ADAMTS-binding immunoglobulin

Abstract

The present invention relates to immunoglobulins that bind to ADAMTS5, and more particularly to polypeptides comprising or consisting essentially of one or more such immunoglobulins. The present invention also relates to constructs comprising such immunoglobulins or polypeptides, such as immunoglobulin single variable domains (ISVDs), and nucleic acids encoding such immunoglobulins or polypeptides; to methods for preparing such immunoglobulins, polypeptides, and constructs; to host cells that express or are capable of expressing such immunoglobulins or polypeptides; to compositions, and in particular pharmaceutical compositions, comprising such immunoglobulins, polypeptides, constructs, nucleic acids, and / or host cells; and in particular to the use of immunoglobulins, polypeptides, constructs, nucleic acids, host cells, and / or compositions for prophylactic and / or therapeutic purposes, such as those described herein. Other aspects, embodiments, advantages, and uses of the present invention will become apparent from the further description herein.

Description

[Technical Field]

[0001] 1. Field of the Invention The present invention relates to immunoglobulins that bind to ADAMTS5, and more particularly to polypeptides comprising or consisting essentially of one or more such immunoglobulins (also referred to herein as "immunoglobulins of the invention" and "polypeptides of the invention," respectively). The present invention also relates to constructs comprising such immunoglobulins or polypeptides, such as immunoglobulin single variable domains (ISVDs), and nucleic acids encoding such immunoglobulins or polypeptides (also referred to herein as "nucleic acids of the invention"); to methods for preparing such immunoglobulins, polypeptides, and constructs; to host cells that express or are capable of expressing such immunoglobulins or polypeptides; to compositions, and particularly pharmaceutical compositions, comprising such immunoglobulins, polypeptides, constructs, nucleic acids, and / or host cells; and particularly to the use of immunoglobulins, polypeptides, constructs, nucleic acids, host cells, and / or compositions for prophylactic and / or therapeutic purposes, such as those described herein. Other aspects, embodiments, advantages, and uses of the present invention will become apparent from the further description herein. [Background technology]

[0002] 2. Background of the invention Osteoarthritis (OA) is one of the most common causes of disability worldwide. It affects 30 million Americans and is the most common joint disorder. More than 20% of the U.S. population is predicted to suffer from it by 2025. The disease is not systemic and is usually limited to a few joints. However, it can occur in any joint, most commonly the knee, hip, hand, shoulder, and spine. OA is characterized by the progressive erosion of articular cartilage (the cartilage that covers bones), resulting in chronic pain and disability. Ultimately, the disease leads to complete destruction of the articular cartilage, hardening of the underlying bone, and osteophyte formation, all of which result in loss of movement and pain. Osteoarthritis can be defined as a heterogeneous group of conditions characterized by a combination of joint symptoms, signs resulting from loss of articular cartilage, and changes in adjacent tissues, including bone, tendons, and muscles. Pain is the most prominent symptom of OA and is often the reason patients seek medical help. There is no cure for OA; current treatments do not slow the structural deterioration of OA joints. Disease management is limited to palliative treatment at best, and rarely addresses the underlying causes of disease progression. While disease initiation may be multifactorial, cartilage destruction is thought to be the result of uncontrolled proteolysis of the extracellular matrix (ECM). The most abundant ECM components of articular cartilage are collagen (primarily collagen II) and proteoglycans, primarily aggrecan (Kiani et al. 2002 Cell Research 12:19-32).

[0003] Aggrecan is important for the proper function of articular cartilage by providing a hydrated gel structure that confers load-bearing properties to cartilage. Aggrecan is a large, multimodular molecule (2,317 amino acids) expressed by chondrocytes. Its core protein consists of three globular domains (G1, G2, and G3) and a large extended region between G2 and G3 for the binding of glycosaminoglycan chains. This extended region is composed of two domains: a keratan sulfate-substituted domain (KS domain) and a chondroitin sulfate-substituted domain (CS domain). The CS domain carries 100–150 glycosaminoglycan (GAG) chains. Aggrecan forms large complexes with hyaluronan, in which 50–100 aggrecan molecules interact with one hyaluronan molecule via the G1 domain and link protein. Upon absorption of water (depending on GAG content), these complexes form reversibly deformable gels that resist compression. The structure, fluid retention, and function of articular cartilage are related to the aggrecan matrix content and the amount of chondroitin sulfate bound to the intact core protein. Structurally, OA is characterized by aggrecan degradation, gradually releasing the G3 and G2 domains (leading to cartilage "shrinkage") and ultimately causing the release of the G1 domain and collagen degradation, irreversibly destroying cartilage structure. The most important aggrecan cleavage site for OA pathogenesis is the sequence TEGE 373 ↓ 374 This cleavage site is located in the interglobular domain (IGD) of aggrecan. 374Antibodies recognizing the ARGS neoepitope led to the discovery of aggrecanase 1, which was proven to be ADAMTS4, and aggrecanase 2, which was proven to be ADAMTS5. Subsequently, other related ADAMTS enzymes, including ADAMTS1, -8, -9, -15, and -20, were shown to have aggrecanase activity. ADAMTS16 and 18 are also weak aggrecanases. ADAMTS and matrix metalloproteinases (MMPs) share a binding site for aggrecan that is highly similar in both sequence and overall shape (El Bakali et al. 2014 Future Medicinal Chemistry (Review) 6:1399).

[0004] ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) enzymes are secreted, multidomain, matrix-associated zinc metalloendopeptidases with diverse roles in tissue morphogenesis and pathophysiological remodeling, as well as in inflammation and vascular biology (Kelwick et al. 2015 Genome Biology 16:113). The human family contains 19 members that can be subgrouped based on their known substrates. Aggrecanases or proteoglycanases include ADAMTS1, -4, -5, -8, -9, -15, and -20, which can cleave hyaluronan-binding chondroitin sulfate proteoglycan (CSPG) extracellular proteins, including aggrecan, versican, brevican, and neurocan. The two most preferred cleavage sites on bovine aggrecan are KEEE. 1667 ↓ 1668 GLGS, then GELE 1480 ↓ 1481 GRGT followed. Then IGD's NITEGE 373 ↓ 374 Cleavage occurs at ARGS (where MMPs do not cleave), releasing the aforementioned neo-epitope, and TAQE in the CS-2 region. 1771 ↓ 1772 AGEG and VSQE 1871 ↓ 1872Cleavage occurs at the LGQR (Fosang et al. 2008 European Cells and Materials 15:11-26). These cleavage sites are highly conserved in humans, cows, mice, and rats. Various lines of evidence indicate that ADAMTS5 is a key enzyme involved in the pathogenesis of osteoarthritis. ADAMTS5 is the major aggrecanase present in cartilage. Knockdown of ADAMTS5 attenuated aggrecan degradation in human cartilage explants and chondrocytes, suggesting that this enzyme may be involved in human tissues. Expression of this enzyme is enhanced by cytokines such as interleukin-1 and oncostatin M, leading to the degradation of aggrecan in tissues. Aggrecan fragments generated by ADAMTS5 have been detected in the synovial fluid and serum of OA patients (Germaschewski et al., 2014 Osteoarthritis Cartilage 22:690-697).

[0005] ADAMTS5 is initially synthesized as an inactive protein containing an N-terminal protease domain and a C-terminal auxiliary domain. The protease domain consists of a signal peptide, a prodomain with a furin recognition sequence, and a catalytic domain. The prodomain is cleaved by a proprotein convertase to produce the active enzyme. ADAMTS5 also contains an auxiliary domain ("exosite") that actively participates in substrate recognition and regulates the affinity of the proteinase for its substrate. The disintegrin-like domain, central thrombospondin type I-like (TS) repeats, cysteine-rich domain, spacer region, and additional TS motifs of ADAMTS5 are auxiliary domains with potential exosite function. The cysteine-rich domain appears to be essential for ADAMTS5 binding and docking to glycosaminoglycans. The greatest variation among ADAMTS members is found in these auxiliary domains (Kelwick et al., 2015 Genome Biology 16:113). Disease-modifying anti-osteoarthritis drugs (DMOADs), which can be defined as drugs that inhibit structural disease progression and ideally improve symptoms and / or function, are highly sought after. Because DMOADs are likely to be prescribed long-term for this chronic disease in an elderly population, excellent safety data are required in a target population with multiple comorbidities and potential drug-drug interactions.

[0006] Several pharmaceutical companies are developing small molecule inhibitors of ADAMTS5. While some of these compounds are claimed to be specific for ADAMTS5, others have effects against other ADAMTS members or even MMPs. This cross-inhibition is thought to be responsible for the musculoskeletal syndrome caused by broad-spectrum inhibitors, including joint pain, muscle pain, joint stiffness, and tendonitis (Santamaria et al., 2015 Biochem J 471:391-401). The Wyeth aggrecanase inhibitor AGG-523 has been used in five Phase I clinical trials in healthy subjects and OA patients but has not progressed further. Other small molecule ADAMTS inhibitors have not yet entered further clinical development as potential DMOADs (Bondeson et al., 2015 Drug Discovery 10:5-14). Indeed, despite numerous recent clinical trials specifically investigating DMOAD, no such treatment has been approved to date (El Bakali et al., 2014 Future Medicinal Chemistry (Review) 6:1399).

[0007] Given the success of targeted biotherapy using antibodies ("Abs"), there has been interest in developing similar therapeutic strategies for OA. A study of Rottapharm's monoclonal antibody (mAb) CRB0017, directed against the spacer domain of ADAMTS5, showed that intra-articular administration of this mAb significantly prevented disease progression in a dose-dependent manner in mice (Chiusaroli et al., 2013 Osteoarthritis Cartilage 21:1807). No comparison with systemic administration was made, and the extent to which the mAb leaked out of the synovial cavity was not assessed. Another study, using systemic administration of the mAb 12F4 in mice, demonstrated both structural disease improvement and a reduction in pain-related behaviors (Miller et al., 2014 Osteoarthritis Cartilage 22iii, S35). However, a single administration of mAb 12F4 to cynomolgus monkeys induced local bleeding, dose-dependent mean arterial pressure and cardiac conductance abnormalities (more specifically, ST-segment elevation on the ECG and ventricular arrhythmias), and cardiac ischemia persisted for up to 8 months after a single administration (Larkin et al., 2014 Osteoarthritis Cartilage 22iii, S483). These side effects led to the halting of further clinical development of mAb 12F4.

[0008] WO2008 / 074840 describes the production of Nanobodies® under the name of Ablynx NV against members of the A disintegrin and metalloproteinase (ADAM) family, including ADAMTS5. Therapeutic intervention in joints is further hindered by the difficulty of targeting drugs to articular cartilage. Because articular cartilage is an avascular and alymphatic tissue, traditional drug delivery routes (oral, intravenous, intramuscular) ultimately rely on transsynovial transfer of drugs from synovial capillaries to the cartilage by passive diffusion. This has prompted the development of intra-articular (IA) delivery of pharmaceuticals. On the other hand, intraarticular delivery of therapeutic proteins is limited by rapid clearance from the joint space and poor retention within the cartilage, limiting it to large joints. The synovial residence time of drugs in joints is often less than 24 hours (Edwards 2011 Vet J 190:15-21; Larsen et al., 2008 J Pham Sci 97:4622-4654). The rapid clearance of most intraarticularly injected drugs necessitates frequent injections to maintain effective concentrations (Owen et al., 1994 Br J Clin Pharmacol 38:349-355). Furthermore, intraarticular delivery of therapeutic proteins is practically impractical for small joints, thereby preventing the treatment of, for example, OA in fingers. There remains a need for effective DMOADs. Summary of the Invention

[0009] 3. Summary of the Invention The present invention aims to provide polypeptides against OA that have improved prophylactic, therapeutic and / or pharmacological properties (e.g., improved ease of preparation, good stability, and / or reduced cost of goods) in addition to other advantageous properties when compared to prior art amino acid sequences and antibodies. In particular, the present invention aims to provide polypeptides that inhibit ADAMTS, and in particular ADAMTS5. ADAMs, ADAMTSs, and MMPs share a binding site for aggrecan, which is highly similar in both sequence and overall shape. Because various other ADAMs (including TACE) and ADAMTS family members have diverse roles in normal physiology and are strongly associated with many common pathological conditions, such as asthma, arthritis, cancer, connective tissue disorders, or thrombotic thrombocytopenic purpura (El Bakali et al., supra), the present inventors recognized the importance of maintaining selectivity. To effectively inhibit only ADAMTS5 activity, targeting the catalytic domain seemed the best option. However, the catalytic domains of ADAMTS4 and ADAMTS5 share a high degree of sequence similarity (see El Bakali et al., supra). Furthermore, high sequence conservation of the catalytic domains across various species has been found to precede robust immune responses.

[0010] Surprisingly, the ISVD of the present invention fulfills these two seemingly mutually exclusive requirements: on the one hand, it maintains selectivity, and on the other hand, it inhibits the (enzymatic) activity of ADAMTS5. Various monovalent ISVDs of the present invention were equipotent to the conventional bivalent antibody mAb 12F4 H4L0 in AlphaLISA enzyme assays. In addition, the ISVDs of the present invention were also equipotent to the conventional bivalent antibody mAb 12F4 H4L0 at high or even excessive aggrecan substrate concentrations reminiscent of joints. Meanwhile, in ex vivo bovine explant assays that more closely resemble physiological conditions, most monovalent ISVDs exhibited superior IC values ​​compared to Rottapharm's comparators mAb 12F4 H4L0 ("12F4") and mAb CRB0017. 50 In human ex vivo explant assays, the ISVDs of the present invention demonstrated IC 50 As shown by the above, the antibody was substantially superior to the conventional antibody CRB0017 of the prior art. After further engineering the ISVDs to take into account a variety of desirable characteristics, including stability, affinity, inhibitory activity, and minimization of immunogenicity, these ISVDs were then evaluated in vivo.

[0011] Systemic administration of the ISVD of the present invention demonstrated potent inhibition of aggrecanase activity in vivo, as assessed in cynomolgus monkeys. Furthermore, the ISVD was safe to use, in contrast to the prior art antibody 12F4. Furthermore, in an in vivo mouse medial meniscus destabilization (DMM) model, the ISVD of the present invention demonstrated up to 50% structural benefit in both preventive and therapeutic treatments administered systemically. Early treatment with the ISVD of the present invention resulted in dose-dependent, significant, and important symptomatic benefits in anterior cruciate ligament resection and medial meniscectomy (ACLT + tMx)-induced OA in rats.

[0012] Since the ultimate goal of ISVD is to inhibit ADAMTS5 in joints, it must be resistant to the synovial fluid, which contains various proteases. In addition to the above-mentioned favorable properties, the isolated ISVD has been shown to be highly stable in synovial fluid. This stability is expected to enable less frequent administration. Thus, the present invention relates to polypeptides comprising at least one immunoglobulin single variable domain (ISVD) that binds to an A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), wherein preferably said ADAMTS is selected from the group consisting of ADAMTS1 to ADAMTS19, preferably ADAMTS5, ADAMTS4, ADAMTS1, ADAMTS8, ADAMTS9, and ADAMTS15 and ADAMTS20, most preferably ADAMTS5. In one aspect, the present invention relates to a polypeptide as described herein, wherein said ISVD that binds to an ADAMTS, preferably ADAMTS5, does not bind to ADAMTS4, MMP1, or MMP14.

[0013] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein the ISVD that specifically binds to ADAMTS5 consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is SEQ ID NO: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; and an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NOs: 21, 35, 20, 22, 25, 33, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; (ii) CDR2 is SEQ ID NO: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49, and 52; and an amino acid sequence having one, two or three amino acid differences from SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and (iii) CDR3 is SEQ ID NO: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67, and 70; and and amino acid sequences having 1, 2, 3 or 4 amino acid differences from SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70.

[0014] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein the ISVD that specifically binds to ADAMTS5 consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein (i) CDR1 is selected from the group consisting of (a) SEQ ID NO: 22; and (b) an amino acid sequence having 1, 2, 3, 4, 5, or 6 amino acid differences from SEQ ID NO: 22, wherein at position 2, S is changed to R; at position 3, A is changed to T; at position 4, V is changed to F; at position 6, V is changed to S; at position 7, N is changed to Y; and / or at position 10, A is changed to G; (ii) CDR2 is SEQ ID NO: 36; and (iii) CDR3 is SEQ ID NO: 54.

[0015] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein the ISVD that specifically binds to ADAMTS5 consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein (i) CDR1 is SEQ ID NO: 33; (ii) CDR2 is selected from the group consisting of (c) SEQ ID NO: 50; and (d) amino acid sequences having one, two, or three amino acid differences from SEQ ID NO: 50, wherein at position 8 M is changed to I; at position 9 P is changed to T; and / or at position 10 Y is changed to F; and (iii) CDR3 is selected from the group consisting of (e) SEQ ID NO: 68; and (f) amino acid sequences having one or two amino acid differences from SEQ ID NO: 68, wherein at position 5 F is changed to L; and / or at position 11 D is changed to E.

[0016] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein the ISVD that specifically binds to ADAMTS5 consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein (i) CDR1 is SEQ ID NO: 28; (ii) CDR2 is selected from the group consisting of (c) SEQ ID NO: 44; and (d) amino acid sequences having one, two, or three amino acid differences from SEQ ID NO: 44, wherein at position 3 S is changed to T; at position 4 R is changed to W; at position 8 T is changed to I; and / or at position 9 T is changed to L; (iii) CDR3 is selected from the group consisting of (e) SEQ ID NO: 62; and (f) amino acid sequences having one or two amino acid differences from SEQ ID NO: 62, wherein at position 1 G is changed to S; and / or at position 14 D is changed to E. In preferred embodiments of all aspects of the invention, immunoglobulin single variable domains (ISVDs) according to the invention preferably consist of or consist essentially of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions CDR1, CDR2, and CDR3, as outlined herein above and below. Preferred framework sequences are disclosed, for example, in Table A-2 below, and can be used in the ISVDs of the invention. Preferably, the CDRs shown in Table A-2 correspond to the respective framework regions of the same ISVD construct.

[0017] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said ISVD is selected from the group of ISVDs, wherein: CDR1 is selected from the group consisting of SEQ ID NOs: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and CDR3 is selected from the group consisting of SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70. In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said ISVD is selected from the group of ISVDs wherein: CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55; CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 118; CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 71; CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54; CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54; CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69; CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62; CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59; CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 119, and CDR3 is SEQ ID NO: 60; CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; CDR1 is SEQ ID NO:29, CDR2 is SEQ ID NO:46, and CDR3 is SEQ ID NO:64; CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66; CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; and CDR1 is SEQ ID NO:34, CDR2 is SEQ ID NO:52, and CDR3 is SEQ ID NO:70.

[0018] In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37 and CDR3 is SEQ ID NO: 55. In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said ISVD is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18. In a further preferred aspect, the present invention relates to a polypeptide as described herein, said polypeptide having a K D is 1E-07 M~1E -13 Between M, for example, 1E -08 M~1E -12 Between M, preferably at most 1E -07 M, preferably 1E -08 M or 1E -09 Less than M or 1E -10 Less than M, for example, 5E -11 M, 4E -11 M, 3E -11 M, 2E -11 M, 1.7E -11 M, 1E -11 M, etc., or 5E -12 M, 4E -12 M, 3E -12 M, 1E -12 M, which binds to ADAMTS5.

[0019] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide exhibits an IC 50 is 1E -07 M~1E -12 Between M, for example, 1E -08 M~1E -11 Inhibiting ADAMTS5 activity among M . In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide is an IC 50 is up to 1E -07 M, preferably 1E -08 M, 5E -09 M, or 4E -9 M, 3E -9 M, 2E -9 M, e.g. 1E -9 M, etc., inhibit the (enzyme) activity of ADAMTS5. In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide is capable of binding to EC 50 is 1E -07 M~1E -12 Between M, for example, 1E -08 M~1E -11Regulating ADAMTS5 among M .

[0020] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide has an off-rate of 1E or less, as determined, for example, by SPR. -04 s -1 It binds to ADAMTS5 at less than 100 kJ / mol. In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said ADAMTS5 is human ADAMTS5 (SEQ ID NO: 149), bovine ADAMTS5 (SEQ ID NO: 150), rat ADAMTS5 (SEQ ID NO: 151), guinea pig ADAMTS5 (SEQ ID NO: 152), mouse ADAMTS5 (SEQ ID NO: 153) or cynomolgus monkey ADAMTS5 (SEQ ID NO: 154), preferably human ADAMTS5, most preferably SEQ ID NO: 149. In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide antagonizes the activity of ADAMTS5, e.g., protease activity, such as cleavage of aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, preferably cleavage of aggrecan; preferably, antagonizes the aggrecanase activity of ADAMTS5.

[0021] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide blocks the binding of ADAMTS5 to aggrecan by at least 20%, such as at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, as determined, for example, by FRET, AlphaLISA or ELISA. In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide inhibits the protease activity of ADAMTS5, e.g., inhibits the proteolysis of substrates such as aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, preferably aggrecan. In a further preferred aspect, the present invention relates to a polypeptide as described herein comprising at least two ISVDs, wherein at least one ISVD specifically binds to ADAMTS, preferably ADAMTS5, and is preferably selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18, wherein preferably at least two ISVDs specifically bind to ADAMTS, preferably ADAMTS5.

[0022] In a further preferred aspect, the present invention relates to a polypeptide comprising two or more ISVDs that specifically bind to ADAMTS5, wherein (a) at least a "first" ISVD specifically binds to a first antigenic determinant, epitope, portion, domain, subunit, or conformation of ADAMTS5; and wherein (b) at least a "second" ISVD specifically binds to a second antigenic determinant, epitope, portion, domain, subunit, or conformation of ADAMTS5, each different from the first antigenic determinant, epitope, portion, domain, subunit, or conformation. and 18, and preferably wherein said "second" ISVD that specifically binds to ADAMTS5 is SEQ ID NO: 118 or 19, and even more preferably said polypeptide is selected from the group consisting of SEQ ID NO: 127 (clone 130 049-093-Alb), SEQ ID NO: 126 (clone 129 2F3-093-ALB), SEQ ID NO: 127 (clone 130 049-093-Alb) and SEQ ID NO: 128 (clone 131 9D3-093-Alb).

[0023] In a further preferred aspect, the present invention relates to a polypeptide as described herein, further comprising a serum albumin-binding ISVD, preferably wherein said serum albumin-binding ISVD consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO: 146; CDR2 is SEQ ID NO: 147; and CDR3 is SEQ ID NO: 148; even more preferably wherein said serum albumin-binding ISVD is selected from the group consisting of ALB8 (SEQ ID NO: 131), ALB23 (SEQ ID NO: 132), ALB 129 (SEQ ID NO: 133), ALB132 (SEQ ID NO: 134), ALB11 (SEQ ID NO: 135), ALB11(S112K)-A (SEQ ID NO: 136), ALB82 (SEQ ID NO: 137), ALB82-A (SEQ ID NO: 138), ALB82-AA (SEQ ID NO: 139), ALB82-AAA (SEQ ID NO: 140), ALB82-G (SEQ ID NO: 141), ALB82-GG (SEQ ID NO: 142), ALB82-GGG (SEQ ID NO: 143), ALB92 (SEQ ID NO: 144), and ALB223 (SEQ ID NO: 145), and even more preferably, said polypeptide is selected from the group consisting of SEQ ID NO: 129 (clone 577 2F3 SO -Alb), SEQ ID NO: 130 (clone 579 2F3 SO -093-Alb), SEQ ID NO: 120 (clone 4 2A12-Alb), Selected from the group consisting of SEQ ID NO: 121 (clone 5 2D7-Alb), SEQ ID NO: 122 (clone 6 2F3-Alb), SEQ ID NO: 123 (clone 69 049-Alb), SEQ ID NO: 124 (clone 70 9D3-Alb), SEQ ID NO: 125 (clone 71 3B2-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb), and SEQ ID NO: 128 (clone 131 9D3-093-Alb).

[0024] In a further preferred aspect, the present invention relates to a polypeptide as described herein, further comprising at least one ISVD that specifically binds to aggrecan, preferably said ISVD selected from the group consisting of SEQ ID NO: 156 (nanobody 00745 PEA114F08) and SEQ ID NO: 157 (nanobody 00747 PEA604F02). In a further preferred aspect, the present invention relates to a polypeptide as described herein, comprising at least two ISVDs that specifically bind to aggrecan, wherein the at least two ISVDs that specifically bind to aggrecan may be identical or different, preferably wherein the at least two ISVDs that specifically bind to aggrecan are independently selected from the group consisting of SEQ ID NOs: 156 and 157, and even more preferably, the ISVDs that specifically bind to aggrecan specifically bind to human aggrecan [SEQ ID NO: 155]. Preferably, the ISVDs that specifically bind to aggrecan specifically bind to dog aggrecan (see also Table 2), bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan; rabbit aggrecan; cynomolgus monkey aggrecan and / or rhesus monkey aggrecan. Preferably, the ISVDs that specifically bind to aggrecan preferably bind to cartilaginous tissue, such as cartilage and / or meniscus.

[0025] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide has stability at 37°C in synovial fluid (SF) for at least 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months. In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein the at least two ISVDs are linked to each other directly or via a linker, preferably the linker is selected from the group consisting of SEQ ID NOs: 158 to 174 (i.e., SEQ ID NOs: 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 and 174), preferably SEQ ID NO: 169. In a further preferred aspect, the present invention relates to a polypeptide as described herein further comprising a C-terminal extension, preferably wherein said C-terminal extension is C-terminal extension (X)n, where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0026] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide has at least 80%, 90%, 95% or 100% sequence identity to any of SEQ ID NOs: 1-19 (i.e. SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19), 116-117 or 120-130 (i.e. SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130). In a further preferred aspect, the present invention relates to a method for treating and / or preventing a disease or disorder in an individual, e.g., one involving ADAMTS5 activity, comprising administering to the individual a polypeptide according to any one of claims 1 to 43 in an amount effective to treat or prevent the symptoms of the disease or disorder, preferably wherein the disease or disorder is selected from the group consisting of arthropathy and chondrodystrophy, arthritis, e.g., osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tear or avulsion, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans, and aggrecanopathy. More preferably, the disease or disorder is arthritis, most preferably osteoarthritis.

