, the value aids in predicting the subject's sensitivity to treatment with an FGF-18 compound.

3. 1. A method for assisting in the selection of subjects having cartilage disorders for inclusion or exclusion from treatment or clinical trials using an FGF-18 compound based on the likelihood of the subject's susceptibility to said treatment or clinical trial, comprising: 1) determining from a sample from the subject the amount of at least one biomarker selected from the group consisting of CTX-II and ProC2, wherein the amount of the at least one biomarker is indicative of the subject's susceptibility to the treatment; and 2) assisting in the selection of the subject for inclusion or exclusion from the treatment or clinical trial according to the subject's susceptibility to the treatment or clinical trial.

4. 1. A method for assisting in the selection of subjects with cartilage disorders to exclude them from treatment or clinical trials using an FGF-18 compound based on the likelihood of the subject being susceptible to said treatment or clinical trial, comprising: 1) determining from a sample from the subject the amount of at least one biomarker selected from the group consisting of CTX-II and ProC2, wherein the amount of the at least one biomarker is indicative of the subject's susceptibility to the treatment; and 2) assisting in the selection of excluding said subject from treatment or clinical trial if a sample from said subject exhibits ProC2 greater than 4.2 ng / mL.

5. 1. A method for assisting in the selection of subjects having cartilage disorders for inclusion in a treatment or clinical trial with an FGF-18 compound based on the likelihood of the subject being susceptible to said treatment or clinical trial, comprising: 1) determining from a sample from the subject the amount of at least one biomarker selected from the group consisting of CTX-II and ProC2, wherein the amount of the at least one biomarker is indicative of the subject's susceptibility to the treatment; and 2) assisting in the selection of said subject for treatment or inclusion in a clinical trial if a sample from said subject exhibits ProC2 lower than 4.2 ng / mL.

6. A kit comprising a reagent for measuring the amount of said at least one biomarker for carrying out the method according to any one of claims 1 to 5, and instructions for use.

7. The method of any one of claims 1 to 5, wherein the FGF-18 compound is sprifermin or a fusion protein comprising an FGF-18 moiety.

8. 7. The kit of claim 6, wherein the FGF-18 compound is sprifermin or a fusion protein containing an FGF-18 moiety.

9. 8. The method of any one of claims 1 to 5 and 7, wherein the cartilage damage is selected from the group consisting of osteoarthritis, cartilage injury, fractures affecting articular cartilage, or cartilage damage due to surgical procedures impacting articular cartilage.

10. 9. The kit of claim 6 or 8, wherein the cartilage damage is selected from the group consisting of osteoarthritis, cartilage injury, fractures affecting articular cartilage, or cartilage damage due to surgical procedures impacting articular cartilage.

Images