[0027] In a further preferred aspect, the present invention relates to a polypeptide as described herein for use as a medicament. In a further preferred aspect, the present invention relates to a polypeptide as described herein for use in the treatment or prevention of symptoms of ADAMTS5-related diseases, such as arthropathy and chondrodystrophy, arthritis such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or avulsions, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated discs, lumbar degenerative disc disease, degenerative joint disease, and symptoms of relapsing polychondritis, osteochondritis dissecans, and aggrecanopathy. More preferably, the disease is arthritis, most preferably osteoarthritis.

[0028] In a further preferred aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide cross-blocks the binding to ADAMTS5 of at least one polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18 and / or is cross-blocked in binding to ADAMTS5 by at least a polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18. In a further preferred aspect, the present invention relates to a polypeptide that cross-blocks binding to ADAMTS5 by a polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18, and / or whose binding to ADAMTS5 is cross-blocked by at least a polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18, wherein the polypeptide comprises at least one VH, VL, dAb, immunoglobulin single variable domain (ISVD) that specifically binds to ADAMTS5, and wherein binding to ADAMTS5 modulates the activity of ADAMTS5.

[0029] Other aspects, advantages, applications, and uses of the polypeptides and compositions will become apparent from the further disclosure herein. Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. [Brief explanation of the drawings]

[0030] 4. Figure legend [Figure 1] FIG. 1 shows that ISVD binding to ADAMTS5 does not inhibit the activity of MMP1 (A) or MMP14 (B). [Figure 2] FIG. 2 shows that ISVD binding to ADAMTS5 does not inhibit ADAMTS4 activity. [Figure 3] FIG. 3 shows the binding levels of pre-existing antibodies (pre-Abs) to nanobody construct 581 (A) and nanobody construct 579 (B) from three donor sample sets derived from a healthy subject, a subject with osteoarthritic disease, and a donor with residual pre-Ab binding. [Figure 4] FIG. 4 shows that nanobody construct 581 (“C011400581”) does not bind to human ADAMTS1 (A) and does not bind to human ADAMTS4 or human ADAMTS15 (B). [Figure 5] Figure 5 shows efficacy in a human explant system, showing GAG accumulation in the supernatant after 7 days.

[0031] [Figure 6]A) Time course of GAG release into the supernatant in a bovine co-culture system. B) Time course (7-28 days) of calculation of the area under the curve (AUC) of GAG release. Replicates n=4. Data are displayed in Box & Whisker format from minimum to maximum. C) Data are displayed as GAG content [ng / mg cartilage] after papain digestion. Replicates n=4. Data are displayed in Box & Whisker format from minimum to maximum. [Figure 7] Figure 7 shows the time course (7-28 days) of area under the curve (AUC) calculation of exAGNx1 release. Number of replicates n=4. Data are displayed from minimum to maximum in Box & Whisker format. [Figure 8] Figure 8 shows the time course (7-28 days) of the area under the curve (AUC) calculation of C2M release. Number of replicates n=4. Data are displayed from minimum to maximum in Box & Whisker format.

[0032] [Figure 9] Figure 9 shows the time course (7-28 days) of the area under the curve (AUC) calculation of C3M release. Number of replicates n=4. Data are displayed from minimum to maximum in Box & Whisker format. [Figure 10] FIG. 10 shows inhibition of aggrecanase activity in NHPs. [Figure 11] Figure 11 shows the inhibition of cartilage degeneration. The total median cartilage degeneration is shown in preventative (A) and therapeutic (B) treatment in the DMM mouse model. [Figure 12] FIG. 12 shows symptomatic behavior in a rat surgical OA model. [Figure 13] FIG. 13 shows the width of medial tibial cartilage degeneration. [Figure 14] Figure 14 shows the effect of anti-ADAMTS5 nanobodies on aggrecanase-mediated degradation of aggrecan in ex vivo cartilage cultures (A, B, C) and in cartilage-synovium cocultures (D, E). The concentrations listed are those of the nanobodies. Statistical analysis was performed using ordinary one-way or two-way ANOVA. Statistical significance was considered at p<0.05.

[0033] 5. Detailed Description There remains a need for safe and effective OA medications, particularly DMOADs. These medications must meet various, often conflicting requirements, especially if a broadly applicable format is intended. Therefore, the format should preferably be useful for a wide range of patients. The format is preferably safe and does not induce infection with frequent administration. In addition, the format is preferably patient-friendly, e.g., it allows for convenient dosing schedules and routes of administration, e.g., systemic administration. For example, upon administration, it is preferred that the format is not rapidly removed from the circulation. However, extended half-life should preferably not result in off-target activity or side effects or limit efficacy. The present invention fulfills at least one of these requirements. Based on unconventional screening, characterization, and combinatorial strategies, the inventors surprisingly observed that immunoglobulin single variable domains (ISVDs) perform very well in in vitro and in vivo experiments.

[0034] Furthermore, the inventors were able to re-engineer the ISVD to perform even better than comparator drugs in improving OA, and the ISVD of the present invention was also demonstrated to be significantly safer than prior art compounds. The present invention aims to provide polypeptides that antagonize ADAMTS, particularly ADAMTS5, which have improved prophylactic, therapeutic and / or pharmacological properties, including a safer profile, compared to prior art amino acid sequences and antibodies. Thus, the present invention relates to ISVDs and polypeptides directed against and / or capable of specifically binding to ADAMTS5 (as defined herein). Thus, the present invention relates to ISVDs and polypeptides that are directed against and / or specifically bind to ADAMTS (as defined herein) and regulate its activity, in particular polypeptides comprising at least one ISVD that specifically binds to ADAMTS5, wherein binding to ADAMTS5 regulates the activity of ADAMTS5.

[0035] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be apparent to those skilled in the art. Reference is made, for example, to standard handbooks such as: Sambrook et al. (Molecular Cloning: A Laboratory Manual (2 nd Ed.) Vols. 1-3, Cold Spring Harbor Laboratory Press, 1989), F. Ausubel et al. (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 1987), Lewin (Genes II, John Wiley & Sons, New York, NY, 1985), Old et al. (Principles of Gene Manipulation: An Introduction to Genetic Engineering (2nd edition) University of California Press, Berkeley, CA, 1981); Roitt et al. (Immunology (6 th Ed.) Mosby / Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's Essential Immunology (10 th Ed.) Blackwell Publishing, UK, 2001), and Janeway et al. (Immunobiology (6 thEd.) Garland Science Publishing / Churchill Livingstone, New York, 2005), as well as the general background art cited therein.

[0036] Unless otherwise indicated, all methods, steps, techniques, and operations not specifically described in detail can be and have been performed in a manner known per se, as would be apparent to one skilled in the art. Reference is again made, for example, to the standard handbooks and general background art mentioned herein and the further references cited therein; reference is also made, for example, to the following reviews: Presta (Adv. Drug Deliv. Rev. 58 (5-6): 640-56, 2006), Levin and Weiss (Mol. Biosyst. 2(1): 49-57, 2006), Irving et al. (J. Immunol. Methods 248(1-2): 31-45, 2001), Schmitz et al. (Placenta 21 Suppl. A: S106-12, 2000), Gonzales et al. (Tumor Biol. 26(1): 31-43, 2005), which describe protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desirable properties of proteins such as immunoglobulins.

[0037] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such different reagents, and reference to a "method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or substituted for the methods described herein. Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0038] The term "and / or" whenever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term". As used herein, the term "about" or "approximately" means within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range. Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises," and variations such as "comprises" and "comprising," mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," or in some cases the term "having" can also be used herein.

[0039] As used herein, the term "sequence" (e.g., "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "V HH "Analytical sequence" or "protein sequence" and other terms should generally be understood to include both the related amino acid sequence as well as the nucleic acid or nucleotide sequence encoding same, unless the context requires a more restrictive interpretation. An amino acid sequence is taken to mean, depending on the context, a single amino acid or an unbranched sequence of two or more amino acids. A nucleotide sequence is taken to mean an unbranched sequence of three or more nucleotides.

[0040] Amino acids are L-amino acids commonly found in natural proteins. Amino acid residues are designated according to the standard three-letter or one-letter amino acid code. See, for example, Table A-2 on page 48 of WO 08 / 020079. Those amino acid sequences containing D-amino acids are not intended to be included in this definition. Amino acid sequences containing post-translationally modified amino acids may be described as the originally translated amino acid sequence using the symbols shown in this Table A-2 and the positions of modifications, such as hydroxylation or glycosylation, but these modifications should not be explicitly shown in the amino acid sequence. All peptides or proteins that can be expressed with sequence-modified bonds, bridges and end caps, non-peptidyl linkages, etc., as known in the art, are included in this definition.

[0041] The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure, and each has the same meaning for purposes of this disclosure. Each term refers to an organic compound made up of a linear chain of two or more amino acids. The compound may have 10 or more amino acids, 25 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, or even 300 or more amino acids. Those of skill in the art will understand that, although polypeptides generally contain fewer amino acids than proteins, there is no art-recognized cutoff point for the number of amino acids that distinguishes a polypeptide from a protein; polypeptides can be made by chemical synthesis or recombinant methods; and proteins are generally made in vitro or in vivo by recombinant methods known in the art. By convention, the amide bonds in the primary structure of a polypeptide are in the order in which the amino acids are written, with the amine end (N-terminus) of the polypeptide always on the left and the acid end (C-terminus) on the right.

[0042] A nucleic acid or amino acid sequence is considered to be "(essentially) isolated" if it has been separated—e.g., compared to the reaction or culture medium from which it was obtained—from at least one other component normally associated with said source or medium, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity, or trace component. In particular, a nucleic acid or amino acid sequence is considered to be "(essentially) isolated" if it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. A nucleic acid or amino acid "in (essentially) isolated form" is preferably essentially homogeneous, as determined using a suitable technique, for example, using a suitable chromatographic technique such as polyacrylamide gel electrophoresis.

[0043] When a nucleotide sequence or amino acid sequence is said to "comprise" or "consist essentially of" another nucleotide sequence or amino acid sequence, respectively, this can mean that the latter nucleotide sequence or amino acid sequence is incorporated into the first-mentioned nucleotide sequence or amino acid sequence, respectively, but more usually it generally means that the first-mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, which has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, regardless of how the first-mentioned sequence was actually produced or obtained (e.g., by any suitable method described herein). By way of non-limiting example, when a polypeptide of the invention is said to comprise an immunoglobulin single variable domain ("ISVD"), this can mean that said immunoglobulin single variable domain sequence has been incorporated into the sequence of the polypeptide of the invention, but more usually it generally means that a polypeptide of the invention comprises within its sequence the sequence of an immunoglobulin single variable domain, regardless of how it was produced or obtained. Also, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, it means that when the first-mentioned nucleic acid or nucleotide sequence is expressed into an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of the expression product (in other words, the latter nucleotide sequence is in the same reading frame as the larger first-mentioned nucleic acid or nucleotide sequence).Also, when a construct of the invention is said to comprise a polypeptide or ISVD, this can mean that the construct at least includes the polypeptide or ISVD, respectively, but more generally it means that the construct includes groups, residues (e.g., amino acid residues), moieties and / or binding units in addition to the polypeptide or ISVD, regardless of how the polypeptide or ISVD is connected to the groups, residues (e.g., amino acid residues), moieties and / or binding units, and regardless of how the construct was produced or obtained.

[0044] "Essentially consisting of" means that the ISVD used in the present invention is either identical to the ISVD of the present invention or corresponds to the following ISVD of the present invention: an ISVD having a limited number of amino acid residues, such as 1 to 20 amino acid residues, for example 1 to 10 amino acid residues, preferably 1 to 6 amino acid residues, for example 1, 2, 3, 4, 5 or 6 amino acid residues, added to the amino terminus, carboxy terminus, or both the amino terminus and carboxy terminus of the ISVD. To compare two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated as follows: [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotide at the corresponding position in the second nucleotide sequence] divided by [the total number of nucleotides in the first nucleotide sequence] multiplied by [100%]; where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings using known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Several other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768. Typically, for purposes of determining the percentage of "sequence identity" between two nucleotide sequences according to the calculation method outlined above, the nucleotide sequence with the greatest number of nucleotides will be referred to as the "first" nucleotide sequence and the other nucleotide sequence will be referred to as the "second" nucleotide sequence.

[0045] For comparing two or more amino acid sequences, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be calculated as follows: [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] divided by [the total number of amino acid residues in the first amino acid sequence] multiplied by [100%]; where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two or more amino acid sequences can be calculated using known computer algorithms, such as those described above for determining the degree of sequence identity of nucleotide sequences, again using standard settings. Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated the "first" amino acid sequence, and the other amino acid sequence is designated the "second" amino acid sequence.

[0046] In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may also consider so-called "conservative" amino acid substitution, which can generally be described as an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, but has little or no effect on the function, activity or other biological properties of polypeptide.Such conservative amino acid substitutions are well known in the art, for example, from WO 04 / 037999, GB 335768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and such (preferred) types and / or combinations of substitutions can be selected based on the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and the further references cited therein.

[0047] Such conservative substitutions are preferably those in which one amino acid residue within the following groups (a) to (e) is replaced with another amino acid residue within the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar positively charged residues: His, Arg, and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Gln to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0048] Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of amino acid mutation frequencies between homologous proteins of different species developed by Schulz et al. ("Principles of Protein Structure", Springer-Verlag, 1978), the structure-forming probability analysis developed by Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45-149, 1978), and the structure-forming probability analysis developed by Eisenberg et al. (Proc. Natl. Acad Sci. USA 81: 140-144, 1984), Kyte and Doolittle (J. Molec. Biol. 157: 105-132, 1981), and Goldman et al. (Ann. Rev. Biophys. Chem. 15: 321-353, 1982). 1986), all of which are incorporated herein by reference in their entirety. Information regarding the primary, secondary and tertiary structure of nanobodies is provided in the description herein and in the general background art cited above. For this purpose, V from llamas may also be used. HH Crystal structures of domains have been given, for example, by Desmyter et al. (Nature Structural Biology, 3: 803, 1996), Spinelli et al. (Natural Structural Biology, 3: 752-757, 1996) and Decaniere et al. (Structure, 7 (4): 361, 1999). H V in the domain H / V L Further information about some of the amino acid residues that form the interface, and potential camelizing substitutions at these positions, can be found in the prior art cited above.

[0049] Amino acid and nucleic acid sequences are said to be "completely identical" if they have 100% sequence identity (as defined herein) over their entire length. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence; it is understood that the two amino acid sequences may contain one, two, or more such amino acid differences. More specifically, in the ISVDs and / or polypeptides of the present invention, the term "amino acid difference" refers to the insertion, deletion, or substitution of a single amino acid residue at the position of the CDR sequence specified in b), d), or f), compared to the CDR sequence of a), c), or e), respectively. It is understood that the CDR sequences of b), d), and f) may contain one, two, three, four, or up to five such amino acid differences compared to the CDR sequence of a), c), or e), respectively.

[0050] "Amino acid differences" can be any one, two, three, four, or up to five substitutions, deletions, or insertions, or any combination thereof, which improve the properties of the ADAMTS5-binding agents of the present invention, such as the polypeptides of the present invention, or at least do not unduly detract from the desired properties, or from the balance or combination of desired properties, of the ADAMTS5-binding agents of the present invention, such as the polypeptides of the present invention. In this regard, the ADAMTS5-binding agents obtained by the present invention, such as the polypeptides of the present invention, should bind to ADAMTS5 with at least the same, approximately the same, or higher affinity than a polypeptide comprising one or more CDR sequences without one, two, three, four, or up to five substitutions, deletions, or insertions. The affinity can be measured by any suitable method known in the art, but is preferably measured by the method described in the Examples section.

[0051] In this respect, the amino acid sequences of the CDRs according to b), d) and / or f) shown below may be amino acid sequences derived from the amino acid sequences according to a), c) and / or e), respectively, by affinity maturation using one or more techniques of affinity maturation known per se or as described in the Examples. Depending for example on the host organism used to express the polypeptide of the invention, such deletions and / or substitutions may be designed to eliminate one or more sites for post-translational modification (such as one or more glycosylation sites), which would be within the capabilities of a person skilled in the art (see Examples). As used herein, "nanobody family," "V HH A "family" or "family" refers to nanobodies and / or Vs that have the same length. HH It refers to a group of sequences (i.e., that have the same number of amino acids in their sequence) in which the amino acid sequences between positions 8 and 106 (according to Kabat numbering) have greater than or equal to 89% amino acid sequence identity.

[0052] The terms "epitope" and "antigenic determinant," which can be used interchangeably, refer to a portion of a macromolecule, e.g., a polypeptide or protein, that is recognized by an antigen-binding molecule, such as an immunoglobulin, a conventional antibody, an immunoglobulin single variable domain, and / or a polypeptide of the invention, more specifically by the antigen-binding site of said molecule. An epitope defines the minimal binding site for an immunoglobulin and thus represents the target of immunoglobulin specificity. The part of an antigen-binding molecule (such as an immunoglobulin, a conventional antibody, an immunoglobulin single variable domain and / or a polypeptide of the invention) that recognizes an epitope is called the "paratope." An amino acid sequence (an immunoglobulin single variable domain, antibody, polypeptide, or generally an antigen-binding protein or polypeptide or fragment thereof of the invention) that is able to "bind" or "specifically bind" or "have affinity for" and / or "have specificity" for a particular epitope, antigen or protein (or at least a portion, fragment or epitope thereof) is said to be "against" or "directed against" said epitope, antigen or protein, or to be a "binding" molecule with respect to such epitope, antigen or protein, or to be an "anti" epitope, "anti" antigen or "anti" protein (e.g., "anti" ADAMTS5).

[0053] Affinity indicates the strength or stability of a molecular interaction. Affinity is usually expressed as K D , or dissociation constant, which has units of moles / liter (or M). Affinity is determined by the association constant, K A can also be expressed as 1 / K D is equal to (moles / liter) -1 (or M -1 ) as used herein, the stability of an interaction between two molecules is primarily determined by the K D It is expressed in terms of the value K A =1 / K D Considering the relationship between K and K, the strength of molecular interaction is calculated by D By specifying the value of A It will be clear to those skilled in the art that the value of K D The value also characterizes the strength of molecular interactions in a thermodynamic sense, which is K D The value corresponds to the change in free energy of binding (DG) according to the well-known relationship DG = RT.ln(K D )(Equivalently, DG=-RT.ln(K A )) where R is the gas constant, T is the absolute temperature, and ln is the natural logarithm. K for biological interactions that are considered important (e.g., specific) D is usually 10 -12 M(0.001nM)~10 -5The stronger the interaction, the higher the K D will be lower.

[0054] K D is k off and the dissociation rate constant of the complex, k on It can also be expressed as a ratio of its association rate, which is given by (hence K D =k off / k on and K. A =k on / k off ). Off-rate k off The unit is s -1 (s is the SI unit for second). on The unit is M -1 s -1 The on-rate is 10 2 M -1 s -1 ~about 10 7 M -1 s -1 The off-rate varies between t and t and approaches the diffusion-limited association rate constant of the bimolecular interaction. 1 / 2 =ln(2) / k off The off-rate is related to the half-life of a particular molecular interaction by -6 s -1 (multiple days of t 1 / 2 (close to an irreversible complex with -1 (t 1 / 2 =0.69s). The specific binding of an antigen-binding protein, such as an ISVD, to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, saturation binding assays and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the various modifications thereof known per se in the art, as well as other techniques mentioned herein.

[0055] The affinity of a molecular interaction between two molecules can be measured by different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (e.g., Ober et al. 2001, Intern. Immunology 13: 1551-1559), where one molecule is immobilized on a biosensor chip and the other molecule is reacted with k on , k off Measurement of K D (or K A A flow of 1000 kJ / min is passed over the immobilized molecule under flow conditions that result in a β-value (β). This can be performed, for example, using the well-known BIACORE® instrument (Pharmacia Biosensor AB, Uppsala, Sweden). Kinetic Exclusion Assay (KINEXA® instrument) (Drake et al. 2004, Analytical Biochemistry 328: 35-43) is based on measuring binding events in solution without labeling of the binding partner and kinetically excluding dissociation of the complex. In-solution affinity analysis can also be performed using the GYROLAB® Immunoassay System (Fraley et al. 2013, Bioanalysis 5: 1765-74), which provides a platform for automated bioanalysis and rapid sample turnaround, or ELISA.

[0056] Also, if the measurement process has any effect on the intrinsic binding affinity of the molecule in question, for example due to artifacts associated with coating the biosensor with a molecule, the measured K D But the apparent K D It will also be apparent to those skilled in the art that when one molecule contains multiple recognition sites for other molecules, the apparent K D In such a situation, the measured affinity may be affected by the avidity of the interaction between the two molecules. In particular, K D Accurate measurement of K can be very labor intensive, resulting in an apparent K DApparent K values ​​are often determined to assess the binding strength of two molecules. As long as all measurements are performed in a consistent manner (e.g., without changing assay conditions), the apparent K D Measurements are true K D can be used as an approximation of K D and apparent K D should be treated with equal importance or relevance.

[0057] The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as a polypeptide or ISVD of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity, for example, as described on pages 53-56 of WO 08 / 020079 (incorporated herein by reference), which also describes some preferred techniques for measuring binding between an antigen-binding molecule (such as a polypeptide or ISVD of the invention) and the relevant antigen. Typically, an antigen-binding protein (such as an ISVD and / or polypeptide of the invention) will have a dissociation constant (K D ) as 10 -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, and more preferably 10 -8 ~10 -12 The number of molecules that bind to their antigens (i.e., the association constant (K A ) as 10 5 ~10 12 liters / mol or more, preferably 10 7 ~10 12 liters / mole or more, and more preferably 10 8 ~10 12 liters / mole). 10 -4 Any K greater than moles / liter D value (or 10 4 Any K less than liters / mole AA value (σ) is generally considered to indicate non-specific binding. Preferably, the monovalent ISVDs of the invention will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM, such as 10-5 pM or less. See also paragraph (n) of pages 53-56 of WO 08 / 020079.

[0058] An ISVD and / or polypeptide is said to be "specific" for a (first) target or antigen compared to another (second) target or antigen if the ISVD and / or polypeptide binds to the first antigen with greater affinity (as described above and K D value, K A value, K off Rate and / or K on For example, an ISVD and / or polypeptide binds to a first target or antigen with an affinity (appropriately expressed as a K , or K ) that is at least 10-fold, e.g., at least 100-fold, preferably at least 1000-fold or greater, than that of said ISVD and / or polypeptide binding to a second target or antigen. D at least 10 times, for example at least 100 times, and preferably at least 1000 times, or even smaller than D Preferably, when an ISVD and / or polypeptide is "specific" for a first target or antigen compared to a second target or antigen, it is directed against said first target or antigen but not against said second target or antigen (as defined herein).

[0059] The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, saturation binding assays and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, as well as different variations thereof known in the art, as well as other techniques mentioned herein. A preferred approach that can be used to assess affinity is the two-step ELISA (enzyme-linked immunosorbent assay) procedure of Friguet et al. 1985 (J. Immunol. Methods 77: 305-19). This method establishes a liquid-phase binding equilibrium measurement and avoids possible artifacts associated with the adsorption of a single molecule to a support, such as plastic. As will be apparent to those skilled in the art, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining dissociation constants will be apparent to those skilled in the art and include, for example, the techniques described on pages 53-56 of WO 08 / 020079.

[0060] Finally, it should be noted that in many situations, an experienced scientist may find it convenient to determine the binding affinity for a reference molecule. For example, to assess the binding strength between molecules A and B, one may use a reference molecule C known to bind to B and suitably labeled with a fluorophore or chromophore or other chemical moiety, such as biotin for ELISA or FACS (fluorescence-activated cell sorting) or other formats (fluorophore for fluorescence detection, chromophore for light absorption detection, biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is maintained at a fixed concentration, and the concentration of A is varied for a particular concentration or amount of B. This results in an IC, corresponding to the concentration of A at which the signal measured for C in the absence of A is halved. 50 The value of K of the reference molecule is obtained. D K Dref , and the total concentration of the reference molecule, c ref If is known, the apparent K of the interaction AB D is obtained from the following equation: D =IC 50 / (1+c ref / K Dref ).c ref < <K Dref If K D ≒IC 50 Note that IC 50If the measurement of is performed in a consistent manner for the binders being compared (e.g., c ref (e.g., immobilizing a molecule), differences in the strength or stability of molecular interactions are measured as IC 50 This measure is referred to throughout this text as K D or apparent K D is judged to be equivalent to .

[0061] Half maximal inhibitory concentration (IC 50 ) is also a measure of a compound's effectiveness in inhibiting a biological or biochemical function, e.g., a pharmacological effect. This quantitative measure indicates the amount of a polypeptide or ISVD (e.g., nanobody) required to inhibit a certain biological process (or component of a process, i.e., enzyme, cell, cell receptor, chemotaxis, anaplasia, metastasis, invasiveness, etc.) by half. In other words, it is the half-maximal (50%) inhibitory concentration (IC) of a substance (50% IC, or IC 50 ) IC 50 The K value can be calculated for a given antagonist, such as a polypeptide or ISVD (e.g., nanobody) of the invention, by determining the concentration required to inhibit half of the maximal biological response of the agonist. D can be determined by generating a dose-response curve and examining the effect of different concentrations of an antagonist, such as a polypeptide or ISVD of the invention (e.g., a nanobody), in reversing agonist activity.

[0062] Median effective concentration (EC 50 The term EC2 (Eq. 1) refers to the concentration of a compound that elicits a response halfway between baseline and maximum after a specified exposure time. In this context, it is used as a measure of the potency of a polypeptide, ISVD (e.g., nanobody). EC2 of a graded dose-response curve 50 represents the concentration of compound at which 50% of the maximal effect is observed. Concentrations are preferably expressed in molar units. In biological systems, small changes in ligand concentration typically result in rapid changes in response that follow a sigmoidal function. The inflection point at which the increase in response with increasing ligand concentration begins to slow is known as the EC 50 This can be determined mathematically by deriving a fitted line. Relying on a graph for estimation is most often convenient. EC 50 is provided in the Examples section, experiments are D It is designed to reflect as accurately as possible the 50 The value is K D The term "average K D " is the average K obtained from at least one, but preferably more than one, e.g., at least two experiments. D Pertaining to values. The term "average" refers to the mathematical term "mean" (the sum of the data divided by the number of items of data).

[0063] This also gives the IC, a measure of the compound's inhibition (50% inhibition). 50 For competitive binding assays and functional antagonist assays, the IC 50 is the most common summary measure for dose-response curves. For agonist / stimulator assays, the most common summary measure is EC 50 is. The inhibition constant (Ki) is a measure of how potent an inhibitor is; it is the concentration required to produce half-maximal inhibition. The IC varies depending on the experimental conditions. 50 Unlike , K is an absolute value and is often called the drug's inhibition constant. The inhibition constant K can be calculated using the Cheng-Prusoff equation:

number

[0064] As used herein, the term "efficacy" of a polypeptide and / or ISVD of the present invention is a function of the amount of the polypeptide and / or ISVD of the present invention required to produce a particular effect. This refers to the ability of the polypeptide and / or ISVD of the present invention to regulate and / or partially or completely inhibit the activity of ADAMTS5. More specifically, it may refer to the ability of the polypeptide and / or ISVD to reduce or completely inhibit ADAMTS5 activity as defined herein. Thus, it may refer to the ability of the polypeptide and / or ISVD to inhibit ADAMTS5 activity, such as enzyme activity, e.g., proteolysis, for example, protease activity and / or endopeptidase activity, and the binding of substrates, including but not limited to aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, preferably the cleavage of aggrecan. The polypeptide and / or ISVD preferably antagonizes the aggrecanase activity of ADAMTS5. Potency can be measured by any suitable assay known in the art or described herein. As used herein, "aggrecanase activity" is defined as the proteolytic cleavage of aggrecan.

[0065] The "efficacy" of a polypeptide of the present invention is measured at the maximal strength of the effect itself at saturating polypeptide concentrations. Efficacy refers to the maximum response achievable from a polypeptide of the present invention. It refers to the ability of a polypeptide to produce a desired (therapeutic) effect. In one aspect, the invention relates to a polypeptide as described herein, wherein the polypeptide binds to ADAMTS5, e.g., as determined by Gyrolab or KinExA, a K D As 1E -07 M~1E -13 Between M, for example, 1E -08 M~1E -12 Between M, preferably at most 1E -07 M, preferably 1E -08 M or 1E -09 Less than M or 1E -10 Less than M, for example, 5E-11 M, 4E -11 M, 3E -11 M, 2E -11 M, 1.7E -11 M, 1E -11 M, etc., or 5E -12 M, 4E -12 M, 3E -12 M, 1E -12 M, etc., to combine

[0066] In one aspect, the invention relates to a polypeptide as described herein, wherein the polypeptide is capable of binding to EC 50 As 1E -07 M~1E -12 Between M, for example, 1E -08 M~1E -11 Regulating ADAMTS5 among M . In one aspect, the invention relates to a polypeptide as described herein, wherein the polypeptide has an off-rate of 5E, as determined, for example, by SPR. -04 s -1 Less than, for example, 1E -04 s -1 or 5E -05 s -1 Less than or even 1E -05 s -1 It binds to ADAMTS5 at less than 100 kJ / kg. In one aspect, the invention relates to a polypeptide as described herein, wherein the polypeptide exhibits an IC 50 As 1E -07 M~1E -12 Between M, for example, 1E -08 M~1E -11 Inhibiting ADAMTS5 activity among M .

[0067] In one aspect, the invention relates to a polypeptide as described herein, the polypeptide comprising: 50 as a maximum of 1E -07 M, preferably 1E -08 M, 5E -09 M, or 4E -9M, 3E -9 M, 2E -9 M, e.g. 1E -9 M and other substances inhibit the enzymatic activity of ADAMTS5. An amino acid sequence, such as an ISVD or polypeptide, is said to be "cross-reactive" with two different antigens or antigenic determinants (e.g., ADAMTS5 from different species of mammals, such as human ADAMTS5, bovine ADAMTS5, rat ADAMTS5, guinea pig ADAMTS5, mouse ADAMTS5, or cynomolgus monkey ADAMTS5) if it is specific (as defined herein) for those different antigens or antigenic determinants. It will be understood that an ISVD or polypeptide may have binding affinities for two different antigens that differ by 2, 5, 10, 50, 100-fold or more, yet be considered cross-reactive if it is specific (as defined herein) for those different antigens or antigenic determinants.

[0068] ADAMTS5 is also known as ADAMTS11, ADMP-2, or aggrecanase 2. Relevant structural information for ADAMTS5 can be found, for example, in the UniProt accession numbers as shown in Table 1 below (see Table B). Table 1 [Table 1]

[0069] "Human ADAMTS5" refers to ADAMTS5 comprising the amino acid sequence of SEQ ID NO: 149. In one aspect, a polypeptide of the invention specifically binds to ADAMTS5 from Human sapiens, Mus musculus, Cavia Porcellus, Bos taurus, Macaca mulatta and / or Rattus norvegicus, preferably human ADAMTS5, preferably SEQ ID NO: 149. The terms "(cross)block," "(cross)blocked," "(cross)blocking," "competitive binding," "(cross)compete," "(cross)competing," and "(cross)competition" are used interchangeably herein to refer to the ability of an immunoglobulin, antibody, ISVD, polypeptide, or other binding agent to interfere with the binding of another immunoglobulin, antibody, ISVD, polypeptide, or binding agent to a given target. The extent to which an immunoglobulin, antibody, ISVD, polypeptide, or other binding agent can interfere with the binding of another to a target, and therefore can be said to cross-block in accordance with the present invention, can be determined using competitive binding assays common in the art, such as by screening purified ISVDs against ISVDs displayed on phage in a competitive ELISA. Particularly suitable quantitative cross-blocking assays include ELISAs.

[0070] Other methods for determining whether an immunoglobulin, antibody, ISVD, polypeptide, or other binding agent directed against a target (cross-)blocking can be (cross-)blocking, competitively binding, or (cross-)competing as defined herein can be evaluated by, for example, an SPR-based "sandwich assay," as described in the Examples section. Other suitable methods are described, for example, in Xiao-Chi Jia et al. (Journal of Immunological Methods 288: 91-98, 2004), Miller et al. (Journal of Immunological Methods 365: 118-125, 2011). Thus, the present invention relates to a polypeptide described herein, e.g., represented by SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18 (see Table A-1), wherein the polypeptide competes with a cross-blocking polypeptide, e.g., as determined by competitive ELISA.

[0071] The present invention relates to a method for determining a competitor, such as a polypeptide, that competes with the polypeptide described herein, wherein the polypeptide described herein is represented by any of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18, wherein the polypeptide described herein competes with or cross-blocks a competitor, such as a polypeptide, for binding to ADAMTS5, for example, human ADAMTS5 (SEQ ID NO: 149), wherein the binding of the competitor to ADAMTS5 in the presence of the polypeptide of the present invention is reduced by at least 5%, for example, 10%, 20%, 30%, 40%, 50% or more, for example, 80%, 90% or 100%, compared to the binding of the competitor to ADAMTS5 in the absence of the polypeptide of the present invention (i.e., is substantially undetectable in a particular assay).Competition and cross-blocking can be determined by any means known in the art, such as competitive ELISA or FACS assay. In one aspect, the present invention relates to a polypeptide of the present invention, wherein said polypeptide cross-blocks the binding to ADAMTS5 of at least one polypeptide set forth in SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18 and / or its binding to ADAMTS5 is cross-blocked by at least one polypeptide set forth in SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18.

[0072] The present invention also relates to competitors that compete with the polypeptides described herein, such as SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18, wherein the competitor competes or cross-blocks the polypeptides described herein for binding to ADAMTS5, and wherein binding of a polypeptide of the present invention to ADAMTS5 in the presence of the competitor is reduced by at least 5%, for example 10%, 20%, 30%, 40%, 50% or more, for example 80% or even more, for example at least 90% or 100% (i.e., substantially undetectable in a particular assay) compared to binding by a polypeptide of the present invention to ADAMTS5 in the absence of the competitor. In one aspect, the present invention relates to a polypeptide that cross-blocks binding to ADAMTS5 by a polypeptide of the invention, such as one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18, and / or whose binding to ADAMTS5 is cross-blocked by a polypeptide of the invention, such as at least one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18, wherein preferably the polypeptide comprises at least one VH, VL, dAb, or ISVD that specifically binds to ADAMTS5, and wherein binding to ADAMTS5 modulates the activity of ADAMTS5.

[0073] "ADAMTS5 activity" and "activity of ADAMTS5" (these terms are used interchangeably herein) include, but are not limited to, enzymatic activity such as proteolysis, e.g., protease activity (also called proteinase or peptidase activity), and endopeptidase activity, as well as activities via exosites, e.g., substrate recognition and / or binding, e.g., via the disintegrin-like domain, central thrombospondin type I-like (TS) repeats, cysteine-rich domain, spacer region, and / or additional TS motifs. ADAMTS5 activity includes binding and / or proteolysis of substrates such as hyaluronan-binding chondroitin sulfate proteoglycans (CSPGs) extracellular proteins, e.g., aggrecan, versican, brevican, neurocan, decorin, and biglycan. As used herein, proteolysis is the breakdown of proteins into smaller polypeptides or amino acids by hydrolysis of the peptide bonds that link the amino acids together in the polypeptide chain.

[0074] In the context of the present invention, "modulating" or "modulating" generally refers to a change in the activity of ADAMTS5, as measured using a suitable in vitro, cellular, or in vivo assay (such as those mentioned herein). In particular, "modulating" or "modulating" can mean reducing or inhibiting the activity of ADAMTS5, or increasing the activity, as measured using a suitable in vitro, cellular, or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, for example at least 10% or at least 25%, for example at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the activity of ADAMTS5 in the same assay under the same conditions but in the absence of the ISVD or polypeptide of the present invention. Thus, the present invention relates to a polypeptide as described herein, wherein said polypeptide modulates the activity of ADAMTS5, preferably inhibits the activity of ADAMTS5. Thus, the present invention relates to polypeptides as described herein, wherein the polypeptides inhibit the protease activity of ADAMTS5, e.g., inhibit the proteolysis of substrates such as aggrecan, versican, brevican, neurocan, decorin, and / or biglycan.

[0075] Thus, the present invention relates to a polypeptide as described herein, wherein the polypeptide blocks the binding of ADAMTS5 to a substrate, such as aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, wherein the substrate is preferably aggrecan. In one aspect, the present invention relates to a polypeptide described herein, wherein the polypeptide blocks the binding of ADAMTS5 to aggrecan by at least 20%, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, e.g., as determined by ELISA. In one aspect, the present invention relates to a polypeptide as described herein, wherein the polypeptide antagonizes or inhibits the activity of ADAMTS5, for example (i) protease activity, preferably cleavage of aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, etc., preferably cleavage of aggrecan; preferably antagonizes or inhibits the aggrecanase activity of ADAMTS5; or (ii) antagonizes or inhibits binding of substrates to ADAMTS5, for example, binding to exosites of ADAMTS5, such as the disintegrin-like domain, central thrombospondin type I-like (TS) repeats, cysteine-rich domain, spacer region or additional TS motifs.

[0076] Accordingly, the present invention relates to a polypeptide as described herein, which inhibits the protease activity of ADAMTS5, preferably by at least 5%, such as 10%, 20%, 30%, 40%, 50% or more, such as at least 60%, 70%, 80%, 90%, 95% or more, as measured, for example, by an enzyme inhibition assay or any suitable method known in the art, such as those described in the Examples section. Although ADAMs, ADAMTSs, and MMPs share aggrecan-binding sites that are highly similar in both sequence and overall shape (e.g., the catalytic domains of ADAMTS4 and ADAMTS5 share a high degree of sequence similarity), we were able to identify target-specific ISVDs, as demonstrated in the Examples section. Target specificity also avoids or at least limits the musculoskeletal syndrome, a side effect caused by broad-spectrum inhibitors.

[0077] In one aspect, the invention relates to ADAMTS5-binding agents, such as ISVDs and polypeptides of the invention, wherein the ADAMTS5-binding agents do not bind to ADAMTS4, ADAMTS1, ADAMTS15, MMP1, and / or MMP14 (membrane form). Preferably, the invention relates to polypeptides as defined herein, wherein the ISVD that binds to ADAMTS5 does not bind to ADAMTS4, MMP1, or MMP14. Unless otherwise specified, the terms "immunoglobulin" and "immunoglobulin sequence", whether used herein to refer to a heavy chain antibody or a traditional four-chain antibody, include a full-size antibody, both its individual chains, and all parts, domains or fragments thereof (e.g., antigen-binding domains or fragments, respectively, e.g., V HH Domain or V H / V L It is used as a general term to include all domains (including but not limited to).

[0078] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that has the ability to retain its tertiary structure independently of the rest of the protein. Domains are generally responsible for distinct functional properties of the protein and can often be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or domain. As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (such as a chain of a conventional four-chain antibody or a heavy-chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by retaining the immunoglobulin fold characteristic of antibody molecules, which consists of a two-layer sandwich of approximately seven antiparallel β-strands arranged in two β-sheets, optionally stabilized by conserved disulfide bonds.

[0079] The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain essentially consisting of four "framework regions," which are referred to in the art and herein as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively; the framework regions are interrupted by three "complementarity-determining regions" or "CDRs," which are referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be depicted as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain that carries the antigen-binding site and thereby confers specificity for the antibody to the antigen.

[0080] The term "immunoglobulin single variable domain" (abbreviated herein as "ISVD" or "ISV") is used interchangeably with "single variable domain" and defines a molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain. Immunoglobulin single variable domains are thus set apart from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. H and V L Both complementarity-determining regions (CDRs) of the nucleotides contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0081] In view of the above definitions, the antigen-binding domain of a traditional four-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or an Fab fragment, an F(ab')2 fragment, an Fv fragment such as a disulfide-linked Fv or scFv fragment, or a diabody derived from such a traditional four-chain antibody (all known in the art) is not typically considered an immunoglobulin single variable domain, because in these cases, binding to each epitope of an antigen is typically not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains, such as a light and heavy chain variable domain, i.e., the V of immunoglobulin domains which cooperatively bind to the respective epitopes of the antigen. H -V L Because it arises from pairs. In contrast, ISVDs can specifically bind to epitopes of antigens without pairing with additional immunoglobulin variable domains. HH , V H or V L The antigen-binding site of an ISVD is therefore formed by three or fewer CDRs.

[0082] Thus, a single variable domain may be composed of a light chain variable domain sequence (e.g., V L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H Array or V HH sequence) or a suitable fragment thereof, so long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit; which consists essentially of a single variable domain and does not require the single antigen-binding domain to interact with another variable domain to form a functional antigen-binding unit). In one embodiment of the invention, the ISVD is a heavy chain variable domain sequence (e.g., V H sequence); more specifically, the ISVD can be a heavy chain variable domain sequence derived from a conventional four-chain antibody, or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0083] For example, the ISVD may be a (single) domain antibody (or amino acids suitable for use as a (single) domain antibody), a "dAb" or dAb (or amino acids suitable for use as a dAb), or a nanobody (as defined herein, including but not limited to VHHs); other single variable domains, or any suitable fragment thereof. In particular, the ISVD may be a Nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx NV.] A general description of nanobodies is provided below in the detailed description and in the prior art cited herein, e.g., WO 08 / 020079 (page 16).

[0084] VHH, V H "VHH" also known as H domain, VHH antibody fragment, and VHH antibody HHThe term "V domain" was originally described as the antigen-binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e., an "antibody lacking light chains"; Hamers-Casterman et al. 1993 Nature 363: 446-448). HH The "V domains" were chosen because they are similar to the heavy chain variable domains (referred to herein as "V" in the present specification) present in conventional four-chain antibodies. H the light chain variable domain (referred to herein as "VH domain") present in conventional four-chain antibodies, LThis is to distinguish them from VL domains (also called "VL domains" or "VL domains"). For further details on VHHs and nanobodies, reference is made to the review by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001) and to the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 from Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 from Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 from Algonomics NV and Ablynx NV; WO 03 / 050531 from Algonomics NV and Ablynx NV; WO 01 / 90190 from the National Research Council of Canada; WO 03 / 025020 (= EP 1433793) from the Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, and further published patent applications by Ablynx NV. See also further prior art cited in these applications, in particular the list of references on pages 41-43 of International Application WO 06 / 040153; that list and references are incorporated herein by reference. As described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) are particularly characterized by the presence of one or more "hallmark residues" in one or more framework sequences.Further description of Nanobodies, including humanization and / or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions", multivalent constructs (including non-limiting examples of linker sequences), and different modifications to increase the half-life of Nanobodies and their preparation can be found, for example, in WO 08 / 101985 and WO 08 / 142164. For a further general description of Nanobodies, reference is made to the prior art cited herein, for example, to the description in WO 08 / 020079 (page 16).

[0085] In particular, framework sequences present in the ADAMTS5-binding agents of the invention, such as the ISVDs and / or polypeptides of the invention, may comprise one or more hallmark residues (e.g., as described in WO 08 / 020079 (Tables A-3 to A-8)), such that the ADAMTS5-binding agents of the invention are nanobodies. Some preferred, but non-limiting, examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein (see, e.g., Table A-2). Generally, nanobodies (especially V HH Humanized nanobodies (sequences and partially humanized nanobodies) can be particularly characterized by the presence of one or more "hallmark residues" in one or more framework sequences (e.g., as further described in WO 08 / 020079, page 61, line 24 to page 98, line 3).

[0086] More particularly, the present invention provides ADAMTS5-binding agents comprising at least one immunoglobulin single variable domain, the amino acid sequence of which has the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and the immunoglobulin single variable domain is: i) have at least 80%, more preferably 90%, even more preferably 95% amino acid identity with at least one of the amino acid sequences of SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18 (see Table A-1), where for purposes of determining the degree of amino acid identity, the amino acid residues forming the CDR sequences are disregarded. Reference is also made in this respect to Table A-2, which lists the framework 1 sequences (SEQ ID NOs: 72 to 84), framework 2 sequences (SEQ ID NOs: 85 to 94), framework 3 sequences (SEQ ID NOs: 95 to 113) of the immunoglobulin single variable domains of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18; or ii) a combination of framework sequences shown in Table A-2; and here: iii) Preferably, one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from hallmark residues, e.g., as set out in Tables A-3 to A-8 of WO 08 / 020079.

[0087] The ADAMTS5-binding agents of the present invention, such as the ISVDs and / or polypeptides of the present invention, may also include specific mutations / amino acid residues described in the following co-pending U.S. provisional applications, all entitled "Improved Immunoglobulin Variable Domains": US 61 / 994552, filed May 16, 2014; US 61 / 014,015, filed June 18, 2014; US 62 / 040,167, filed August 21, 2014; and US 62 / 047,560, filed September 8, 2014 (all assigned to Ablynx NV). In particular, the ADAMTS5 binding agents of the present invention, such as the ISVDs and / or polypeptides of the present invention, may suitably contain: (i) K or Q at position 112; or (ii) K or Q at position 110 in combination with V at position 11; or (iii) T at position 89; or (iv) L at position 89 and K or Q at position 110; or (v) V at position 11 and L at position 89; or an appropriate combination of (i) to (v).

[0088] As also described in the co-pending U.S. provisional application, when the ADAMTS5-binding agent of the present invention, such as the ISVD and / or polypeptide of the present invention, comprises a mutation according to one of (i) to (v) above (or a suitable combination thereof): the amino acid residue at position 11 is preferably selected from L, V, or K (and V is most preferred); and / or the amino acid residue at position 14 is suitably selected from A or P; and / or the amino acid residue at position 41 is suitably selected from A or P; and / or the amino acid residue at position 89 is preferably selected from T, V or L; and / or the amino acid residue at position 108 is suitably selected from Q or L; and / or the amino acid residue at position 110 is suitably selected from T, K or Q; and / or The amino acid residue at position 112 is preferably selected from S, K or Q, as appropriate.

[0089] As described in the co-pending US provisional application, the mutations are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to the immunoglobulins and compounds of the invention. To this end, the ADAMTS5-binding agents of the invention, such as the ISVDs and / or polypeptides of the invention, may also (optionally in combination with the mutations) comprise a C-terminal extension (X)n, where n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I). See, e.g., US provisional application and WO 12 / 175741. In particular, the ADAMTS5 binding agents of the present invention, such as the ISVDs and / or polypeptides of the present invention, may include such C-terminal extensions when they form the C-terminus of proteins, polypeptides, or other compounds or constructs comprising them (see, e.g., the aforementioned U.S. provisional applications and WO 12 / 175741).

[0090] The ADAMTS5-binding agents of the invention can be immunoglobulins, e.g., immunoglobulin single variable domains, derived in any suitable manner from any suitable source, e.g., naturally occurring V HH sequences (i.e., derived from an appropriate species of Camelidae) or synthetic or semi-synthetic amino acid sequences, including but not limited to "humanized" (as defined herein) Nanobodies or VHH sequences, "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or natural immunoglobulin sequences), CDR grafting, veneering, combining fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art; or any suitable combination of the foregoing, as further described herein. Also, the immunoglobulin may be a VHH sequence. HHIf the immunoglobulin comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), the immunoglobulin may be suitably humanized to provide one or more further (partially or fully) humanized immunoglobulins of the invention, as further described herein. Similarly, if the immunoglobulin comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), the immunoglobulin may be optionally further suitably humanized to provide one or more further (partially or fully) humanized immunoglobulins of the invention, again as described herein.

[0091] "Domain antibodies", also known as "Dabs", "Domain Antibodies" and "dAbs" ("Domain Antibodies" and "dAbs" are trademarks of the GlaxoSmithKline group of companies), are described, for example, in EP 0368684, Ward et al. (Nature 341: 544-546, 1989), Holt et al. (Tends in Biotechnology 21: 484-490, 2003) and WO 03 / 002609 and other published patent applications, such as WO 04 / 068820, WO 06 / 030220, WO 06 / 003388; and other published patent applications of Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammals, in particular human four-chain antibodies. Binding an epitope as a single antigen-binding domain, i.e., without pairing with a VL or VH domain, respectively, requires specific selection of such antigen-binding properties, for example, by using a library of human single VH or VL domain sequences. Domain antibodies, like VHHs, have a molecular weight of about 13 to about 16 kDa, and if derived from entirely human sequences, do not require humanization, for example, for therapeutic use in humans.

[0092] It should also be noted that single variable domains may be derived from certain sharks (e.g., so-called "IgNAR domains", see e.g., WO 05 / 18629), although these are not of mammalian origin and therefore less preferred in the context of the present invention. The present invention particularly relates to an ISVD, wherein said ISVD is selected from the group consisting of a VHH, a humanized VHH and a camelized VH. The numbering of amino acid residues in the VHH domain is based on the V numbering scheme according to Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). H The domains are numbered in accordance with a common numbering scheme similar to that applied to camelid VHH domains, as shown, for example, in Figure 2 of Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999); all of which are known in the art. H Alternative methods for numbering the amino acid residues of a domain can be applied to VHH domains in a similar manner and are known in the art, however in this specification, claims and figures, unless otherwise stated, the Kabat numbering system applied to VHH domains as described above will be followed.

[0093] V H It should be noted that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This generally means that the Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH and VHH domains will usually be in the range of 110-120, often 112-115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0094] With regard to CDRs, as is well known in the art, there are several rules for defining and describing the CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of the structural loop regions). See, for example, the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, CDRs are most preferably defined based on the Abm definition (which is based on Oxford Molecular's AbM antibody modeling software), which is considered to be the best compromise between the Kabat and Chothia definitions (see: http: / / www.bioinf.org.uk / abs / ). As used herein, FR1 comprises amino acid residues at positions 1-25, CDR1 comprises amino acid residues at positions 26-35, FR2 comprises amino acid residues at positions 36-49, CDR2 comprises amino acid residues at positions 50-58, FR3 comprises amino acid residues at positions 59-94, CDR3 comprises amino acid residues at positions 95-102, and FR4 comprises amino acid residues at positions 103-113. In the sense of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from a non-human source, preferably a camelid, preferably a camelid heavy chain antibody, which may be humanized as described herein. Furthermore, the term includes polypeptides derived from a non-camelid source, for example a mouse or a human, which are "camelized" as described herein.

[0095] Thus, ISVDs, such as domain antibodies and nanobodies (comprising VHH domains), may be subject to humanization. In particular, a humanized ISVD, such as a nanobody (comprising a VHH domain), may be an ISVD as generally defined herein, but in which at least one amino acid residue (particularly at at least one of the framework residues) is present which is and / or corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions include those that are not part of naturally occurring VHH domains.HH The sequences of the framework regions of the sequences were compared with one or more closely related human V H The V sequence can then be compared to the corresponding framework sequence of the V sequence, and one or more potentially useful humanizing substitutions (or combinations thereof) thus determined can then be incorporated into the V sequence. HH sequence (by any manner known per se, as further explained herein), resulting in a humanized V HH The sequences can be tested for affinity to the target, stability, ease and level of expression, and / or other desired properties. In this way, with a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by those skilled in the art based on the disclosure herein. Also based on the above, (the framework regions of) ISVDs such as nanobodies (including VHH domains) can be partially or fully humanized.

[0096] Another particularly preferred class of ISVDs of the present invention are naturally occurring V H The ISVDs include those having amino acid sequences that correspond to the amino acid sequences of the V domains, but that are "camelized," i.e., the naturally occurring V domains from conventional four-chain antibodies. H One or more amino acid residues in the amino acid sequence of the domain are HH By substituting one or more amino acid residues at corresponding positions in the V domain. This can be done in a manner known per se and will be clear to the skilled artisan, for example, based on the description herein. Such "camelizing" substitutions are preferably made at positions corresponding to the V domain, as defined herein. H -V L The amino acid residues are inserted at amino acid positions that form and / or are present in the interface and / or at the so-called camelid hallmark residues (see also, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the V domains used as starting material or starting points for generating or designing camelized immunoglobulin single variable domains are HThe sequence is preferably a V from a mammal H sequence, more preferably V H Human V3 sequences H However, such camelized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se and therefore have the same or similar structure as the naturally occurring V H It should be noted that the present invention is not strictly limited to polypeptides obtained using a polypeptide containing the domain as a starting material, see Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13: 475-479, 1995; Prot. Eng. 9: 531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999).

[0097] For example, as further described herein, both "humanization" and "camelization" can be performed as follows: HH Domain or V H providing a nucleotide sequence encoding each of the domains, and then altering one or more codons in said nucleotide sequence in a manner known per se, so that the new nucleotide sequence encodes a "humanized" or "camelized" ISVD of the invention, respectively. This nucleic acid can then be expressed in a manner known per se to provide the desired ISVD of the invention. Alternatively, naturally occurring V HH Domain or V H Based on the amino acid sequences of the respective domains, the amino acid sequences of the desired humanized or camelized ISVDs of the present invention can be designed and synthesized de novo using known peptide synthesis techniques. HH Domain or V HBased on the amino acid or nucleotide sequence of the domain, respectively, a nucleotide sequence encoding the desired humanized or camelized ISVD of the invention, respectively, can then be designed and synthesized de novo using techniques for nucleic acid synthesis known per se, and the nucleic acid thus obtained can then be expressed in a manner known per se to provide the desired ISVD of the invention.

[0098] ISVDs such as domain antibodies and nanobodies (including VHH domains and humanized VHH domains) can also be affinity matured by introducing one or more changes in the amino acid sequence of one or more CDRs, which result in improved affinity of the resulting ISVD for its respective antigen compared to the respective parent molecules. Affinity-matured ISVD molecules of the present invention can be prepared by methods known in the art, such as those described in Marks et al. (Biotechnology 10:779-783, 1992), Barbas, et al. (Proc. Nat. Acad. Sci. USA 91:3809-3813, 1994), Shier et al. (Gene 169:147-155, 1995), Yelton et al. (Immunol. 155:1994-2004, 1995), Jackson et al. (J. Immunol. 154:3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226:889-896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996).

[0099] V H , V L , V HHThe process of designing / selecting and / or preparing a polypeptide starting from an ISVD, such as a domain antibody or nanobody, is also referred to herein as "formatting" said ISVD; an ISVD that makes part of a polypeptide is said to be a "formatted" or "formatted" polypeptide. Examples of how an ISVD can be formatted and examples of such formats will be apparent to the skilled person based on the disclosure herein; such formatted immunoglobulin single variable domains form a further aspect of the present invention. Preferred CDRs are shown in Table A-2.

[0100] In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is SEQ ID NO: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; and an amino acid sequence having 1, 2 or 3 amino acid differences from SEQ ID NOs: 21, 35, 20, 22, 25, 33, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; (ii) CDR2 is SEQ ID NO: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49, and 52; and an amino acid sequence having one, two or three amino acid differences from SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and (iii) CDR3 is SEQ ID NO: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67, and 70; and and amino acid sequences having 1, 2, 3 or 4 amino acid differences from SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70.

[0101] In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is (a) SEQ ID NO: 22; and (b) an amino acid sequence having 1, 2, 3, 4, 5, or 6 amino acid differences from SEQ ID NO: 22; wherein - in position 2 S is changed to R; - in position 3 A is changed to T; - in position 4 V is changed to F; - in position 6 V is changed to S; - in position 7 N is changed to Y; and / or - in position 10 A is changed to G; (ii) CDR2 is SEQ ID NO: 36; and (iii) CDR3 is SEQ ID NO: 54.

[0102] In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is SEQ ID NO: 33; (ii) CDR2 is (c) SEQ ID NO: 50; and (d) an amino acid sequence having 1, 2, or 3 amino acid differences from SEQ ID NO: 50; wherein: - in position 8 M is changed to I; - at position 9 P is changed to T; and / or - in position 10 Y is changed to F; and (iii) CDR3 is (e) SEQ ID NO: 68; and (f) an amino acid sequence having one or two amino acid differences from SEQ ID NO: 68: wherein - in position 5 F is changed to L; and / or - In position 11, D is changed to E.

[0103] In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: (i) CDR1 is SEQ ID NO: 28; (ii) CDR2 is (c) SEQ ID NO: 44; and (d) an amino acid sequence that differs by 1, 2, or 3 amino acids from SEQ ID NO: 44; wherein - in position 3 S is changed to T; - in position 4 R is changed to W; - at position 8 T is changed to I; and / or - in position 9 T is changed to L; (iii) CDR3 is (e) SEQ ID NO: 62; and (f) an amino acid sequence having one or two amino acid differences from SEQ ID NO: 62; wherein - at position 1 G is changed to S; and / or - In position 14, D is changed to E.

[0104] In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 is selected from the group consisting of SEQ ID NOs: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; - CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and - CDR3 is selected from the group consisting of SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70.

[0105] In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein said ISVD is selected from the group of ISVDs, wherein: CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55; CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 118; CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 71; CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54; CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54; CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69; CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62; CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59; CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 119, and CDR3 is SEQ ID NO: 60; CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; CDR1 is SEQ ID NO:29, CDR2 is SEQ ID NO:46, and CDR3 is SEQ ID NO:64; CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66; CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; and CDR1 is SEQ ID NO:34, CDR2 is SEQ ID NO:52, and CDR3 is SEQ ID NO:70.

[0106] In certain preferred embodiments, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is or comprises SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55. In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein said ISVD is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18.

[0107] Without limitation, it will be understood that the immunoglobulin single variable domains of the invention can be used as "building blocks" for the preparation of polypeptides, which can optionally include one or more further immunoglobulin single variable domains that can function as building blocks (i.e., against the same or another epitope on ADAMTS5 and / or against one or more other antigens, proteins, or targets other than ADAMTS5). The polypeptides of the present invention (also referred to herein as "nanobody constructs") comprise at least one ISVD that binds to ADAMTS, preferably ADAMTS5, for example, two ISVDs that bind to ADAMTS5, and preferably an ISVD that binds to albumin. In the polypeptides of the present invention, the ISVDs may be directly linked or linked via a linker. Even more preferably, the polypeptides of the present invention comprise a C-terminal extension. As detailed herein, the C-terminal extension essentially prevents / eliminates binding of pre-existing antibodies / factors in most samples from human subjects / patients. The C-terminal extension is present C-terminal to the last amino acid residue (usually a serine residue) of the last (most C-terminal) ISVD.

[0108] As further detailed below, ISVD HH , V H or V L The ISVD may be derived from a V domain, but the ISVD may be derived from a V domain in the polypeptides of the present invention. H and V L The domains are selected so that they do not form complementary pairs. HH , and humanized V HH are unusual in that they are derived from natural camelid antibodies that lack light chains; in fact, these domains associate with camelid light chains to form complementary V HH and V L Thus, the polypeptides of the present invention do not include a complementary ISVD and / or do not include, for example, a complementary V H / V L They do not form complementary ISVD pairs such as pairs. Generally, polypeptides or constructs comprising or consisting essentially of a single building block, a single ISVD, or a single Nanobody are referred to herein as "monovalent" polypeptides and "monovalent constructs," respectively. Polypeptides or constructs comprising two or more building blocks (e.g., ISVDs) are also referred to herein as "multivalent" polypeptides or constructs, and the building blocks / ISVDs present in such polypeptides or constructs are referred to herein as being in a "multivalent format." For example, a "bivalent" polypeptide may comprise two ISVDs optionally linked via a linker sequence, whereas a "trivalent" polypeptide may comprise three ISVDs optionally linked via two linker sequences; whereas a "tetravalent" polypeptide may comprise four ISVDs optionally linked via three linker sequences, etc.

[0109] In a multivalent polypeptide, the two or more ISVDs may be the same or different and may be directed against the same antigen or antigenic determinant (e.g., against the same portion of the epitope or different portions of the epitope), or alternatively, may be directed against different antigens or antigenic determinants, or any suitable combination thereof. Polypeptides and constructs comprising at least two building blocks (e.g., ISVDs), where at least one building block is directed against a first antigen (i.e., ADAMTS5) and at least one building block is directed against a second antigen (i.e., different from ADAMTS5), are also referred to as "multispecific" polypeptides and constructs, and the building blocks (e.g., ISVDs) present in such polypeptides and constructs are also referred to herein as "multispecific formats." Thus, for example, a "bispecific" polypeptide of the present invention is a polypeptide comprising at least one ISVD directed against a first antigen (i.e., ADAMTS5) and at least one ISVD directed against a second antigen (i.e., different from ADAMTS5), whereas a "trispecific" polypeptide of the present invention is a polypeptide comprising at least one ISVD directed against a first antigen (i.e., ADAMTS5), at least one further ISVD directed against a second antigen (i.e., different from ADAMTS5), and at least one further ISVD directed against at least a third antigen (i.e., different from both ADAMTS5 and the second antigen), etc.

[0110] In one aspect, the present invention relates to a polypeptide comprising at least two ISVDs, wherein at least one ISVD specifically binds to ADAMTS, preferably ADAMTS5, and more preferably is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18. In one aspect, the present invention relates to a polypeptide comprising at least two ISVDs, wherein said at least two ISVDs specifically bind to ADAMTS, preferably ADAMTS5, and more preferably each ISVD of said two ISVDs is independently selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18. "Multiple paratopic" polypeptides and "multiple paratopic" constructs, such as "double paratopic" polypeptides or constructs and "triple paratopic" polypeptides or constructs, comprise or consist essentially of two or more building blocks, each having a different paratope.

[0111] Thus, an ISVD of the invention that binds ADAMTS5 may be in essentially isolated form (as defined herein) or may form part of a construct or polypeptide which may comprise or essentially consist of one or more ISVDs that bind ADAMTS5, and may optionally further comprise one or more additional amino acid sequences (all optionally linked via one or more suitable linkers). The present invention relates to polypeptides or constructs which comprise or essentially consist of at least one ISVD according to the invention, such as one or more ISVDs of the invention (or suitable fragments thereof) that bind ADAMTS5. One or more ISVDs of the invention are used as building blocks in such polypeptides or constructs to provide monovalent, multivalent, or multiparatopic polypeptides or constructs of the invention, all as described herein. Thus, the invention also relates to polypeptides that are monovalent constructs, comprising or consisting essentially of a monovalent polypeptide or ISVD of the invention.

[0112] The present invention therefore also relates to polypeptides or constructs that are multivalent polypeptides or multivalent constructs, such as, for example, bivalent or trivalent polypeptides or constructs comprising or consisting essentially of two or more ISVDs of the invention (for multivalent and multispecific polypeptides comprising one or more VHH domains and their preparation see Conrath et al. (J. Biol. Chem. 276: 7346-7350, 2001), and also, for example, WO96 / 34103, WO99 / 23221 and WO 2010 / 115998).

[0113] In one aspect, in its simplest form, a multivalent polypeptide or construct of the invention is a bivalent polypeptide or construct of the invention comprising a first ISVD, such as a Nanobody directed against ADAMTS5, and an identical second ISVD, such as a Nanobody directed against ADAMTS5, wherein said first and said second ISVD, such as a Nanobody, may optionally be linked via a linker sequence (defined herein). In another form, a multivalent polypeptide or construct of the invention is a trivalent polypeptide or construct of the invention comprising a first ISVD, such as a Nanobody directed against ADAMTS5, an identical second ISVD, such as a Nanobody directed against ADAMTS5, and an identical third ISVD, such as a Nanobody, wherein said first, second and third ISVD, such as a Nanobody, may optionally be linked via one or more, in particular two, linker sequences. In one aspect, the present invention relates to a polypeptide or construct comprising or consisting essentially of at least two ISVDs according to the invention, such as two, three or four ISVDs (or suitable fragments thereof), that bind to ADAMTS5. The two or more ISVDs may optionally be linked via one or more peptide linkers.

[0114] In another aspect, a multivalent polypeptide or construct of the invention may be a bispecific polypeptide or construct of the invention comprising a first ISVD, such as a Nanobody, directed against ADAMTS5 and a second ISVD, such as a Nanobody, directed against a second antigen, such as aggrecan, where said first and second ISVDs, such as Nanobodies, may optionally be linked via a linker sequence (defined herein); on the other hand, a multivalent polypeptide or construct of the invention may also be a trispecific polypeptide or construct of the invention comprising a first ISVD, such as a Nanobody, directed against ADAMTS5, a second ISVD, such as a Nanobody, directed against a second antigen, such as aggrecan, and a third ISVD, such as a Nanobody, directed against a third antigen, where said first, second and third ISVDs, such as Nanobodies, may optionally be linked via one or more, in particular two, linker sequences. The present invention further relates to a multivalent polypeptide comprising or (essentially) consisting of at least one ISVD (or a suitable fragment thereof) that binds to ADAMTS5, preferably human ADAMTS5, and one additional ISVD, such as an ISVD that binds to aggrecan.

[0115] Particularly preferred bivalent, bispecific polypeptides or constructs according to the present invention are those shown in the Examples and Table A-1 herein (see SEQ ID NOs: 120-130 (i.e., SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 and 130), and most preferably SEQ ID NOs: 129 and 130). In a preferred aspect, the polypeptide or construct of the present invention comprises or essentially consists of at least two ISVDs, wherein the at least two ISVDs may be identical or different, but at least one ISVD is directed against ADAMTS5, and preferably the ISVD that binds to ADAMTS5 is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

[0116] The two or more ISVDs present in a multivalent polypeptide or construct of the invention may be light chain variable domain sequences (e.g., V L sequence) or heavy chain variable domain sequence (e.g., V H They may be composed of heavy chain variable domain sequences derived from conventional four-chain antibodies, or heavy chain variable domain sequences derived from heavy chain antibodies. In preferred aspects, they are domain antibodies (or amino acids suitable for use as a domain antibody), single domain antibodies (or amino acids suitable for use as a single domain antibody), or "dAbs" (or amino acids suitable for use as a dAb), Nanobodies® (V HH including, but not limited to, humanized V HH Sequence, camelization V H sequence; or V obtained by affinity maturation HH The two or more immunoglobulin single variable domains can consist of a partially or fully humanized Nanobody or a partially or fully humanized VHH.

[0117] In one aspect of the present invention, the first and second ISVDs present in a multiparatopic (preferably biparatopic or triparatopic) polypeptide or construct of the present invention do not (cross-)compete with each other for binding to ADAMTS5 and therefore belong to different families. Thus, the present invention relates to a multiparatopic (preferably biparatopic) polypeptide or construct comprising two or more ISVDs, each ISVD belonging to a different family. In one aspect, the first ISVD of this multiparatopic (preferably biparatopic) polypeptide or construct of the present invention does not cross-block the binding to ADAMTS5 of the second ISVD of this multiparatopic (preferably biparatopic) polypeptide or construct of the present invention and / or the first ISVD is not cross-blocked from binding to ADAMTS5 by the second ISVD. In another aspect, a first ISVD of a multiparatopic (preferably biparatopic) polypeptide or construct of the present invention cross-blocks the binding of a second ISVD of the multiparatopic (preferably biparatopic) polypeptide or construct of the present invention to ADAMTS5, and / or the first ISVD is cross-blocked from binding to ADAMTS5 by the second ISVD.

[0118] In a particularly preferred aspect, the polypeptide or construct of the invention comprises or consists essentially of three or more ISVDs, of which at least two ISVDs are directed against ADAMTS5. It will be understood that said at least two ISVDs directed against ADAMTS5 may be identical or different, may be directed against the same epitope or different epitopes of ADAMTS5, may belong to the same epitope bin or different epitope bins, and / or may bind to the same domain or different domains of ADAMTS5.

[0119] The relative affinity may depend on the position of the ISVDs within the polypeptide. It will be understood that the order (orientation) of the ISVDs in a polypeptide of the invention can be selected according to the needs of one skilled in the art. The order of individual ISVDs and whether the polypeptide includes a linker are matters of design choice. Some orientations, with or without a linker, may provide preferential binding properties compared to other orientations. For example, the order of the first ISVD (e.g., ISVD1) and the second ISVD (e.g., ISVD2) in a polypeptide of the invention can be (from N-terminus to C-terminus): (i) ISVD1 (e.g., Nanobody1)-[linker]-ISVD2 (e.g., Nanobody2)-[C-terminal extension]; or (ii) ISVD2 (e.g., Nanobody2)-[linker]-ISVD1 (e.g., Nanobody1)-[C-terminal extension]; (where the parts in square brackets, i.e., the linker and C-terminal extension, are optional). All orientations are encompassed by the present invention. Polypeptides containing an ISVD orientation that provides the desired binding properties can be readily identified by routine screening, e.g., as illustrated in the Examples section. The preferred order is, from N-terminus to C-terminus: ADAMTS5-bound ISVD-[linker]-albumin or aggrecan-bound ISVD-[C-terminal extension], where the portion in square brackets is optional.

[0120] In one aspect, the present invention relates to a polypeptide comprising two or more ISVDs that specifically bind to ADAMTS5, wherein: a) at least a "first" ISVD specifically binds to a first antigenic determinant, epitope, portion, domain, subunit or conformation of ADAMTS5, preferably said "first" ISVD that specifically binds to ADAMTS5 is selected from the group consisting of SEQ ID NOs: 2, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18; and b) At least the "second" ISVD specifically binds to a second antigenic determinant, epitope, part, domain, subunit or three-dimensional structure of ADAMTS5, which is different from the first antigenic determinant, epitope, part, domain, subunit or three-dimensional structure, and preferably, the "second" ISVD that specifically binds to ADAMTS5 is sequence number 116 or 19.

[0121] In a preferred aspect, the polypeptide or construct of the present invention comprises or essentially consists of at least two ISVDs that bind to ADAMTS5, wherein the at least two ISVDs can be identical or different and are independently selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18. In a further aspect, the present invention relates to a multiparatopic (preferably biparatopic) polypeptide or construct comprising two or more ISVDs directed against ADAMTS5, wherein the ISVDs bind to the same epitope as bound by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18. In a further aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide has at least 80%, 90%, 95% or 100% sequence identity to any of SEQ ID NOs: 1-19 (i.e., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19), 116-117 or 120-130 (i.e., SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130).

[0122] In one aspect, the invention relates to a polypeptide described herein selected from the group consisting of SEQ ID NO: 127 (clone 130 049-093-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb) and SEQ ID NO: 128 (clone 131 9D3-093-Alb). The art is in need of more effective treatments for disorders affecting articular cartilage, such as osteoarthritis. Most drugs have insufficient residence times, especially when administered systemically. The inventors hypothesized that the efficacy of therapeutic agents, such as the constructs, polypeptides, and ISVDs of the invention, could be significantly increased by conjugating the therapeutic agent to a moiety that extends the half-life of the drug, thereby increasing drug retention, but does not interfere with the efficacy of the therapeutic agent.

[0123] In certain aspects of the present invention, constructs or polypeptides of the present invention may have a moiety that confers increased half-life compared to the corresponding constructs or polypeptides of the present invention lacking the moiety. Some preferred, but non-limiting, examples of such constructs and polypeptides of the present invention will be apparent to those skilled in the art based on the further disclosure herein, and include, for example: an ISVD or polypeptide of the present invention that has been chemically modified to increase its half-life (e.g., by pegylation); an ADAMTS5-binding agent of the present invention, such as an ISVD and / or polypeptide of the present invention, that comprises at least one additional binding site for binding to a serum protein (such as serum albumin); or a polypeptide of the present invention comprising at least one ISVD of the present invention linked to at least one moiety (particularly at least one amino acid sequence) that extends the half-life of the amino acid sequence of the present invention.Examples of constructs of the invention, such as polypeptides of the invention comprising such half-life extending moieties or ISVDs, will become clear to the skilled person based on the further disclosure herein; examples include, but are not limited to, the following polypeptides: a polypeptide in which one or more ISVDs of the invention are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof), or to one or more binding units capable of binding to serum proteins (examples of serum proteins include, for example, domain antibodies, immunoglobulin single variable domains suitable for use as domain antibodies, single domain antibodies, immunoglobulin single variable domains suitable for use as ... the immunoglobulin single variable domain of the invention is a single variable domain, a dAb, an immunoglobulin single variable domain suitable for use as a dAb, or a Nanobody capable of binding to a serum protein such as serum albumin (such as human serum albumin), a serum immunoglobulin such as IgG, or transferrin; see further description and references herein); a polypeptide in which an amino acid sequence of the invention is linked to an Fc portion (such as human Fc) or a suitable portion or fragment thereof; or a polypeptide in which one or more immunoglobulin single variable domains of the invention are suitably linked to one or more small proteins or peptides capable of binding to a serum protein, such as, but not limited to, the proteins and peptides described in WO 91 / 01743, WO 01 / 45746, WO 02 / 076489, WO2008 / 068280, WO2009 / 127691 and PCT / EP2011 / 051559.

[0124] In one aspect, the present invention provides a construct or polypeptide of the present invention, wherein said construct or said polypeptide further comprises a serum protein binding moiety or serum protein. Preferably, said serum protein binding moiety binds to serum albumin, such as human serum albumin. In one aspect, the invention relates to a polypeptide described herein that comprises an ISVD that binds serum albumin. In general, a construct or polypeptide of the invention with an increased half-life preferably has a half-life that is at least 1.5 times longer, preferably at least 2 times longer, such as at least 5 times longer, for example at least 10 times or 20 times longer than the half-life of the corresponding construct or polypeptide of the invention itself, i.e., in the absence of the moiety that provides the increased half-life. For example, a construct or polypeptide of the invention with an increased half-life may have an increased half-life of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, e.g., in humans, compared to the corresponding construct or polypeptide of the invention itself, i.e., in the absence of the moiety that provides the increased half-life.

[0125] In a preferred, but non-limiting aspect of the invention, the constructs of the invention and the polypeptides of the invention have a serum half-life, e.g. in humans, that is increased by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding construct or polypeptide of the invention itself, i.e., without the moiety that confers the increased half-life. In another preferred, but non-limiting, aspect of the present invention, such constructs of the present invention, including polypeptides of the present invention, exhibit a serum half-life in humans of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 72 hours or more. For example, a construct or polypeptide of the present invention may have a serum half-life of at least 5 days (e.g., about 5-10 days), preferably at least 9 days (e.g., about 9-14 days), more preferably at least about 10 days (e.g., about 10-15 days), or at least about 11 days (e.g., about 11-16 days), more preferably at least about 12 days (e.g., about 12-18 days or more), or 14 days or more (e.g., about 14-19 days).

[0126] In a particularly preferred, but non-limiting aspect, the present invention provides constructs and polypeptides of the present invention, which comprise, in addition to one or more building blocks that bind to ADAMTS5, at least one building block that binds to serum albumin, e.g., an ISVD that binds to serum albumin, such as human serum albumin, as described herein. Preferably, the ISVD that binds to serum albumin comprises or consists essentially of four framework regions (FR1-FR4, respectively) and three complementarity determining regions (CDR1-CDR3, respectively), where CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR. Preferably, the ISVD that binds to serum albumin is selected from the group consisting of Alb8, Alb23, Alb129, Alb132, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG, Alb92, or Alb223 (see Table D).

[0127] In one aspect, the present invention relates to a polypeptide as described herein, wherein the polypeptide comprises at least one ISVD that binds to ADAMTS5 and an ISVD that binds to serum albumin, preferably selected from the group consisting of: SEQ ID NO: 129 (clone 577 2F3 * -Alb), SEQ ID NO: 130 (clone 579 2F3 * -093-Alb), SEQ ID NO:120 (clone 4 2A12-Alb), SEQ ID NO:121 (clone 5 2D7-Alb), SEQ ID NO:122 (clone 6 2F3-Alb), SEQ ID NO:123 (clone 69 049-Alb), SEQ ID NO:124 (clone 70 9D3-Alb), SEQ ID NO:125 (clone 71 3B2-Alb), SEQ ID NO:126 (clone 129 2F3-093-Alb), SEQ ID NO:127 (clone 130 049-093-Alb), and SEQ ID NO:128 (clone 131 9D3-093-Alb) (see Table A-1). In one aspect, the invention relates to a construct of the invention, such as a polypeptide comprising a serum protein binding moiety, wherein said serum protein binding moiety is a non-antibody-based polypeptide.

[0128] The art is in need of more effective treatments for disorders affecting articular cartilage, such as osteoarthritis. Even when administered intra-articularly, the residence time of most drugs is insufficient to treat diseased cartilage. The inventors hypothesized that the efficacy of therapeutic agents, such as the constructs, polypeptides, and ISVDs of the present invention, could be modulated by attaching the therapeutic agent to a moiety that "anchors" the drug within the joint, thereby increasing drug retention but not interfering with therapeutic agent efficacy (this moiety is also referred to herein as a "cartilage anchoring protein" or "CAP"). This anchoring concept not only modulates drug efficacy, but also allows for the specificity of drug action in diseased joints by reducing toxicity and side effects, thereby increasing the number of potentially useful drugs.

[0129] It is expected that a molecular format for clinical use will include one or two building blocks, such as an ISVD, that bind to ADAMTS5, and one or more building blocks, such as an ISVD, that have such a retention mode of action, and possibly further moieties. In a co-pending application, it has been demonstrated that such a format retains both ADAMTS5 binding and therapeutic effects, such as inhibitory activity, as well as retention properties. The one or more building blocks, such as an ISVD, that have a retention mode of action are any building blocks that have a retention effect in diseases involving ADAMTS5, such as, for example, arthritis, osteoarthritis, spondyloepiphyseal dysplasia, lumbar disc degenerative disease, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, and aggrecanopathy. A "CAP building block" is used to guide, fix, and / or retain other, e.g., therapeutic, building blocks, e.g., ISVDs that bind to ADAMTS5, at a desired site, e.g., a joint, where the other, e.g., therapeutic, building block exerts its effect, e.g., binding and / or inhibiting ADAMTS5.

[0130] The inventors further hypothesized that aggrecan binders, such as ISVDs, that bind to aggrecan could potentially function as such anchors, even though aggrecan is heavily glycosylated and degraded in various disorders affecting articular cartilage. Furthermore, given the cost and extensive testing in various animal models required before a drug can enter the clinic, such aggrecan binders should preferentially have broad cross-reactivity, e.g., they should bind to aggrecan from various species. Using various inventive immunization, screening, and characterization methods, the inventors were able to identify a variety of aggrecan-binding agents with excellent selectivity, stability, and specificity that allow for extended retention time and activity within the joint. In one aspect the present invention relates to a method for reducing and / or inhibiting the efflux of a composition, polypeptide or construct from a joint, said method comprising administering to a person in need thereof a pharmaceutically active amount of at least one polypeptide according to the invention, a construct according to the invention or a composition according to the invention.

[0131] In the present invention, the term "reducing and / or inhibiting outflow" refers to reducing and / or inhibiting the outward flow of a composition, polypeptide, or construct from within a joint to the outside. Preferably, the outflow of the composition, polypeptide, or construct within the joint is reduced and / or inhibited by at least 10%, for example, at least 20%, 30%, 40%, or 50%, or more, for example, at least 60%, 70%, 80%, 90%, or even 100%, compared to the outflow under the same conditions but without the presence of an aggrecan-binding agent of the present invention, such as an ISVD that binds to aggrecan. In addition to diseases in which ADAMTS5 is involved, such as arthritis, osteoarthritis, spondyloepiphyseal dysplasia, lumbar degenerative disc disease, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, and aggrecanopathy, it is expected that the aggrecan binding agents of the present invention can also be used in a variety of other diseases that affect cartilage, such as arthropathy and chondrodystrophy, arthritis (e.g., osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tear or avulsion), achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis (generally referred to herein as "aggrecan-related diseases").

[0132] The above-mentioned CAP building block, for example, the ISVD that binds to aggrecan, preferably binds to cartilage tissues such as cartilage and / or meniscus. In a preferred aspect, the CAP building block is cross-reactive with other species and specifically binds to one or more of the following: human aggrecan (SEQ ID NO: 155), dog aggrecan, bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and / or rhesus monkey aggrecan. Related structural information of aggrecan is listed in (UniProt) accession numbers, for example, as shown in Table 2 below. A preferred CAP building block is ISVD-linked aggrecan, preferably human aggrecan, preferably represented by SEQ ID NO: 155 as shown in Table B. Table 2 [Table 2]

[0133] The present invention therefore relates to a polypeptide or construct according to the invention, which further comprises at least one CAP building block. The present invention therefore relates to a polypeptide or construct according to the invention, which further comprises at least one ISVD that specifically binds to aggrecan, preferably selected from the ISVDs represented by SEQ ID NOs: 156 and 157. In one aspect, the invention relates to a polypeptide as described herein, comprising at least two ISVDs that specifically bind to aggrecan. In one aspect, the present invention relates to a polypeptide described herein comprising at least two ISVDs that specifically bind to aggrecan, wherein the at least two ISVDs that specifically bind to aggrecan may be the same or different.

[0134] In one aspect, the present invention relates to a polypeptide described herein comprising at least two ISVDs that specifically bind to aggrecan, wherein the at least two ISVDs that specifically bind to aggrecan are independently selected from the group consisting of SEQ ID NOs: 156-157. In one aspect, the present invention relates to a polypeptide described herein comprising at least two ISVDs that specifically bind to aggrecan, wherein the at least two ISVDs that specifically bind to aggrecan are represented by SEQ ID NOs: 156-157. In one aspect, the invention relates to a polypeptide described herein comprising an ISVD that specifically binds to aggrecan, wherein the ISVD that specifically binds to aggrecan specifically binds to human aggrecan [SEQ ID NO: 155].

[0135] In one aspect, the present invention relates to a polypeptide as described herein, wherein the ISVD that specifically binds to aggrecan specifically binds to human aggrecan (SEQ ID NO: 155), dog aggrecan, bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and / or rhesus monkey aggrecan. In one aspect, the invention relates to a polypeptide as described herein, wherein said ISVD that specifically binds to aggrecan preferably binds to cartilaginous tissue, such as cartilage and / or meniscus. It will be understood that the ISVDs, polypeptides, and constructs of the present invention are preferably stable. The stability of a polypeptide, construct, or ISVD of the present invention can be measured by routine assays known to those skilled in the art. Exemplary assays include (but are not limited to) assays in which the activity of a polypeptide, construct, or ISVD is measured, followed by incubation in synovial fluid for a desired period of time, after which activity is measured again.

[0136] In one aspect, the present invention relates to an ISVD, polypeptide or construct of the invention having stability in synovial fluid (SF) at 37°C for at least 7 days, such as at least 14 days, 21 days, 1 month, 2 months, or even 3 months. The desired activity of a therapeutic building block, such as an ISVD that binds to ADAMTS5 in a multivalent polypeptide or construct of the invention, can be measured by routine assays known to those skilled in the art. In one aspect, the invention relates to a construct as described herein, comprising at least one ISVD or polypeptide and one or more other groups, residues, moieties or binding units, preferably selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc portions, and small proteins or peptides capable of binding to serum proteins, further amino acid residues, tags or other functional moieties, such as toxins, labels, radioactive chemicals, etc.

[0137] In one aspect, as mentioned below, the present invention relates to a construct of the invention, such as a polypeptide, comprising a moiety that confers half-life extension, wherein said moiety is PEG. Thus, the present invention also relates to a construct or polypeptide of the invention comprising PEG. The additional amino acid residues may or may not change, alter or otherwise affect other (biological) properties of the polypeptides of the invention and may or may not add additional functionality to the polypeptides of the invention. For example, such amino acid residues may be: a) may contain an N-terminal Met residue, for example as a result of expression in a heterologous host cell or host organism; b) A signal or leader sequence may be formed that directs secretion of the polypeptide from the host cell upon synthesis (e.g., to provide a pre-, pro-, or prepro-form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention). Suitable secretory leader peptides will be apparent to those skilled in the art and may be as further described herein. Typically, such a leader sequence is linked to the N-terminus of the polypeptide, although the invention in its broadest sense is not limited thereto;

[0138] c) "Tags", i.e., amino acid sequences or residues that allow or facilitate, for example, the purification of the polypeptide, may be formed, for example, using affinity techniques directed against said sequences or residues. Said sequences or residues can then be removed (e.g., by chemical or enzymatic cleavage) to provide the polypeptide (for this purpose, the tag can optionally be linked to the amino acid or polypeptide sequence via a cleavable linker sequence or can comprise a cleavable motif). Some preferred, but non-limiting, examples of such residues are multiple histidine residues, glutathione residues, and myc tags such as AAAEQKLISEEDLNGAA; d) one or more amino acid residues that are functionalized and / or can serve as attachment sites for functional groups. Suitable amino acid residues and functional groups will be apparent to those skilled in the art and include, but are not limited to, the amino acid residues and functional groups referred to herein for derivatives of the polypeptides of the invention.

[0139] Further encompassed by the present invention are constructs comprising the polypeptides and / or ISVDs of the present invention, which further comprise other functional moieties, such as toxins, labels, radiochemicals, and the like. The other groups, residues, moieties, or binding units may be, for example, chemical groups, residues, moieties, which may or may not be biologically and / or pharmacologically active in their own right. For example, but not limited to, such groups may be linked to one or more ISVDs or polypeptides of the invention to provide "derivatives" of the polypeptides or constructs of the invention. Thus, the present invention in its broadest sense also includes constructs and / or polypeptides that are derivatives of the constructs and / or polypeptides of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and / or biological (e.g. enzymatic) modification, of the constructs and / or polypeptides of the invention and / or of one or more amino acid residues forming the polypeptides of the invention.

[0140] Examples of such modifications, as well as examples of amino acid residues within a polypeptide sequence that can be modified in such a manner (i.e., on the protein backbone or preferably on the side chains), methods and techniques that can be used for such modifications, and the potential uses and advantages of such modifications will be apparent to those skilled in the art (see also Zangi et al., Nat Biotechnol 31(10):898-907, 2013). For example, such modifications may involve the introduction (e.g., by covalent bonding or in other suitable manner) of one or more (functional) groups, residues or moieties into or onto the polypeptides of the invention, and in particular the introduction of one or more (functional) groups, residues or moieties which impart one or more desired properties or functionalities to the constructs and / or polypeptides of the invention. Examples of such functional groups will be apparent to those skilled in the art.

[0141] For example, such modifications may include the introduction (e.g., by covalent bonding or other suitable means) of one or more functional moieties that increase the half-life, solubility, and / or absorption of the constructs or polypeptides of the invention, reduce the immunogenicity and / or toxicity of the constructs or polypeptides of the invention, eliminate or attenuate undesirable side effects of the constructs or polypeptides of the invention, and / or confer other advantageous properties and / or reduce undesirable properties on the constructs or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional moieties and techniques for introducing them will be clear to those skilled in the art and may generally include all functional moieties and techniques mentioned in the general background art cited herein above, as well as functional moieties and techniques known per se for the modification of pharmaceutical proteins; and in particular, for the modification of antibodies or antibody fragments (including ScFvs and single-domain antibodies), see, for example, Remington (Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA, 1980). Such functional moieties may, for example, be linked to the polypeptides of the invention directly (eg covalently) or, optionally via a suitable linker or spacer, which will again be apparent to those skilled in the art.

[0142] One specific example is a derivative polypeptide or construct of the present invention, in which the polypeptide or construct has been chemically modified to increase its half-life (e.g., by pegylation). This is one of the most widely used techniques for extending the half-life and / or reducing the immunogenicity of pharmaceutical proteins, and involves the attachment of a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as PEGylation used in the art for antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv); see, for example, Chapman (Nat. Biotechnol. 54: 531-545, 2002), Veronese and Harris (Adv. Drug Deliv. Rev. 54: 453-456, 2003), Harris and Chess (Nat. Rev. Drug. Discov. 2: 214-221, 2003) and WO 04 / 060965. Various reagents for protein PEGylation are also commercially available, for example from Nektar Therapeutics, USA.

[0143] Preferably, site-specific pegylation, especially via cysteine ​​residues, is used (see, for example, Yang et al. (Protein Engineering 16: 761-770, 2003)). For example, for this purpose, PEG can be attached to cysteine ​​residues naturally present in the polypeptide of the invention; the construct or polypeptide of the invention can be modified to appropriately introduce one or more cysteine ​​residues for PEG attachment; or an amino acid sequence comprising one or more cysteine ​​residues for PEG attachment can be fused to the N-terminus and / or C-terminus of the construct or polypeptide of the invention, all using protein engineering techniques known per se to those skilled in the art. Preferably, for constructs or polypeptides of the invention, PEG is used having a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example, a molecular weight in the range of 20,000 to 80,000.

[0144] Another, usually less preferred modification, includes N-linked or O-linked glycosylation, usually as part of co- and / or post-translational modification, depending on the host cell used to express the polypeptide of the invention. Further modifications may include the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the polypeptide or construct of the invention. Suitable labels and techniques for attaching, using, and detecting them will be apparent to those of skill in the art and include, for example, but are not limited to, fluorescent labels (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine) and 152 fluorescent metals such as Eu, or other metals of the lanthanide series), phosphorescent labels, chemiluminescent or bioluminescent labels (e.g., luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, dioxetane or GFP and its analogs), radioisotopes (e.g., 3 H, 125 I, 32 P, 35 S, 14 C. 51 Cr, 36 Cl, 57 Co, 58 Co, 59 Fe, and 75 Se), metals, metal chelates, or metal cations, e.g. 99m Tc, 123 I, 111 In, 131 I, 97 Ru, 67 Cu, 67 Ga, and 68Ga, or other metals or metal cations particularly suitable for use in in vivo, in vitro or in situ diagnostics and imaging, e.g. 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe), as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinylated peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase). Other suitable labels will be apparent to those of skill in the art and include, for example, moieties detectable using NMR or ESR spectroscopy.

[0145] Such labeled polypeptides and constructs of the invention can be used, for example, for in vitro, in vivo or in situ assays (including immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays", etc.), as well as for in vivo diagnostic and imaging purposes, depending on the choice of the particular label. As will be apparent to those skilled in the art, another modification may involve the introduction of a chelating group, for example, to chelate one of the metals or metal cations listed above. Suitable chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0146] Yet another modification may include the introduction of a functional moiety that is part of a specific binding pair, such as a biotin-(streptavidin) binding pair. Such a functional moiety can be used to link the polypeptide of the present invention to another protein, polypeptide, or chemical compound that binds to the other half of the binding pair, for example, by forming a binding pair. For example, a construct or polypeptide of the present invention can be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such a conjugated construct or polypeptide of the present invention can be used as a reporter, for example, in a diagnostic system in which a detectable signal-generating agent is conjugated to avidin or streptavidin. Such a binding pair can also be used, for example, to bind the construct or polypeptide of the present invention to a carrier, including a carrier suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described in Cao and Suresh (Journal of Drug Targeting 8: 257, 2000). Such binding pairs can also be used to link therapeutically active agents to the polypeptides of the invention.

[0147] Other possible chemical and enzymatic modifications will be apparent to those skilled in the art. Such modifications can also be introduced for research purposes (e.g., to study function-activity relationships). See, e.g., Lundblad and Bradshaw (Biotechnol. Appl. Biochem. 26: 143-151, 1997). Preferably, the constructs, polypeptides and / or derivatives bind to ADAMTS5 with affinity as defined herein (K as further described herein). D value (actual or apparent), K A value (actual or apparent), k on rate or on rate, and / or k off or off-rate, or alternatively IC 50(as defined for the polypeptide of the invention) as appropriately measured and / or displayed as a value. Such constructs and / or polypeptides of the invention and derivatives thereof may also be in essentially isolated form (as defined herein).

[0148] In one aspect, the invention relates to a construct of the invention comprising or consisting essentially of an ISVD of the invention or a polypeptide of the invention, optionally further comprising one or more other groups, residues, moieties or binding units linked via one or more peptide linkers. In one aspect, the present invention relates to a construct of the present invention, wherein the one or more other groups, residues, moieties or binding units are selected from the group consisting of a polyethylene glycol molecule, a serum protein or fragment thereof, a binding unit capable of binding to a serum protein, an Fc portion, and a small protein or peptide capable of binding to a serum protein.

[0149] In constructs of the present invention, such as polypeptides of the present invention, two or more building blocks, such as ISVDs, and optionally one or more other groups, drugs, agents, residues, moieties, or binding units, may be directly linked to each other (e.g., as described in WO 99 / 23221) and / or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof. Spacers or linkers suitable for use in multivalent and multispecific polypeptides will be apparent to those skilled in the art and may generally be any linkers or spacers used in the art to link amino acid sequences. Preferably, the linkers or spacers are suitable for use in the construction of constructs, proteins, or polypeptides intended for pharmaceutical use.

[0150] For example, the polypeptide of the invention may be a trivalent, trispecific polypeptide, comprising, for example, one building block such as an ISVD that binds to ADAMTS5, an ISVD that binds to albumin, and potentially another building block such as a third ISVD, wherein said first, second and third building blocks such as ISVDs may optionally be linked via one or more, in particular two, linker sequences. The invention also provides a construct or polypeptide of the invention comprising a first ISVD that binds to ADAMTS5, and optionally a second ISVD that binds to albumin, and / or optionally a third ISVD and / or optionally a fourth ISVD, wherein said first ISVD and / or optionally said second ISVD and / or optionally said third ISVD and / or optionally said fourth ISVD are linked via linkers, in particular three linkers.

[0151] Some particularly preferred linkers include those used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art above, as well as linkers used in the art to construct, for example, diabodies or ScFv fragments (in this regard, however, it should be noted that in diabodies and ScFv fragments, the linker sequences used should be those that are suitable V H and V L It should have length, some flexibility, and other properties that allow the domains to come together to form a complete antigen-binding site; there are no particular restrictions on the length or flexibility of linkers used in the polypeptides of the invention, since each ISVD, such as a nanobody, forms a complete antigen-binding site by itself.

[0152] For example, the linker may be a suitable amino acid sequence, particularly an amino acid sequence of 1 to 50, preferably 1 to 30, e.g., 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include, for example, (gly x sery )2 type gly-ser linkers, such as (gly4ser)3 or (gly3ser2)3 described in WO 99 / 42077, and the GS30, GS15, GS9, and GS7 linkers described in the Ablynx applications cited herein (see, e.g., WO 06 / 040153 and WO 06 / 122825), as well as hinge-like regions, such as those of naturally occurring heavy chain antibodies or similar sequences (described in WO 94 / 04678). Preferred linkers are shown in Table C. Some other particularly preferred linkers are polyalanine (such as AAA), as well as linkers GS30 (see also SEQ ID NO: 85 of WO 06 / 122825) and GS9 (see also SEQ ID NO: 84 of WO 06 / 122825). Other suitable linkers generally include organic compounds or polymers, particularly those suitable for use with proteins for pharmaceutical applications, for example, poly(ethylene glycol) moieties have been used to link antibody domains; see, e.g., WO 04 / 081026.

[0153] It is within the scope of the present invention that the length, degree of flexibility, and / or other properties of the linker used (although typically less important than for linkers used with ScFv fragments) may have some effect on the properties of the final construct of the present invention, such as the polypeptide of the present invention, including but not limited to its affinity, specificity, or avidity for a chemokine or one or more other antigens. Based on the disclosure herein, one skilled in the art will be able to determine the optimal linker for use in a particular construct of the present invention, such as the polypeptide of the present invention, optionally after some limited routine experimentation. For example, in a multivalent polypeptide of the invention comprising building blocks, ISVDs or nanobodies directed against ADAMTS5 and another target, the length and flexibility of the linker is preferably such that each building block, such as an ISVD of the invention in the polypeptide, can bind to its cognate target, e.g., an antigenic determinant of each target. Again, based on the disclosure herein, one skilled in the art will be able to determine the optimal linker for use in a particular construct of the invention, such as a polypeptide of the invention, optionally after some limited routine experimentation.

[0154] It is further within the scope of the present invention that the linker used imparts one or more other desirable properties or functionalities to the constructs of the invention, such as the polypeptides of the invention, and / or provides one or more sites for the formation of derivatives and / or the attachment of functional groups (e.g., as described herein for derivatives of the ISVDs of the invention). For example, a linker comprising one or more charged amino acid residues can provide improved hydrophilicity, while a linker that forms or includes a small epitope or tag can be used for detection, identification, and / or purification purposes. Again, based on the disclosure herein, one of skill in the art will be able to determine the optimal linker for use with a particular polypeptide of the invention, optionally after some limited routine experimentation.

[0155] Finally, when two or more linkers are used in a construct, such as a polypeptide, of the invention, these linkers may be the same or different. Again, based on the disclosure herein, one of skill in the art will be able to determine the optimal linker for use in a particular construct or polypeptide of the invention, optionally after some limited routine experimentation. Typically, for ease of expression and production, constructs of the present invention, such as polypeptides of the present invention, are linear polypeptides. However, the present invention is not limited thereto in its broadest sense. For example, when constructs of the present invention, such as polypeptides of the present invention, include three or more building blocks, ISVDs, or nanobodies, they can be linked by using a linker with three or more "arms," ​​each "arm" being linked to a building block, ISVD, or nanobody to provide a "star-shaped" construct. Although generally less preferred, circular constructs can also be used.

[0156] The present invention therefore relates to constructs of the invention, such as polypeptides of the invention, wherein said ISVDs are linked to each other directly or via a linker. The present invention therefore relates to constructs of the invention, such as polypeptides of the invention, in which a first ISVD and / or a second ISVD and / or an ISVD that possibly binds to serum albumin are linked via a linker. The invention therefore relates to constructs of the invention, such as polypeptides of the invention, wherein said linker is selected from the group consisting of 3A, 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, 35GS, poly-A, 8GS, 40GS, G1 hinge, 9GS-G1 hinge, llama upper long hinge region, and G3 hinge linkers, e.g. as shown in Table C (SEQ ID NOs: 158-174).

[0157] The present invention therefore relates to a construct of the invention, such as a polypeptide of the invention, wherein said polypeptide is selected from the group consisting of SEQ ID NOs: 120-130. The present invention further relates to methods of preparing the constructs, polypeptides, ISVDs, nucleic acids, host cells, and compositions described herein. Multivalent polypeptides of the invention can generally be prepared by a method comprising suitably linking at least an ISVD and / or a monovalent polypeptide of the invention to one or more further ISVDs, optionally via one or more suitable linkers, thereby providing a multivalent polypeptide of the invention. Polypeptides of the invention can also be prepared by a method comprising at least the steps of providing a nucleic acid encoding a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such a method can be carried out in a manner known per se and will be clear to the skilled person, for example based on the methods and techniques further described herein.

[0158] Methods for preparing multivalent polypeptides of the invention may include at least the step of linking together two or more ISVDs of the invention and, for example, one or more linkers in a suitable manner. The ISVDs (and linkers) of the invention can be joined by any method known in the art and further described herein. A preferred technique involves linking nucleic acid sequences encoding the ISVDs (and linkers) of the invention to prepare a genetic construct that expresses the multivalent polypeptide. Techniques for linking amino acids or nucleic acids will be clear to those skilled in the art, and reference is again made to standard handbooks, such as Sambrook et al. and Ausubel et al., supra, as well as the examples below.

[0159] Thus, the present invention also relates to the use of an ISVD of the present invention in preparing a multivalent polypeptide of the present invention. The method for preparing a multivalent polypeptide comprises linking an ISVD of the present invention to at least one additional ISVD of the present invention, optionally via one or more linkers. The ISVD of the present invention is then used as a binding domain or building block in providing and / or preparing a multivalent polypeptide comprising two (e.g., in a bivalent polypeptide), three (e.g., in a trivalent polypeptide), four (e.g., in a tetravalent polypeptide), or more (e.g., in a multivalent polypeptide) building blocks. In this regard, the ISVD of the present invention can be used as a binding domain or binding unit in providing and / or preparing a bivalent, trivalent, or tetravalent, etc. multivalent polypeptide of the present invention comprising two, three, four, or more building blocks.

[0160] Thus, the present invention also relates to the use of an ISVD polypeptide of the invention (as described herein) in the preparation of a multivalent polypeptide, the method of preparing a multivalent polypeptide comprising linking an ISVD of the invention to at least one further ISVD of the invention, optionally via one or more linkers. The polypeptides and nucleic acids of the present invention can be prepared in a manner known per se, as will be clear to those skilled in the art from the further description herein.For example, the polypeptides of the present invention can be prepared in any manner known per se for preparing antibodies, particularly antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments).Some preferred, but non-limiting, methods for preparing polypeptides and nucleic acids include the methods and techniques described herein.

[0161] A method for producing a polypeptide of the present invention may comprise the following steps: expression of a nucleic acid encoding said polypeptide of the present invention (herein also referred to as "nucleic acid of the present invention") in a suitable host cell or host organism (herein also referred to as "host of the present invention") or in another suitable expression system; optionally followed by isolation and / or purification of the polypeptide of the present invention thus obtained. In particular, such a method may comprise the steps of: culturing and / or maintaining a host of the invention under conditions such that the host of the invention expresses and / or produces at least one polypeptide of the invention; optionally followed by isolating and / or purifying the polypeptide of the invention thus obtained.

[0162] The present invention therefore also relates to nucleic acids or nucleotide sequences (also called "nucleic acids of the invention") that encode the polypeptides, ISVDs or constructs of the invention. The nucleic acid of the present invention can be in the form of single-stranded or double-stranded DNA or RNA. According to one aspect of the present invention, the nucleic acid of the present invention is essentially isolated as defined herein. The nucleic acid of the present invention can also be in the form of, present in, and / or part of a vector, such as an expression vector, for example, a plasmid, a cosmid, or a YAC (which can also be essentially isolated). Therefore, the present invention also relates to an expression vector comprising the nucleic acid or nucleotide sequence of the present invention.

[0163] The nucleic acids of the present invention can be prepared or obtained in a manner known per se based on the information on the polypeptides of the present invention described herein, and / or isolated from a suitable natural source. Furthermore, as will be clear to those skilled in the art, to prepare the nucleic acids of the present invention, several nucleotide sequences, such as at least two nucleic acids encoding the ISVDs of the present invention and, for example, nucleic acids encoding one or more linkers, can be linked together in a suitable manner. Techniques for generating the nucleic acids of the present invention will be clear to those skilled in the art and may include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more parts thereof), introducing mutations that result in the expression of truncated expression products; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using appropriate restriction enzymes), and / or introducing mutations by PCR using one or more "mismatched" primers. These and other techniques will be clear to those skilled in the art, and again, reference is made to standard handbooks, such as Sambrook et al. and Ausubel et al., supra, as well as the examples below.

[0164] In a preferred but non-limiting embodiment, the genetic construct of the present invention comprises: a) at least one nucleic acid of the invention; b) operably linked to one or more regulatory elements, e.g., a promoter, and optionally, a suitable terminator; and optionally also c) one or more further elements of the genetic construct, known per se; Here, the terms "regulatory element," "promoter," "terminator," and "operably linked" have their ordinary meanings in the art. Genetic constructs of the present invention can generally be provided by appropriately linking a nucleotide sequence of the present invention to one or more of the above-mentioned further elements, using techniques described in general handbooks such as, for example, Sambrook et al. and Ausubel et al., supra.

[0165] The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform host cells or host organisms, i.e., for the expression and / or production of the polypeptides of the invention. Suitable hosts or host cells will be clear to those skilled in the art and can, for example, be any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or (non-human) eukaryotic organism, as well as host cells or (non-human) hosts known per se for the expression and production of antibodies and antibody fragments, including, but not limited to, (single) domain antibodies and ScFv fragments, and will be clear to those skilled in the art. See also the general background art cited above, as well as, for example, WO 94 / 29457; WO 96 / 34103; WO 99 / 42077; Frenken et al. (Res Immunol. 149: 589-99, 1998); Riechmann and Muyldermans (1999) supra; van der Linden (J. Biotechnol. 80: 261-70, 2000); Joosten et al. (Microb. Cell Fact. 2: 1, 2003); Joosten et al. (Appl. Microbiol. Biotechnol. 66: 384-92, 2005); and further references cited therein. Furthermore, the polypeptides of the present invention can also be expressed and / or produced in cell-free expression systems; suitable examples of such systems will be apparent to those skilled in the art. Suitable techniques for transforming the host or host cells of the present invention will be clear to those skilled in the art and may depend on the intended host cell / host organism and the genetic construct used. Again, reference is made to the handbooks and patent applications mentioned above. Transformed host cells (which may be in the form of a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form a further aspect of the present invention. Thus, the present invention relates to a host or host cell comprising a nucleic acid according to the present invention or an expression vector according to the present invention.Preferably, these host cells or host organisms express or are (at least) capable of expressing (e.g., under appropriate conditions) the polypeptide of the invention (and in the case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and / or descendants of the host cells or host organisms of the invention, which may be obtained, for example, by cell division or sexual or asexual reproduction.

[0166] To produce / obtain expression of the polypeptide of the present invention, the transformed host cell or transformed host organism may generally be kept, maintained and / or cultured under conditions such that the (desired) polypeptide of the present invention is expressed / produced. Suitable conditions will be clear to those skilled in the art and will usually depend on the host cell / host organism used, as well as on the regulatory elements controlling the expression of the (relevant) nucleotide sequence of the present invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraph concerning the genetic constructs of the present invention.

[0167] The polypeptides of the invention can then be isolated from the host cells / host organisms and / or the medium in which said host cells or host organisms have been cultivated using protein isolation and / or purification techniques known per se, such as (preparative) chromatographic and / or electrophoretic techniques, differential precipitation techniques, affinity techniques (e.g. using specific cleavable amino acid sequences fused to the polypeptides of the invention) and / or preparative immunological techniques (i.e. using antibodies against the polypeptides to be isolated). In one aspect, the present invention relates to a method for producing a construct, polypeptide or ISVD according to the invention, comprising at least the following steps: (a) expressing a nucleic acid sequence according to the invention in a suitable host cell or host organism or another suitable expression system; optionally followed by (b) isolating and / or purifying the construct, polypeptide, ISVD according to the invention.

[0168] In one aspect, the invention relates to a composition comprising a construct, a polypeptide, an ISVD or a nucleic acid according to the invention. As described above, there remains a need for safe and effective OA therapeutics. Based on unconventional screening, characterization, and combination strategies, the present inventors have identified ISVDs that bind to and inhibit ADAMTS5. These ADAMTS5-binding agents demonstrated excellent performance in in vitro and in vivo experiments. Furthermore, the ISVDs of the present invention have been demonstrated to be significantly more effective than prior art compounds. Thus, the present invention provides ISVDs and polypeptides that antagonize ADAMTS, particularly ADAMTS5, and have improved prophylactic, therapeutic, and / or pharmacological properties, including a safer profile, compared to prior art amino acid sequences and antibodies. Furthermore, when these ADAMTS5-binding agents are linked to ISVDs that bind to albumin, they are retained in the subject, allowing for systemic administration while retaining activity.

[0169] In one aspect, the present invention relates to a method for treating or preventing a disease or disorder in an individual, for example one involving ADAMTS5 activity, comprising administering to the individual an ISVD or polypeptide according to the present invention in an amount effective to treat or prevent the symptoms of the disease or disorder. In one aspect, the present invention relates to a composition according to the invention, an ISVD according to the invention, a polypeptide according to the invention, and / or a construct according to the invention for use as a medicament. In another aspect, the present invention relates to the use of the ISVD, polypeptide and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least an ADAMTS5-related disease and / or for use in one or more of the treatment methods described herein.

[0170] The present invention also relates to the use of the ISVD, polypeptide and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least one disease or disorder that can be prevented and / or treated by modulating the activity of ADAMTS, preferably ADAMTS5, for example by inhibiting the degradation of aggrecan. The present invention also relates to the use of an ISVD, polypeptide, compound and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least one disease, disorder or condition that can be prevented and / or treated by administering an ISVD, polypeptide, compound and / or construct of the present invention to a patient.

[0171] The present invention further relates to the ISVD, polypeptide, compound and / or construct of the invention, or a pharmaceutical composition comprising same, for use in the prevention and / or treatment of at least one ADAMTS5-related disease. It is expected that the ADAMTS5-binding agents of the present invention can be used in a variety of diseases affecting cartilage, such as arthropathy and chondrodystrophy, arthritis such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or avulsions, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated discs, lumbar degenerative disc disease, degenerative joint diseases, and relapsing polychondritis, osteochondritis dissecans and aggrecanopathy and non-alcoholic steatohepatitis (NASH) (generically referred to herein as "ADAMTS5-associated diseases").

[0172] In one aspect, the invention relates to a composition, ISVD, polypeptide and / or construct according to the invention for use in treating or preventing symptoms of an ADAMTS5-associated disease, such as arthropathy and chondrodystrophy, arthritis, e.g. osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or avulsions, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and aggrecanopathy and NASH. In one aspect, the present invention relates to a method for preventing or treating arthropathies and chondrodystrophies, arthritis such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or avulsions, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis and NASH, wherein said method comprises administering to a subject in need thereof at least a pharmaceutically active amount of a composition, immunoglobulin, polypeptide, or construct according to the present invention.

[0173] In one aspect, the invention relates to the use of an ISVD, polypeptide, composition or construct according to the invention, for example in the preparation of a pharmaceutical composition for treating or preventing arthropathy and chondrodystrophy, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or avulsions, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and aggrecanopathy and NASH. It is also expected that by binding to aggrecan, the constructs and / or polypeptides of the present invention may reduce or inhibit the activity of not only members of the serine protease family, cathepsins, e.g., matrix metalloproteinases (MMPs) such as MMP20, but also ADAMTS4 (aggrecanase 1) and / or ADAMTS11 in the degradation of aggrecan.

[0174] In the context of the present invention, the term "prevention and / or treatment" not only includes the prevention and / or treatment of disease, but also generally includes: preventing the onset of disease, delaying or reversing the progression of disease, preventing or delaying the disease, preventing or delaying the onset of one or more symptoms associated with the disease, alleviating and / or mitigating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or symptoms associated therewith, and / or preventing a further increase in the severity of the disease and / or any symptoms associated therewith, preventing, alleviating, or reversing any physiological damage caused by the disease, and generally any pharmacological effect beneficial to the treated patient.

[0175] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient will depend on many factors, including, for example, the patient's size, weight, body surface area, age, the specific compound being administered, the activity of the polypeptide (including antibody) used, the time and route of administration, general health, and combination with other therapies or treatments. Proteinaceous pharmaceutically active agents may be present in amounts of 1 g to 100 mg / kg body weight per dose; however, doses below or above this exemplary range are also contemplated. If the regimen is a continuous infusion, the range may be 1 pg to 100 mg per kilogram of body weight per minute.

[0176] The ISVDs, polypeptides or constructs of the present invention may be used at concentrations of, for example, 0.01, 0.1, 0.5, 1, 2, 5, 10, 20 or 50 pg / ml to inhibit and / or neutralize the biological function of ADAMTS5 by at least about 50%, preferably 75%, more preferably 90%, 95% or up to 99%, and most preferably about 100% (essentially completely), when assayed by methods well known in the art. Generally, a treatment regimen involves the administration of one or more pharmaceutically effective amounts or doses of one or more ISVDs, polypeptides, and / or constructs of the present invention, or one or more compositions comprising the same. The specific amount or dose to be administered can be determined by a clinician, again based on the factors described above. Useful doses of the constructs, polypeptides, and / or ISVDs of the present invention can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art; see, e.g., U.S. Pat. No. 4,938,949.

[0177] Generally, the clinician will be able to determine an appropriate dosing regimen depending on the particular disease, disorder or condition being treated, the potency of the particular ISVD, polypeptide and / or construct of the invention used, the particular route of administration and the particular pharmaceutical formulation or composition used. The amount of the constructs, polypeptides, and / or ISVDs of the invention required for therapeutic use will vary depending not only on the particular immunoglobulin, polypeptide, compound, and / or construct selected, but also on the route of administration, the nature of the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the attending physician or clinician. Doses of the constructs, polypeptides, and / or ISVDs of the invention will also vary depending on the target cell, tumor, tissue, graft, or organ.

[0178] The desired dose may conveniently be presented in a single dose or as divided dose administered at appropriate intervals, e.g., as two, three, four or more sub-doses per day, which sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. The dosing regimen may include chronic daily treatment. "Chronic" means a period of at least two weeks, preferably several weeks, months, or years. Necessary modifications to this dosage range can be determined by one of ordinary skill in the art using only the teachings herein and routine experimentation. See Remington's Pharmaceutical Sciences (Martin, EW, ed. 4), Mack Publishing Co., Easton, PA. Dosages can also be adjusted by the individual physician in the event of any complications.

[0179] Typically, the above methods use an ISVD, polypeptide and / or construct of the invention, however, it is within the scope of the invention to use a combination of two or more ISVDs, polypeptides and / or constructs of the invention. The ISVDs, polypeptides and / or constructs of the present invention can be used in combination with one or more additional pharmaceutically active compounds or ingredients, i.e., as a combination treatment regimen, which may or may not produce a synergistic effect. The pharmaceutical composition may also include at least one additional active agent, such as one or more additional antibodies or antigen-binding fragments thereof, peptides, proteins, nucleic acids, organic and inorganic molecules.

[0180] Again, the clinician will be able to select such additional compounds or components, as well as the appropriate combination treatment regimen, based on the above factors and their own professional judgment. In particular, the ISVDs, polypeptides and / or constructs of the present invention can be used in combination with other pharmaceutically active compounds or ingredients that are or can be used in the prevention and / or treatment of the diseases, disorders and conditions cited herein, which may or may not result in a synergistic effect. Examples of such compounds and ingredients, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

[0181] When two or more substances or components are used as part of a combination treatment regimen, they can be administered via the same route of administration or different routes of administration, at essentially the same time or at different times (e.g., essentially simultaneously, sequentially, or according to an alternating regimen). When substances or components are administered simultaneously via the same route of administration, they can be administered as part of different pharmaceutical formulations or compositions, or combined pharmaceutical formulations or compositions, as will be apparent to those skilled in the art.

[0182] In addition, when two or more active substances or components are used as part of a combined treatment plan, each of the substances or components can be administered in the same amount and according to the same schedule as when the compound or component is used by itself, and such a combination may or may not produce a synergistic effect.However, when the combined use of two or more active substances or components produces a synergistic effect, it is also possible to reduce the amount of one, more, or all of the substances or components administered, and still achieve the desired therapeutic effect.This can, for example, help to avoid, limit, or reduce any undesirable side effects associated with the use of one or more substances or components when used in normal amounts, and still achieve the desired medicinal or therapeutic effect.

[0183] The effectiveness of the treatment regimen used in accordance with the present invention can be determined and / or followed in any manner known per se for the disease, disorder, or condition involved, as will be apparent to the clinician. The clinician can also modify or alter the particular treatment regimen as needed and on a case-by-case basis to achieve the desired therapeutic effect and avoid, limit, or reduce undesirable side effects, and / or achieve an appropriate balance between achieving the desired therapeutic effect, on the one hand, and avoiding, limiting, or reducing undesirable side effects, on the other hand.

[0184] Generally, a treatment regimen will be continued until the desired therapeutic effect is achieved and / or for as long as the desired therapeutic effect is maintained, again, this can be determined by the clinician. Thus, in a further aspect, the present invention relates to a pharmaceutical composition comprising at least one construct of the invention, at least one polypeptide of the invention, at least one ISVD of the invention, or at least one nucleic acid of the invention, and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances. In a particular aspect, the present invention relates to a pharmaceutical composition comprising at least one construct, polypeptide, ISVD or nucleic acid according to the invention, preferably at least one of SEQ ID NO: OOO, and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances.

[0185] The subject to be treated may be any warm-blooded animal, but is particularly a mammal, more particularly a human.In veterinary applications, the subject to be treated includes any animal that is commercially bred or kept as a pet.As will be clear to those skilled in the art, the subject to be treated is particularly a person who is suffering from or at risk of the diseases, disorders and conditions mentioned herein.Therefore, in a preferred aspect of the present invention, the pharmaceutical composition comprising the polypeptide of the present invention is for use in medicine or diagnosis.Preferably, the pharmaceutical composition is for use in human medicine, but can also be used for veterinary purposes.

[0186] Again, in such pharmaceutical compositions, one or more immunoglobulins, polypeptides, compounds and / or constructs of the invention, or nucleotides encoding same, and / or pharmaceutical compositions comprising same, may be suitably combined with one or more other active ingredients, such as those described herein. The present invention also relates to compositions (such as, without limitation, pharmaceutical compositions or formulations further described herein) for use either in vitro (e.g., in in vitro or cellular assays) or in vivo (e.g., in single cells or multicellular organisms, and particularly in mammals, more particularly humans, e.g., humans at risk of or suffering from a disease, disorder or condition of the invention).

[0187] Unless otherwise specified, references to treatment will be understood to include both treatment of established symptoms and prophylactic treatment. Generally, for pharmaceutical use, the constructs, polypeptides, and / or ISVDs of the present invention can be formulated as pharmaceutical formulations or compositions comprising at least one construct, polypeptide, and / or ISVD of the present invention and at least one pharmaceutically acceptable carrier, diluent or excipient, and / or adjuvant, and optionally one or more pharmaceutically active polypeptides and / or compounds. By way of non-limiting example, such formulations can be in a form suitable for oral administration, parenteral administration (such as intravenous, intramuscular, or subcutaneous injection or intravenous infusion), topical administration, administration by inhalation, skin patch, implant, suppository, etc., with parenteral administration being preferred. Such suitable dosage forms—which may be solid, semi-solid, or liquid depending on the mode of administration—and the methods and carriers used for their preparation will be apparent to those skilled in the art and are further described herein. Such pharmaceutical formulations or compositions are generally referred to herein as "pharmaceutical compositions."

[0188] Examples of excipients include disintegrants, binders, fillers, and lubricants.Examples of disintegrants include agar, algin, calcium carbonate, cellulose, colloidal silicon dioxide, gum, magnesium aluminum silicate, methylcellulose, and starch.Examples of binders include microcrystalline cellulose, hydroxymethylcellulose, hydroxypropylcellulose, and polyvinylpyrrolidone.Examples of fillers include calcium carbonate, calcium phosphate, tribasic calcium sulfate, carboxymethylcellulose calcium, cellulose, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrin, maltose, sorbitol, starch, sucrose, sugar, and xylitol. Examples of lubricants include agar, ethyl oleate, ethyl laurate, glycerin, glyceryl palmitostearate, hydrogenated vegetable oils, magnesium oxide, stearates, mannitol, poloxamer, glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc. Conventional stabilizers, preservatives, wetting agents and emulsifiers, viscosity improvers, flavor improvers, salts for varying osmotic pressure, buffer substances, solubilizers, diluents, emollients, colorants, and masking agents, and antioxidants are considered as pharmaceutical adjuvants.

[0189] Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, water, alcohol, polyol, glycerol, vegetable oils, and the like. In general, the constructs, polypeptides, and / or ISVDs of the invention can be formulated and administered in any suitable manner known per se, for example as described in the general background art cited above (and in particular WO 04 / 041862, WO 04 / 041863, WO 04 / 041865, WO 04 / 041867 and WO 08 / 020079), as well as in standard handbooks, for example Remington's Pharmaceutical Sciences, 18 th Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see, e.g., pages 252-255).

[0190] In a particular aspect, the present invention relates to a pharmaceutical composition comprising a construct, polypeptide, ISVD or nucleic acid according to the invention, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more further pharmaceutically active polypeptides and / or compounds. The constructs, polypeptides, and / or ISVDs of the present invention can be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFvs and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing them will be apparent to those skilled in the art and include, for example, formulations that are preferably suitable for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial, intraspinal, intranasal, or intrabronchial administration), as well as for topical (i.e., transdermal or intradermal) administration.

[0191] Formulations for parenteral administration may be, for example, sterile solutions, suspensions, dispersions, or emulsions suitable for infusion or injection. Suitable carriers or diluents for such formulations include, but are not limited to, those described on page 143 of WO 08 / 020079. Usually, aqueous solutions or suspensions are preferred. The constructs, polypeptides, and / or ISVDs of the invention can also be administered using delivery methods known from gene therapy, see, e.g., U.S. Patent No. 5,399,346, which is incorporated by reference for its gene therapy delivery methods. Using gene therapy delivery methods, primary cells transfected with genes encoding the constructs, polypeptides, and / or ISVDs of the invention can be further transfected with tissue-specific promoters that target specific organs, tissues, grafts, tumors, or cells, and can further be transfected with signal and stabilizing sequences for intracellularly localized expression.

[0192] According to another aspect of the present invention, the polypeptide of the present invention can be used for further in vivo and in vitro applications.For example, the polypeptide of the present invention can be used for diagnostic purposes, for example, in assays designed to detect and / or quantify the presence of ADAMTS5 and / or purify ADAMTS5.The polypeptide can also be tested in animal models of specific diseases to conduct toxicology, safety, and dosage studies. Finally, the present invention relates to a kit comprising at least one polypeptide according to the present invention, at least one nucleic acid sequence encoding said component, a vector or vector system according to the present invention, and / or a host cell according to the present invention. It is contemplated that the kit may be provided in different forms, for example as a diagnostic kit.

[0193] The present invention is further illustrated by the following non-limiting preferred aspects, examples and figures. The entire contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated by reference, particularly with respect to the teachings referenced above.

[0194] The sequences are set forth in the body of the specification and in a separate sequence listing according to WIPO Standard ST.25. A sequence designated by a particular number should be identical in the body of the specification and in the separate sequence listing. For example, SEQ ID NO: 1 should define the same sequence in both the body of the specification and the separate sequence listing. In the event of a discrepancy between the sequence definitions in the body of the specification and the separate sequence listing (e.g., if SEQ ID NO: 1 in the body of the specification erroneously corresponds to SEQ ID NO: 2 in the separate sequence listing), the reference to a particular sequence in the present application, particularly in a particular embodiment, should be understood as a reference to the sequence in the body of the present application, not to the separate sequence listing. In other words, a discrepancy in sequence definition / designation between the body of the specification and the separate sequence listing will be resolved by amending the separate sequence listing to the sequences and their designations disclosed in the body of the application, including the specification, examples, figures, and claims. [Example]

[0195] example Example 1. Production of recombinant ADAMTS5 protein from different species Various formats of recombinant ADAMTS5 proteins from bovine, rat, guinea pig, mouse, and cynomolgus monkey were produced in-house using the HEK293 Flp-In™ expression system with FuGENE® HD transfection reagent (Promega). Two days after transfection, selection medium containing 100 μg / ml hygromycin B was added to the cells to select for stable expressing cells. Stably expressing cells were expanded. Conditioned medium containing secreted ADAMTS5 was collected daily from the cells and purified by HisTrap chromatography, followed by buffer exchange with 50 mM Hepes buffer, pH 7.5. Protein purity was confirmed by SDS-PAGE.

[0196] Example 2 Immunization of llamas with ADAMTS5 protein, cloning of heavy chain-only antibody fragment repertoires, and phage preparation 2.1 Immunization After approval by the Ethics Committee of the Faculty of Veterinary Medicine (University Ghent, Belgium), three llamas were immunized with recombinant human ADAMTS5 protein (R&D Systems, Minneapolis, US; cat # 2198-AD).

[0197] 2.2 Cloning of heavy chain-only antibody fragment repertoires and phage preparation Following the final immunogen injection, blood samples were collected. Peripheral blood mononuclear cells (PBMCs) were prepared from these blood samples using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, US). Total RNA was extracted from PBMCs and used as starting material for RT-PCR to amplify DNA segments encoding VHHs / nanobodies, essentially as described in WO 05 / 044858. Phages were then prepared according to standard protocols (see, e.g., the prior art and the applications of Ablynx NV cited herein) and stored for further use after filter sterilization at 4°C.

[0198] Example 3. Selection of human ADAMTS5-specific VHHs via phage display Human ADAMTS5-binding nanobodies were selected using the VHH repertoire obtained from all llamas and cloned into a phage library. Recombinant human ADAMTS5 protein (R&D Systems, Minneapolis, US; cat # 2198-AD) was immobilized on Maxisorp plates (Nunc, Wiesbaden, Germany) at 5 μg / ml, next to a negative control antigen at 0 μg / ml. After incubation with the phage library and extensive washing, bound phages were eluted with trypsin (1 mg / ml) and used to infect E. coli cells. The infected E. coli cells were used to prepare phages for the next selection round or to plate agar plates for analysis. Additionally, a synthetic library was used in three consecutive selection rounds. The output of every selection round was analyzed for enrichment factor, which is the number of phage present in the eluate compared to the control. The optimal selection conditions were selected for further analysis. To screen the selection output of specific binders, single colonies were picked from agar plates and grown in 1 mL 96-deep-well plates. LacZ-controlled VHH expression was induced by the addition of IPTG. Periplasmic extracts were prepared according to standard protocols (see, e.g., the prior art and the applications of Ablynx NV cited herein).

[0199] Example 4. Screening periplasmic extracts for functional blocking nanobodies In a first step, periplasmic extracts were tested for binding to recombinant human ADAMTS5 by binding ELISA. Briefly, recombinant human ADAMTS5 (R&D Systems, Minneapolis, US; cat # 2198-AD) was coated onto 384-well MaxiSorp plates (Nunc, Wiesbaden, Germany) at a concentration of 1 μg / ml. Wells were blocked with casein solution (1%). After addition of 10-fold dilutions of periplasmic extracts, nanobody binding was detected using a mouse anti-Flag-HRP conjugate (Sigma, St. Louis, US) and a subsequent enzymatic reaction in the presence of the substrate esTMB (3,3',5,5'-tetramentylbenzidine) (SDT, Brussels, Belgium). Clones showing an ELISA signal higher than the sum of the mean signal of the irrelevant nanobody control and three times the standard deviation of the irrelevant nanobody control were considered to encode positive human ADAMTS5-binding nanobodies.

[0200] To identify nanobodies capable of preventing ADAMTS5-mediated cleavage of aggrecan, clones were tested in a FRET-based human ADAMTS5 enzyme assay using 50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM CaCl *2H2O, 0.1% CHAPS, 5% glycerol were used as the assay buffer. Briefly, periplasmic extracts containing ADAMTS5-binding nanobodies were incubated with 10 μl of 25 μM quenched fluorescent human peptide substrate (Abz-TEGEARGSVI-Dap(Dnp)-KK-NH2, Anaspec, Serain Belgium, cat# 60431-1) and 10 μl of 25 nM human ADAMTS5 (R&D Systems, Minneapolis, US; cat# 2198-AD) in a 384-well OptiPlate (PerkinElmer, Waltham, MA, US). The ability of the nanobodies to prevent ADAMTS5-mediated cleavage was monitored every minute for 2 hours using a Tecan Infinite M1000 plate reader. Data were analyzed using Graphpad Prism software. From the enzymatic progress curve, the initial velocity of the negative control (v0) as well as the velocities of all nanobody clones in the plate (v i ) and background signal (S b ) was determined. * (1-(v i -S b ) / (v0-S b )) was used to calculate the percent inhibition.

[0201] Periplasmic extracts containing irrelevant nanobodies were used as negative controls. Periplasmic extracts containing ADAMTS5 nanobodies that were able to reduce the fluorescent signal by 50% or more compared to the signal of the negative control were considered inhibitory nanobodies. Subsequently, the DNA sequences of positive clones were determined. A summary of the periplasmic extract screening data is shown in Table 4.1. The amino acid sequences of the anti-ADAMTS5 nanobodies are shown in Table A-1. Table 4.1 Screening results of periplasmic extracts containing anti-ADAMTS5 nanobodies [Table 3]

[0202] After cloning and sequencing, various families were identified, consisting of clones that differ in their CDRs but exhibit similar binding and inhibitory properties. After cloning and sequencing, nanobody 02A12 was identified as a family member of clone 09D03 (see Tables A-1 and A-2). The sequence diversity of the CDR regions is shown in Tables 4.1A, 4.1B, and 4.1C below. The amino acid sequences of the CDRs of clone 09D03 were used as a reference to compare the CDRs of family members (CDR1 starts at Kabat position 26, CDR1 starts at Kabat position 50, and CDR3 starts at Kabat position 95). Table 4.1A(09D03 CDR1) [Table 4] *Maximum of 6 CDR1 mutations in one clone (SEQ ID NO: 22)

[0203] Table 4.1B(09D03 CDR21) [Table 5] *Maximum of 0 CDR2 mutations in one clone (SEQ ID NO: 36) Table 4.1C(09D03 CDR3) [Table 6] *Maximum of 0 CDR mutations in one clone (SEQ ID NO: 54)

[0204] After cloning and sequencing, clone 09A05 was identified as a family member of clone 03B02 (see Tables A-1 and A-2). The sequence diversity of the CDR regions is shown in Tables 4.1D, 4.1E, and 4.1F below. The amino acid sequences of the CDRs of clone 03B02 were used as a reference to compare the CDRs of family members (CDR1 begins at Kabat position 26, CDR1 begins at Kabat position 50, and CDR3 begins at Kabat position 95). Table 4.1D(03B02 CDR1) [Table 7] *Maximum of 0 CDR1 mutations in one clone (SEQ ID NO: 33)

[0205] Table 4.1E(03B02 CDR2) [Table 8] *Maximum of 3 CDR2 mutations in one clone (SEQ ID NO: 50) Table 4.1F (03B02 CDR3) [Table 9] *Maximum of 2 CDR mutations in one clone (SEQ ID NO: 68)

[0206] After cloning and sequencing, clones 03D02 and 02C10 were identified as family members of clone 03D01 (see Tables A-1 and A-2). The sequence diversity of the CDR regions is shown in Tables 4.1G, 4.1H, and 4.1I below. The amino acid sequences of the CDRs of clone 03D01 were used as a reference for comparing the CDRs of family members (CDR1 starts at Kabat position 26, CDR1 starts at Kabat position 50, and CDR3 starts at Kabat position 95). Table 4.1G(03D01 CDR1) [Table 10] *Maximum of 0 CDR1 mutations in one clone (SEQ ID NO: 28)

[0207] Table 4.1H(03D01 CDR2) [Table 11] *Maximum of 3 CDR1 mutations in one clone (SEQ ID NO: 44) Table 4.1I(03D01 CDR3) [Table 12] *Maximum of 1 CDR mutation (SEQ ID NO: 62) in one clone These results demonstrate that several amino acid changes are tolerated while retaining binding and inhibitory properties. Notably, binding to ADAMTS5 is not a predictor of inhibition of ADAMTS5 activity. For example, clone 3F04 is a strong binder but a weak inhibitor.

[0208] Example 5. Characterization of purified monovalent ADAMTS5 nanobodies Clones selected from the screening described in Example 4 were further characterized. Upon transformation of selected nanobodies into E. coli (TG-1), expression was induced by the addition of 1 mM IPTG and continued for 4 hours at 37°C. After spinning the cell culture, periplasmic extracts were prepared by freeze-thawing the pellet followed by centrifugation. These extracts were then used as starting material for purification via IMAC on a HisTrap FF crude 1 ml column (GE healthcare, Buckinghamshire, United Kingdom), followed by desalting on a Zeba spin column (Pierce, Rockford, IL, USA) to obtain at least 95% purity as assessed by SDS-PAGE.

[0209] 5.1 Evaluation of ADAMTS5-blocking nanobodies in the enzymatic ADAMTS5 assay The ability of the nanobodies to prevent ADAMTS5-mediated cleavage of aggrecan was confirmed in a FRET-based enzyme assay essentially as described in Example 4. Data were analyzed with Graphpad Prism software. Initial velocities were determined from enzyme progress curves (v i ), plot these values ​​as a function of inhibitor concentration to obtain the IC 50 The IC values ​​were calculated. Essentially, the human FRET assay using purified nanobodies confirmed the results described in Example 4 using periplasmic extracts. 50 The IC ranged from 1.8E-09M to 3.7E-08M. In particular, the conventional mAb 12F4 had a higher IC than the monovalent nanobody. 50 was slightly superior at 1.0E-09M, but all nanobodies showed excellent efficacy (data not shown).

[0210] Following the FRET-based assay, an AlphaLISA (Perkin Elmer, Waltham, MA, US)-based human ADAMTS5 assay was performed using a biotinylated 43-mer aggrecan oligopeptide as a substrate. ADAMTS5 cleavage of this substrate releases a biotinylated ARGSV neoepitope product, which can be detected by an anti-neoepitope ("ARGSV") antibody captured on streptavidin-AlphaScreen donor beads and anti-mouse IgG-coated AlphaLISA acceptor beads, resulting in the generation of an emissive AlphaScreen signal upon laser excitation.

[0211] To determine the inhibitory potency of nanobodies, serial dilutions of purified nanobodies (along with positive and negative irrelevant nanobody controls) were incubated with human ADAMTS5 (R&D Systems, Minneapolis, US; cat # 2198-AD) in a 384-well Optiplate (Perkin Elmer, Waltham, MA, US) for 15 min at room temperature. Biotinylated 43-mer aggrecan oligopeptide substrate (Biosyntan, Berlin, Germany) was added, followed by a 3-h incubation at 37°C before the reaction was stopped. The first detection step was performed by adding 5 μl of detection solution 1, which contained an aggrecan antibody against the neoepitope "ARGSV," mouse BC3 mAb (MDBioproducts, Egg, Switzerland), and streptavidin AlphaScreen donor beads (Perkin Elmer). After a 1-h incubation at room temperature, 5 μl of the second detection solution, containing anti-mouse AlphaLISA acceptor beads (Perkin Elmer), was added. The plates were incubated in the dark at room temperature for 2 hours, and measurements were performed by reading the plates on an Envision Multi-label Plate reader (Perkin Elmer, Waltham, MA, US).

[0212] To determine the ability of nanobodies to prevent ADAMTS5-mediated substrate cleavage, the signal reduction was analyzed as a function of nanobody concentration and the IC 50 The value was calculated. The results are summarized in Table 5.1. Table 5.1 ADAMTS5 potency (IC) for nanobodies and reference compounds in the human AlphaLISA enzyme assay 50 ) and percent inhibition (%). [Table 13]

[0213] In the AlphaLISA assay, at least three monovalent nanobodies (2F03, 11B06, and 9D03) had IC values ​​similar to those of the conventional mAb 12F4. 50 The rest show higher values. However, all nanobodies show almost 100% inhibition. Surprisingly, they have IC values ​​at least 100-fold higher than mAb 12F04. 50 Nanobody 3B03, with a value of 0.01, still has 100% inhibition.

[0214] 5.2 Evaluation of ADAMTS5-blocking nanobodies in a bovine explant assay To assess the ability of nanobodies to block cartilage degradation in an ex vivo assay (where the substrate is presented under conditions more similar to physiological conditions compared to biochemical assays), a bovine explant assay was performed. Briefly, bovine cartilage explants (4 mm diameter) were freshly prepared from bovine knee joints and incubated in 96-well plates in the presence of IL-1α to induce cartilage degradation. As a measure of cartilage / aggrecan degradation, GAG release was detected in the supernatant after 5 days of incubation (37°C, 7.5% CO2) via the metachromatic dye 1,9-dimethylmethylene blue (emission at 633 nm). Chondroitin sulfate was included as an assay standard. Efficacy was defined by IL-1α-induced controls without compound (0%) and in the presence of MSC2310852A (100% effect). The results are summarized in Table 5.2.

[0215] Table 5.2 IC of monovalent nanobodies in bovine explant assay 50 value. [Table 14] *Results are only comparable within one assay using the same explants and the same experiment ID.

[0216] 5.3 Epitope binning An SPR-based "sandwich assay" was performed on a Biacore T100 instrument to group ADAMTS5 nanobodies into distinct epitope bins. To this end, each anti-ADAMTS5 nanobody was immobilized on a CM5 sensor chip via amine coupling using EDC and NHS chemistry. After a capture step of 100 nM ADAMTS5, purified nanobodies at 1 μM concentration were injected at a flow rate of 45 μL / min with a surface contact time of 120 s, each in one cycle without regeneration. Curves were processed using the Biacore T100 evaluation software and evaluated with (different bins) or without (same bins) additional binding to the captured ADAMTS5. The nanobodies could be classified into two groups: one large bin ("Bin 1") contains all inhibitory nanobodies with similar or overlapping epitopes, and a second group contains the binding and non-functional nanobody 3F04, which recognizes a different epitope of ADAMTS5 ("Bin 2"). Table 5.3 summarizes the binding epitopes of the tested nanobodies. Table 5.3 Epitope bins of anti-ADAMTS5 nanobodies [Table 15]

[0217] 5.4 Species cross-reactivity Species cross-reactivity was first assessed by SPR-based off-rate analysis on a Biacore T100 instrument. Nanobodies were tested for binding to human, cynomolgus monkey (cyno), guinea pig, mouse, and bovine ADAMTS5. For this purpose, recombinant ADAMTS5 was immobilized on a CM5 chip by amine coupling using EDC and NHS. Purified nanobodies were injected at a concentration of 100 nM for 2 min and allowed to dissociate for 15 min at a flow rate of 45 μl / min. The off-rates of individual nanobodies were determined using BIA evaluation software by fitting a 1:1 interaction model (Langmuir model) to the individual dissociation curves. As a reference, the off-rate for human ADAMTS5 was determined in each experiment. The results are summarized in Table 5.4A.

[0218] Table 5.4A Summary of species cross-reactivity data for monovalent anti-ADAMTS5 nanobodies [Table 16]

[0219] For each nanobody tested, similar off-rates were observed in all species tested. Additionally, the cross-reactivity of cynomolgus monkey and guinea pig ADAMTS5 was also investigated by potency measurements using AlphaLISA essentially as described in Example 5.1, but using biotinylated cynomolgus monkey 43-aggrecan oligopeptide substrate and biotinylated guinea pig 43-aggrecan oligopeptide substrate. To determine the ability of nanobodies to prevent ADAMTS5-mediated substrate cleavage, the signal reduction was analyzed as a function of nanobody concentration, and the IC 50 The value was calculated. The efficacies obtained are summarized in Table 5.4B. Table 5.4B Summary of species cross-reactivity data for monovalent anti-ADAMTS5 nanobodies [Table 17]

[0220] All nanobodies and control compounds blocked human and cynomolgus monkey ADAMTS5 with similar potency. In guinea pig AlphaLISA, the most potent nanobodies showed slightly lower potency than in human AlphaLISA, likely a result of reduced assay sensitivity due to increased enzyme concentration. The cross-reactivity of the nanobodies 2A12, 2F03, 093, and 049 was assessed using a Biacore T100 with SPR-based affinity measurements. For reference, the affinity for human ADAMTS5 was also determined. For this purpose, rat and human ADAMTS5 were immobilized on a CM5 chip by amine coupling using EDC and NHS chemistry. Purified nanobodies were injected at different concentrations (between 1 and 1000 nM) for 2 min and allowed to dissociate for 25 min at a flow rate of 45 μl / min. Kinetic constants were calculated from the sensorgrams using BIAEvaluation software (1:1 interaction).

[0221] As shown in Table 5.4C, similar affinities for human and rat ADAMTS5 were obtained for all nanobodies tested. The affinity of the half-life-extended nanobody 069 (see below) was similar to that of its monovalent counterpart (nanobody 049), indicating that the addition of ALB11 to the C-terminus does not affect affinity. The affinity of the half-life-extended biparatopic nanobody 130 was higher in both species (15-fold higher for human ADAMTS5 and 10-fold higher for rat ADAMTS5) compared to the affinity of the half-life-extended nanobody 069 (Table 5.4C). Table 5.4C Summary of affinities for human ADAMTS5 and rat ADAMTS5 [Table 18]

[0222] 5.5 Inhibition of MMP1 and MMP14 activity To confirm the selectivity of the nanobodies for ADAMTS5, inhibition of MMP1 and MMP14 enzymatic activity was assessed using a FRET-based assay with each enzyme. Briefly, activated human MMP1 or MMP14 was incubated with 10 μl of a dilution series of nanobodies for 30 minutes at room temperature. After incubation, 20 μl of 5 μM or 2.5 μM fluorescent peptide substrate (Mca-PLGL-Dpa-AR-NH2 fluorescent MMP substrate (R&D Systems cat #ES001)) was added. The ability of the nanobodies to prevent MMP1- and MMP14-mediated cleavage was monitored every minute for 2 hours at 37°C using a Tecan Infinite M1000 plate reader. The titration curve obtained is shown in FIG. The natural inhibitors TIMP2 and TIMP3 inhibited MMP1 and MMP14 activity, whereas none of the nanobodies tested showed any inhibition.

[0223] 5.6 Inhibition of human ADAMTS4 activity To assess inhibition of human ADAMTS4, an assay similar to the AlphaLISA for human ADAMTS5 was performed essentially as described in Example 5.1, but using human ADAMTS4 (R&D Systems, Minneapolis, US; cat # 4307-AD). To determine the ability of nanobodies to prevent human ADAMTS4-mediated substrate cleavage, the signal reduction was analyzed as a function of nanobody concentration, and the IC 50 The value was calculated. The results are shown in Figure 2. The small molecule MSC2310852A inhibited human ADAMTS4 activity, whereas none of the tested nanobodies or mAb 12F4 showed inhibitory activity (see Figure 2). The monoclonal antibody 12F4 (H4L0) is described in WO 2011 / 002968 as being selective over ADAMTS4.

[0224] Example 6: Formatting a nanobody 6.1 Knockout of N-glycosylation motifs The N-glycosylation motif present in nanobody 11B06 (position N52) and nanobody 3F04 (position N110f) was knocked out prior to formatting. This site was randomized using an NNK codon library. Because these positions are in the CDRs, mutations in the motif could affect binding properties. Therefore, the library was screened for possible substitutions that do not affect binding properties by SPR-based off-rate analysis of human ADAMTS5 using a Biacore T100 instrument, essentially as described in Example 5.4. Surprisingly, the substitution of amino acid N52 with serine in nanobody 11B06 and amino acid N100 in nanobody 3F04 F Substitution of with glutamine did not affect binding (data not shown). Therefore, nanobodies 049 (= 11B06 (N52S)) and 093 (= 3F04 (N100)) were selected as building blocks for further formatting. F Q)) were used in place of nanobodies 11B06 and 3F04, respectively (see Table A-1).

[0225] 6.2 Generation of formatted nanobodies To generate half-life-extending nanobody constructs that block human ADAMTS5 aggrecanase activity, nanobodies 2A12, 2D07, 2F03, 049, 9D03, and 3B02 were fused to the anti-human serum albumin (HSA)-nanobody ALB11 (see Table 6.3). Additionally, ALB11 half-life-extending constructs were generated from the combination of two nanobodies from different binding epitopes (a bin 1 member and a bin 2 member). The formatted nanobody constructs were expressed in Pichia pastoris, secreted into the culture medium, and affinity-purified with Poros MabCaptureA Protein A beads (Applied Biosystems, Bleiswijk, Netherlands, cat. # 4374729). The integrity of the nanobody constructs was confirmed.

[0226] 6.3 Efficacy in AlphaLISA in the Absence and Presence of HSA The formatted nanobody constructs were tested in AlphaLISA as described in Example 5.1. Furthermore, the effect of HSA binding on nanobody potency was investigated by performing experiments in the presence of excess HSA (final concentration 4.2 μM). IC of all half-life extended nanobody constructs 50 The values ​​are shown in Table 6.3. Table 6.3. AlphaLISA-determined potency of formatted nanobody constructs (+ / - HSA) [Table 19] ND=Not decided;093 * =3F04(N100fQ);049 2* =11B06(N52S) The results show that neither the ALB11 format nor HSA conjugation affects the potency of the nanobody constructs.

[0227] 6.4 Binding to HSA The affinity of the ALB11 nanobody for HSA was determined via SPR on a Biacore T100. For this purpose, HSA was immobilized on a CM5 chip by amine coupling and treated essentially as described in Example 5.3. As a reference, the affinity of monovalent ALB11 was also measured, which was 3.2 nM. The results are summarized in Table 6.4. Table 6.4 Affinity of HLE nanobody constructs to HSA [Table 20] ND=Not determined; * FNb = functional nanobody; 2* =11B06(N52S);093 3* =3F04(N100fQ) All half-life extended ADAMTS5 nanobody constructs had similar affinities (see Table 6.4), which were lower than the affinity of monovalent ALB11 for HSA.

[0228] 6.5 KinExA affinity determination of human ADAMTS5 The affinities of the formatted nanobody constructs 069 (049-35GS-ALB11) and 130 (049-35GS-093-ALB11) (see also Table 6.5) were determined in solution by the equilibrium exclusion method on a KinExA3000 instrument (Sapidyne, Boise, USA). For this purpose, human ADAMTS5 was coupled to PMMA beads according to the manufacturer's instructions, as detailed in Darling and Brault (2004 Assay Drug Dev Technol. 2:647-57). A fixed concentration of nanobody construct (50 pM nanobody construct 069 or 5 pM nanobody construct 130) was added to a series of human ADAMTS5 concentrations ranging from 2 nM to 0.2 pM and incubated at room temperature for 16 h to reach equilibrium. The mixture was then injected via the KinExA autosampler onto a column packed with PMMA beads conjugated to human ADAMTS5. Free nanobody constructs on the beads were captured and detected with Alexa647-labeled HSA. The percentage of free nanobody constructs was plotted as a function of titrated human ADAMTS5 and fitted using KinExA Pro software v3.2.6.

[0229] The results are shown in Table 6.5. Table 6.5 Affinity of two nanobody constructs for binding to human ADAMTS5 determined via KinExA [Table 21] 049 * =11B06(N52S);093 2* =3F04(N100 f Q) Approximately 20-fold higher affinity is observed with nanobody construct 130 compared to nanobody construct 069. These data confirm the strong binding of nanobody construct 130 to human ADAMTS5.

[0230] Example 7: Sequence optimization of nanobodies (SO) Exemplary nanobodies include nanobody 2F03, nanobody 049 (11B06(N52S)) and nanobody 093 (3F04(N100 f Q)) were subjected to a sequence optimization process in which the parent nanobody sequence is mutated, covering nanobody humanization (i) and knockout post-translational modifications (ii) as well as epitopes for potential pre-existing antibodies (iii). Nanobody ALB11 was also mutated to knockout epitopes for potential pre-existing antibodies. (i) Mutation of the parent Nanobody sequence for humanization purposes to generate a Nanobody sequence more identical to the human IGHV3-IGHJ germline consensus sequence. Specific amino acids in framework regions that differ between the Nanobody and the human IGHV3-IGHJ germline consensus (excluding so-called hallmark residues) are altered to their human counterparts while ensuring that the protein's structure, activity, and stability remain intact. Some hallmark residues are not mutated because they are known to be important for Nanobody stability, activity, and affinity.

[0231] (ii) Amino acids present in the CDRs and with experimental evidence of being sensitive to post-translational modifications (PTMs) are altered such that the PTM sites are inactivated but the protein structure, activity, and stability remain maintained. (iii) The sequence of the nanobody is optimized without affecting the protein structure, activity, and stability to minimize binding of any pre-existing naturally occurring antibodies and reduce the likelihood of causing an immunogenic response following treatment. To generate the sequence-optimized and formatted nanobody construct 581 and nanobody construct 579, the respective building blocks were connected via a 35GS linker. This resulted in nanobody construct 581 (2F3 SO -35GS linker-Alb11) and nanobody construct 579 (2F3 SO -35GS Linker-093 SO The resulting nanobody constructs (-35GS linker-Alb11) were synthesized as described in Table A-1. The constructs were produced as untagged proteins in Pichia Pastoris, purified by Protein A affinity chromatography, and then desalted. The integrity of the nanobody constructs was confirmed.

[0232] 7.1 Affinity for human ADAMTS5 Nanobody construct 581(2F3 SO The affinity of the nanobody construct 581 (-35GS linker-Alb11) was determined using KinExA as described in Example 6.5, with minor adaptations to the protocol. A fixed concentration of 20 pM of nanobody construct 581 was incubated with a dilution series of human ADAMTS5 (2.2-fold serial dilutions ranging from 20 nM to 0.32 pM and a blank without ADAMTS5). To test for albumin interference, HSA was added to this preincubation in a selection experiment using human ADAMTS5 at a concentration 100-fold higher than the KD of nanobody construct 581. The mixture was incubated and allowed to reach equilibrium for 24 h before injection into a column packed with PMMA beads conjugated to human ADAMTS5 via the autosampler of KinExA. The captured nanobody construct 581 was detected using an AF647-labeled anti-nanobody tool (produced by Ablynx) that recognizes nanobody construct 581.

[0233] The results are shown in Table 7.1. Table 7.1 Affinity of nanobody construct 581 to human ADAMTS5 with and without HSA [Table 22] The results show that the affinity of nanobody construct 581 for human ADAMTS5 is 3.65 pM without HSA and 4.84 pM with HSA.

[0234] 7.2 Affinity for serum albumin Nanobody construct 581(2F3 SO The affinity of the ALB11 building block in (-35GS linker-Alb11) for binding to human, cynomolgus monkey, guinea pig, mouse, and rat serum albumin (SA) was determined using SPR as described in Example 5.3. The results are shown in Table 7.2. Table 7.2 Affinity (K ) of nanobody construct 581 for binding to human, cynomolgus monkey, guinea pig, mouse, and rat serum albumin. D , nM) [Table 23] *Off rate is outside the detection limit: >5.0E-01 (1 / s)

[0235] 7.3 Pre-Ab binding Nanobody construct 581(2F3 SO -35GS linker-Alb11) and nanobody construct 579 (2F3 SO -35GS Linker-093 SOThe -35GS linker-Alb11 was screened for pre-existing antibody (pre-Ab) binding from three sets of related donor serum samples (a sample from a healthy subject, an OA sample, and a biased set of samples with known residual pre-Ab bound to other nanobodies) using SPR technology on a ProteOn XPR36 instrument. Blood samples were analyzed for binding to the anti-ADAMTS5 nanobody construct captured on HSA. Pre-existing antibody binding levels were determined after double-referencing the sensorgrams, with a report point set to 125 seconds (5 seconds after the end of association). Analysis was performed using ProteOn manager 3.1 (Bio-Rad Laboratories, Inc.). The results are shown in Figure 3. Both nanobody constructs had low residual pre-Ab binding levels, with nanobody construct 581 clearly showing the least residual pre-Ab binding (see Figure 3).

[0236] 7.4 Function in Human AlphaLISA AlphaLISA was run on nanobody construct 581 (2F3 SO The procedure was carried out essentially as described in Example 5.1 using human ADAMTS5 with -35GS linker-Alb11). The results are shown in Table 7.4. Table 7.4 Potency (pM) of nanobody construct 581 determined by AlphaLISA. [Table 24] The results show that the potency of nanobody construct 581 is 188 pM.

[0237] 7.5 Immunogenicity Profiling in Dendritic Cell T Cell Assays Relative immunogenicity was determined in a dendritic cell T cell proliferation assay. Essentially, the nanobodies were tested against cell samples from 50 healthy donors containing the most abundant HLA class II alleles, as these represent the majority of the world's population. The immune response was assessed using T cell proliferation as a surrogate marker of anti-drug antibody formation. Keyhole limpet hemocyanin (KLH) was used as a positive control. The positive control KLH produced positive responses in all 50 donors. All donors tested were immunocompetent with nanobody construct 581 (2F3 SO -35GS linker-Alb11). SO -35GS Linker-093 SO The -35GS linker-Alb11) gave positive responses in 3 donors (6%). In the blank condition (medium only), 2 out of 50 donors (4%) gave positive responses. These results were used to set an overall response threshold of greater than 3 out of 50 donors, which corresponds to an overall response threshold of greater than 6%. The overall immunogenic potential of the tested nanobodies was considered low because the rate of significant positive responses was below 6% (see Table 7.5).

[0238] Table 7.5 Immunogenicity profiling results measured by DC-T cell assay [Table 25]

[0239] 7.6 Binding to human ADAMTS1 Binding to human ADAMTS1 was tested by binding ELISA (see Figure 4A). Briefly, 18.5 nM of human ADAMTS1 (R&D Systems, Minneapolis, US; cat # 2197-AD) and human ADAMTS5 (R&D Systems, Minneapolis, US; cat # 2198-AD) were coated onto 384-well MaxiSorp plates (Nunc, Wiesbaden, Germany). The wells were blocked with casein solution (1%) and the nanobody construct 581 (2F3 SO A dilution series of anti-ADAMTS1 mAb (-35GS linker-Alb11) and anti-human ADAMTS1 mAb (R&D Systems, Minneapolis, US; cat. # MAB2197) was tested for binding to both coated proteins. Detection was performed with an HRP-conjugated anti-nanobody tool recognizing the nanobody construct 581 (produced by Ablynx) and an HRP-conjugated anti-mouse antibody (Abcam, Cambridge, UK), respectively, followed by enzymatic reaction in the presence of the substrate esTMB (3,3',5,5'-tetramentylbenzidine) (SDT, Brussels, Belgium), and the OD was measured. Dose-response curves were observed for the nanobody construct 581 for human ADAMTS5h and anti-human ADAMTS1 mAb for human ADAMTS1, showing that the nanobody construct 581 does not bind to human ADAMTS1.

[0240] 7.7 Binding to human ADAMTS4 and ADAMTS15 Binding to human ADAMTS4 and ADAMTS15 was tested by binding ELISA (see Figure 4B). Briefly, 1 μg / mL of human ADAMTS4 (R&D Systems, Minneapolis, US; cat # 4307-AD) or human ADAMTS15 (R&D Systems, Minneapolis, US; cat # 5149-AD) was coated onto 96-well Maxisorp ELISA plates (Nunc, Wiesbaden, Germany) overnight. After blocking the plates with SuperBlock T20 (PBS) (Thermo Scientific Pierce; cat # 37516), nanobody construct 581 ("C011400581"), or a positive control anti-ADAMTS4 mAb (R&D Systems, Minneapolis, US; cat # MAB4307), or a positive control anti-ADAMTS15 mAb (R&D Systems, Minneapolis, US; cat # MAB5149) was applied to the coated plates starting at 1 μM and incubated for 1 h at room temperature. Bound C011400581 nanobody was detected with a biotinylated anti-nanobody tool that recognizes nanobody construct 581 (generated by Ablynx), while the positive control mAb was detected with a biotinylated goat anti-mouse pAb (Jackson Immuno Research; cat # 115-065-062). Both streptavidin-HRP assays were used as secondary detection tools (Thermo Scientific Pierce; cat # 21126). Plates were then visualized by adding sTMB (SDT, Brussels, Belgium). All dilutions and detection tools were prepared in assay diluent (=PBS + 10% Superblock + 0.05% Tween 20). The color reaction was stopped after 2.5 minutes by adding 1 M HCl. Optical density was measured at 450 nm with a reference wavelength of 620 nm.

[0241] 7.8 Species Cross-Reactivity by KinExA Affinity Determination Nanobody construct 581(2F3 SOThe affinity of the -35GS linker-Alb11) for ADAMTS5 in several species was determined using KinExA as described in Example 7.1. For all species, PMMA beads conjugated to human ADAMTS5 were prepared and used to capture free nanobody construct 581 as described above. The captured nanobody construct 581 was detected using an AF647-labeled anti-nanobody tool that recognizes nanobody construct 581 (produced by Ablynx). The results are shown in Table 7.7. Table 7.7 Affinity of nanobody construct 581 to ADAMTS5 in cynomolgus monkey, rat, mouse, and bovine [Table 26]

[0242] Example 8: Potency in human explant assays Nanobody construct 581(2F3 SO The -35GS linker-Alb11) was evaluated for its ability to block cartilage degradation in an ex vivo assay essentially as described in Example 5.2, but this time in a human explant assay. Briefly, human cartilage explants (4 mm diameter) were freshly prepared from human knee joints and incubated in 96-well plates in the presence of 10 ng / ml IL-1α to induce cartilage degradation. As a measure of cartilage / aggrecan degradation, GAG release was detected in the supernatant after 7 days of incubation (37°C, 7.5% CO2) via the metachromatic dye 1,9 dimethylmethylene blue (emission at 633 nm). Chondroitin sulfate was included as an assay standard. Efficacy was defined by IL-1β and OSM induction controls in the presence of SC2310852A without compound. CRB0017 is an anti-ADAMTS5 mAb used in a phase I clinical trial (Rottapharm).

[0243] The results are summarized in Figure 5 and Table 8. Table 8 [Table 27] The nanobodies show high potency and 100% inhibition of cartilage degradation (GAG loss) in the human explant assay. As shown in Table 8, the nanobodies are at least 10-fold superior to CRB0017 in the bovine explant assay.

[0244] Example 9 Regulation of C2M, C3M and exAGNX1 in a bovine co-culture model We investigated the effect of nanobody construct 581 (labeled "581" in the figure) on the release and content of GAGs, C2M (a marker of collagen II degradation), C3M (a marker of collagen III degradation), and exAGNx1 (a marker of aggrecan degradation characterizing the neoepitope TEGE) in a bovine coculture system. In this coculture system, cartilage explants from bovine knees were incubated with synovial membrane from the same animal for 28 days. The highest GAG release was detected after 7 days of culture (Figure 6A). Nanobody construct 581 was tested at three concentrations: 1 nM, 10 nM, and 100 nM. Incubation of cartilage explants with synovial membrane increased GAG release. Nanobody construct 581 inhibited GAG release (Figure 6B).

[0245] Along with GAG release, analysis of the remaining GAG content of cartilage explants (after papain digestion) revealed a decrease in GAG content after co-incubation of explants with synovium when compared to explants alone ( Figure 6C ). In parallel with the analysis of GAG release, we measured the release of exAGNx1 as a specific marker of aggrecan degradation. Area under the curve analysis (AUC) showed a strong induction of exAGNx1 release when cartilage explants and synovium were incubated together. Furthermore, the nanobody construct 581 inhibited exAGNx1 release in a concentration-dependent manner, with complete inhibition at 100 nM (Figure 7).

[0246] The effect of nanobody construct 581 on the collagen network of cartilage in a coculture system was measured using C2M as a marker of collagen II degradation. In contrast to markers of aggrecan network degradation (GAG and exAGNx1), C2M began to increase between days 14 and 21. Incubation of nanobody construct 581 in the coculture system inhibited the release of C2M (Figure 8). As an additional marker of synovial inflammation, C3M (i.e., a marker of type III collagen degradation) was determined. Time course analysis of C3M release showed that C3M began to increase after 14 days and peaked around day 21. Treatment with nanobody construct 581 inhibited C3M at all three concentrations (Figure 9).

[0247] Example 10. Inhibition of Aggrecanase Activity in NHPs Nanobody construct 581(2F3 SO The ability of the nanobody construct 581 (-35GS linker-Alb11; "C011400581") to inhibit aggrecanase activity in vivo was evaluated in the non-human primate (NHP) model, cynomolgus monkeys. Briefly, groups of three male and three female cynomolgus monkeys were given subcutaneous (sc) administration once weekly for four weeks at dose levels of 6, 30, or 150 mg / kJ. A combination control group was treated with vehicle: 20 mM histidine, 8% sucrose, 0.01% Tween 20, pH 6.0.

[0248] Inhibition of aggrecanase activity was measured by determining the levels of aggrecan degradation fragments, characterized by the presence of the neoepitope ARGS, in serum samples collected at several time points. All animals treated with nanobody construct 581 showed a similar profile; ARGS levels decreased with the first dose, reaching very low levels around the lower limit of the assay's measuring range (LLMR) (0.08 nM) between 48 and 120 hours. Thereafter, ARGS levels showed a sustained maximal decrease around the LLMR and did not return to baseline by the end of the 4-week study. A summary of the results is shown in Figure 10. In conclusion, the ADAMTS5-specific nanobody engages its target after systemic administration and potently modulates ARGS neoepitope levels in vivo at all dose levels tested. Furthermore, in contrast to prior art mAbs, no test-item-related pathological arrhythmias were observed. Furthermore, there was no evidence of test-related ST-segment elevation in male and female monkeys treated with the nanobody at any of the dose levels tested.

[0249] Example 11: Inhibition of cartilage degeneration To further evaluate the ability of nanobodies and nanobody constructs to inhibit cartilage degeneration in vivo, a murine DMM (medial meniscus destabilization) model was used. Briefly, the medial meniscus was surgically destabilized. The exemplary nanobody construct 581 (2F3 SO Various concentrations of α-35GS linker-Alb11 were administered subcutaneously 3 days before (prophylactic) or 3 days after (therapeutic) induction of DMM. After 8 weeks of treatment, the animals were sacrificed and the knees were removed. The knees were embedded in paraffin blocks, sectioned, and stained with toluidine blue. The sections were then scored for several parameters, including total medial cartilage degeneration. The results are shown in Figure 11. The results show that both prophylactic and therapeutic treatment (sc) with nanobodies in the DMM mouse model has a structural benefit of up to 50%.

[0250] Example 12 Symptomatic benefit in a surgical OA model in rats To evaluate the ability of nanobodies to establish symptomatic benefit, a surgical OA model in rats was used. Briefly, rats were treated with ACLt and tMx surgery to induce OA on day 0. In the ACLt and tMx surgical model (anterior cruciate ligament resection augmented with medial meniscectomy), nanobody construct 581 was administered subcutaneously every other day starting on day 3. Weekly symptomatic benefit was determined by gait analysis (on a CatWalk) and reduction in joint diameter. The results are shown in Figure 12.

[0251] Osteoarthritis was induced in adult male rats (Lister Hooded; mean body weight 346 ± 20 g) by anterior cruciate ligament resection (ACLT) plus medial meniscus resection (tMx) in the right knee joint (day 0). Treatment with vehicle or test item (sc) began on postoperative day 3 and continued every other day until day 42. A healthy control group did not undergo surgical intervention but received sc vehicle at the same time points as the treatment groups. (A) Gait impairment over time was calculated as "% benefit vs. vehicle." Mean for "healthy + placebo" group = 100% benefit. Mean for "ACLT tMx + vehicle" group = 0% benefit. (B) Mean "benefit vs. vehicle" at all time points examined after treatment initiation. Shown is the mean ± SEM of 14–15 rats / group. * = p<0.05, calculated using one-way ANOVA and Dunnett's. Early treatment with nanobodies resulted in dose-dependent, significant and important symptomatic benefits during ACLT+tMx-induced OA.

[0252] Example 13 Toxicity study in cynomolgus monkeys A 4-week subchronic toxicity study was conducted in cynomolgus monkeys to obtain information on the systemic toxicity, local tolerability and safety pharmacology of the nanobodies. SO -35GS linker-Alb11) was administered subcutaneously to groups of three male and three female cynomolgus monkeys (Macaca fascicularis) at doses of 6, 30, or 150 mg / kg once weekly for four weeks in the dorsal region. Concurrent control groups were treated with vehicle, 20 mM histidine, 8% sucrose, 0.01% Tween 20, pH 6.0. No signs of local intolerance related to the test article were observed at any of the dose levels tested. No test article-related effects were observed at any dose level on behavior, body weight, food consumption, hematological and biochemical parameters, lymphocyte typing, CRP levels, urinalysis parameters, ophthalmologic and auditory function, and organ weights in all animals (data not shown). Gross and histopathological examination at necropsy also did not reveal any local or systemic organ changes related to treatment with the test article in any of the animals examined at any of the dose levels tested.

[0253] Because mAb 12F4 has been reported to cause focal intracardiac hemorrhage, a dose-dependent increase in blood pressure without evidence of reversibility, and cardiac conductance abnormalities (ST-segment elevation and ventricular arrhythmias) (Larkin, et al., "The highs and lows of translational drug development: Antibody-mediated inhibition of ADAMTS-5 for osteoarthritis disease modification," OARSI conference 2014: Paris; Renninger et al., "Identification of Altered Cardiovascular Function Produced by a Novel Biologic Compound in a Stand Alone Safety Pharmacology Primate Study," in SPS meeting, 2013), extensive noninvasive telemetry was performed on animals using the EMKA system. No test article-related effects were observed in telemetry parameters (using the non-invasive EMKA system) in any animal at any dose level. In particular, no pathological arrhythmias were observed and no evidence of test article-related ST-segment elevation was found in male or female monkeys treated with the nanobody at any of the dose levels tested (data not shown). These studies confirm that ADAMTS5-specific nanobodies can be considered safe.

[0254] Example 14. DMOAD study of an in vivo rat MMT model To demonstrate the in vivo efficacy of the ADAMTS5 inhibitor fused to the CAP binder of the present invention, a surgically induced medial meniscus tear (MMT) model was used in rats. Briefly, an anti-ADAMTS5 nanobody was conjugated to a CAP binder (designated as nanobody construct C010100954 or nanobody construct 954). One knee of the rat was operated on to induce OA-like symptoms. Treatment was initiated by intravenous injection on day 3 after surgery. Histopathology was performed on day 42 after surgery. Intermediate and final serum samples were collected for exploratory biomarker analysis. The width of medial and total parenchymal cartilage degeneration, as well as the rate of reduction of cartilage degeneration, were determined. 20 animals were used per group.

[0255] A subchondral defect of the medial tibia is shown in Figure 13 . The results show that cartilage width was significantly reduced by the ADAMTS5-CAP construct compared to vehicle after 42 days. These results further suggest the following: (a) The CAP moiety does not adversely affect the activity of anti-ADAMTS5 nanobodies; (b) the CAP portion allows retention of the anti-ADAMTS5 nanobody; and (c) Anti-ADAMTS5 nanobodies have a positive effect on cartilage width even when bound to the CAP moiety.

[0256] Example 15 Methods: Bovine cartilage explants (BEX) from four animals, as well as human cartilage explants from eight surgically replaced knee joints (HEX) and one healthy human knee joint (hHEX), were cultured in media alone (w / o), with pro-inflammatory cytokines (oncostatin M [10 ng / mL] + TNFα [20 ng / mL] (O+T)), or with an exemplary nanobody construct 581 (2F3 SOCartilage and synovium from bovine (bCC) and four displaced osteoarthritic human knee joints (hCC) were cultured for up to 21 days in the presence of α-35GS linker-Alb11 [1 μM–1 nM]. Ex vivo cartilage and synovium from bovine (bCC) and four displaced osteoarthritic human knee joints (hCC) were cultured for up to 28 days in medium alone (w / o), O+T, or O+T plus the exemplary nanobody construct 581 (2F3 SO The cartilage was co-cultured with α-35GS linker-Alb11 (1 μM-0.6 nM). Cartilage was cut into equal-sized explants using a biopsy puncher. Synovial membrane was cut into equal-sized explants (30 mg [±3 mg]) using a scalpel. Metabolic activity of the explants was assessed by Alamar Blue. Cartilage turnover was assessed by measuring well-characterized biomarkers of degradation (huARGS, exAGNxI, C2M) and formation (ProC2) in conditioned medium using an enzyme-linked immunosorbent assay (ELISA). ProC2 and C2M are metabolites of type II collagen formation and degradation, respectively, and exAGNxI and huARGS are metabolites of ADAMTS5-degraded aggrecan. Means and standard errors of the mean (SEM) are reported. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Dunn's multiple comparison test or two-way ANOVA with Dunn's multiple comparison test assuming normal distribution.

[0257] Results: The metabolic activity of BEX, HEX, and bCC remained stable throughout the culture period, while that of hCC and hHEX was significantly reduced from day 14 in the O / T treatment condition compared to w / o. In O+T-stimulated cultures, ADAMTS5-degraded aggrecan metabolites peaked during the first week of culture, except for hHEX, where huARGS and exAGNxI increased slightly later. C2M, which is responsible for type II collagen degradation by O+T, peaked after 19 days. Pro-C2, which is responsible for type II collagen formation, remained relatively stable throughout the culture compared to w / o controls.

[0258] Exemplary nanobody construct 581 (2F3 SOTreatment with -35GS linker-Alb11 in combination with O+T dose-dependently reduced huARGS in BEX (highest dose: 8% of O+T), HEX (highest dose: 40% of O+T), bCC (highest dose: 10% of O+T), hCC (highest dose: 40% of O+T), and hHEX (highest dose: 24% of O+T) (Figure 14). SO -35GS linker-Alb11) IC 50 ranged from 300 nM for BEX to <15 nM for HEX, hHEX, bCC, and hCC (Figure 14). Exemplary nanobody construct 581 (2F3 SO The effect of the -35GS linker-Alb11) on exAGNxI was similar to that of huARGS in the cultures tested. SO The -35GS linker-Alb11) also significantly and dose-dependently reduced C2M (a marker of type II collagen degradation), although the effect was less than that of the aggrecan degradation marker. SO The -35GS linker-Alb11) showed no effect on the type II collagen-forming metabolite Pro-C2 under any of the conditions tested.

[0259] Conclusion: Here, the exemplary nanobody construct 581 (2F3 SO We demonstrated that -35GS linker-Alb11) has chondroprotective effects through dose-dependent inhibition of ADAMTS5-mediated aggrecan degradation and MMP-mediated type II collagen degradation in the proinflammatory state of ex vivo cultures of bovine and human cartilage and in co-cultures of cartilage and synovium. SO -35GS linker-Alb (SEQ ID NO: 129) is one of the preferred embodiments of the present invention.

[0260] Example 16: The present nanobody constructs are superior to prior art nanobodies WO 2008 / 074840 describes various ADAMTS5 nanobodies. In this experiment, 26 nanobodies described in WO 2008 / 074840 and the exemplary nanobody 2F3 SO (2F3 * ) and the nanobodies of the invention represented by All constructs were tested in the AlphaLISA assay as described in Example 5.1. Nanobody 2F3 SO (2F3 * ) is an ADAMTS5-binding monovalent building block of C011400581 (SEQ ID NO: 129). As a negative control, the irrelevant nanobody IRR00028 was used.

[0261] First, the activity of prior art nanobodies was determined by AlphaLISA and the results are shown in Table 16.1. Table 16.1. Activity of prior art nanobodies determined by AlphaLISA [Table 28]

[0262] The results show that only seven prior art nanobodies have blocking activity, the remaining prior art nanobodies either had no activity or had enhanced activity. In the second part of this comparative experiment, the blocking nanobody was compared with the exemplary monovalent nanobody 2F3 SO (2F3 * ) were compared. IC of nanobody constructs 50 The values ​​are shown in Table 16.2. Exemplary monovalent nanobody 2F3 SO (2F3 * ) are also shown for ease of comparison.

[0263] Table 16.2 Nanobody 2F3 determined by AlphaLISA SO (2F3 * ) Efficacy of prior art nanobody constructs against [Table 29] It can be concluded that the nanobody constructs of the present invention are superior to prior art ADAMTS5 nanobodies.

[0264] Table A-1: ​​Names and short descriptions ("ID"), sequence numbers ("SEQ") and amino acid sequences of monovalent and multivalent anti-ADAMTS5 nanobodies [Table 30-1] [Table 30-2] [Table 30-3] *SO (array optimization) version

[0265] Table A-2: CDR and framework sequences and preferred combinations provided in formula I, i.e., FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (the term "ID" below refers to the given SEQ ID NO.) [Table 31-1] [Table 31-2]

[0266] Table B: Various amino acid sequences: Name and short description ("ID"), sequence number ("SEQ") and amino acid sequence ("Seq") [Table 32-1] [Table 32-2]

[0267] Table C: Various linker sequences ("ID" refers to SEQ ID NO as used herein) [Table 33]

[0268] Table D: Serum albumin binding ISVD sequences, including CDR sequences ("ID" refers to SEQ ID NO as used herein) [Table 34] [1] A polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that binds to an A disintegrin and metalloproteinase with thrombospondin motif (ADAMTS). [2] The polypeptide according to Item 1, wherein the ADAMTS is selected from the group consisting of ADAMTS1 to ADAMTS19, preferably ADAMTS5, ADAMTS4, ADAMTS1, ADAMTS8, ADAMTS9, ADAMTS15, and ADAMTS20, and most preferably ADAMTS5. [3] The polypeptide according to item 2, wherein the ISVD that binds to ADAMTS, preferably ADAMTS5, does not bind to ADAMTS4, MMP1, or MMP14. [4] The polypeptide according to Item 3, wherein the ISVD that binds to ADAMTS5 comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein (i) CDR1 is selected from the group consisting of SEQ ID NOs: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32, and 34; and amino acid sequences having one, two, or three amino acid differences from SEQ ID NOs: 21, 35, 20, 22, 25, 33, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32, and 34; and (ii) CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49, and 5 and an amino acid sequence having one, two, or three amino acid differences from SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49, and 52; and (iii) CDR3 is selected from the group consisting of SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67, and 70; and an amino acid sequence having one, two, three, or four amino acid differences from SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67, and 70. [5] The polypeptide according to Item 3, wherein the ISVD that specifically binds to ADAMTS5 comprises three complementarity-determining regions (CDR1 to CDR3), respectively, wherein (i) CDR1 is selected from the group consisting of: (a) SEQ ID NO: 22; and (b) an amino acid sequence having 1, 2, 3, 4, 5, or 6 amino acid differences from SEQ ID NO: 22, wherein the amino acid differences are defined as follows: at position 2, S is changed to R; and / or at position 3, A is changed to T; and / or at position 4, V is changed to F; and / or at position 6, V is changed to S; and / or at position 7, N is changed to Y; and / or at position 10, A is changed to G; (ii) CDR2 is SEQ ID NO: 36; and (iii) CDR3 is SEQ ID NO: 54. [6] The polypeptide according to Item 3, wherein the ISVD that specifically binds to ADAMTS5 comprises three complementarity-determining regions (CDR1 to CDR3), wherein (i) CDR1 is SEQ ID NO: 33; (ii) CDR2 is selected from the group consisting of: (c) SEQ ID NO: 50; and (d) an amino acid sequence having one, two, or three amino acid differences from SEQ ID NO: 50, wherein the amino acid differences are defined as follows: at position 8, M is changed to I; and / or at position 9, P is changed to T; and / or at position 10, Y is changed to F; and (iii) CDR3 is selected from the group consisting of: (e) SEQ ID NO: 68; and (f) an amino acid sequence having one or two amino acid differences from SEQ ID NO: 68, wherein the amino acid differences are defined as follows: at position 5, F is changed to L; and / or at position 11, D is changed to E; and / or - at position 8, T is changed to I; and / or - at position 9, T is changed to L; and (iii) CDR3 is selected from the group consisting of: (e) SEQ ID NO: 62; and (f) an amino acid sequence having one or two amino acid differences from SEQ ID NO: 62, wherein the amino acid difference is defined as follows: - at position 1, G is changed to S; and / or - at position 14, D is changed to E; [8] The polypeptide of any one of Aspects 1 to 7, wherein CDR1 is selected from the group consisting of SEQ ID NOs: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32, and 34; CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49, and 52; and CDR3 is selected from the group consisting of SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67, and 70. [9] The polypeptide according to any one of items 1 to 7, wherein the ISVD comprises a combination of CDR1, CDR2, and CDR3 selected from the group consisting of: CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55; CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 118; CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 71; CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO:54; CDR1 is SEQ ID NO:22, CDR2 is SEQ ID NO:36, and CDR3 is SEQ ID NO:54; CDR1 is SEQ ID NO:25, CDR2 is SEQ ID NO:40, and CDR3 is SEQ ID NO:58; CDR1 is SEQ ID NO:33, CDR2 is SEQ ID NO:50, and CDR3 is SEQ ID NO:68; CDR1 is SEQ ID NO:33, CDR2 is SEQ ID NO:51, and CDR3 is SEQ ID NO:69; CDR1 is SEQ ID NO:28, CDR2 is SEQ ID NO:44, and CDR3 is SEQ ID NO:62; CDR1 is SEQ ID NO: CDR1 is SEQ ID NO:28, CDR2 is SEQ ID NO:43, and CDR3 is SEQ ID NO:61; CDR1 is SEQ ID NO:24, CDR2 is SEQ ID NO:39, and CDR3 is SEQ ID NO:57; CDR1 is SEQ ID NO:23, CDR2 is SEQ ID NO:38, and CDR3 is SEQ ID NO:56; CDR1 is SEQ ID NO:26, CDR2 is SEQ ID NO:41, and CDR3 is SEQ ID NO:59; CDR1 is SEQ ID NO:27, and CDR2 is SEQ ID NO:119 and CDR3 is SEQ ID NO:60; CDR1 is SEQ ID NO:27, CDR2 is SEQ ID NO:42, and CDR3 is SEQ ID NO:60; CDR1 is SEQ ID NO:29, CDR2 is SEQ ID NO:46, and CDR3 is SEQ ID NO:64; CDR1 is SEQ ID NO:30, CDR2 is SEQ ID NO:47, and CDR3 is SEQ ID NO:65; CDR1 is SEQ ID NO:31, CDR2 is SEQ ID NO:48, and CDR3 is SEQ ID NO:66; CDR1 is SEQ ID NO:32, CDR2 is SEQ ID NO:49, and CDR3 is SEQ ID NO:67;and CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70;

[10] The polypeptide of any one of items 1 to 9, wherein the ISVD consists of or essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions CDR1, CDR2, and CDR3.

[11] The polypeptide of any one of Items 1 to 10, wherein the polypeptide is SEQ ID NO: 129 or 130, or a polypeptide having at least 95% sequence identity to SEQ ID NO: 129 or 130.

[12] The polypeptide of any one of items 4, 8, and 9, wherein CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55.

[13] The polypeptide of any one of Items 4 to 12, wherein the ISVD is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15, and 18.

[14] If the polypeptide is determined, for example, by KinExA or alternatively by Gyrolab, the K D is 1E -07 M~1E -13 Between M, for example, 1E -08 M~1E -12 Between M, preferably at most 1E -07 M, preferably 1E -08 M or 1E -09 Less than M or 1E -10 Less than M, for example, 5E -11 M, 4E -11 M, 3E -11 M, 2E -11 M, 1.7E -11 M, 1E -11 M, etc., or 5E -12 M, 4E-12 M, 3E -12 M, 1E -12 14. The polypeptide according to any one of items 1 to 13, wherein M binds to ADAMTS5.

[15] The IC of a polypeptide, as determined, for example, by a human FRET assay or human AlphaLISA. 50 is 1E -07 M~1E -12 Between M, for example, 1E -08 M~1E -11 15. The polypeptide according to any one of items 1 to 14, which inhibits the activity of ADAMTS5 between M.

[16] The polypeptide is IC 50 is up to 1E -07 M, preferably 1E -08 M, 5E -09 M, or 4E -9 M, 3E -9 M, 2E -9 M, e.g. 1E -9 16. The polypeptide according to item 15, which inhibits the (enzymatic) activity of ADAMTS5, such as M.

[17] When a polypeptide is determined, for example, by binding ELISA, EC 50 is 1E -07 M~1E -12 Between M, for example, 1E -08 M~1E -11 17. The polypeptide according to any one of items 1 to 16, which regulates ADAMTS5 between M.

[18] The polypeptide has an off-rate of 1E, as determined, for example, by SPR. -04 s -1 15. The polypeptide of any one of items 1 to 14, which binds to ADAMTS5 with less than 1 kJ / s.

[19] The polypeptide of any one of Aspects 1 to 18, wherein the ADAMTS5 is human ADAMTS5 (SEQ ID NO: 149), bovine ADAMTS5 (SEQ ID NO: 150), rat ADAMTS5 (SEQ ID NO: 151), guinea pig ADAMTS5 (SEQ ID NO: 152), mouse ADAMTS5 (SEQ ID NO: 153), or cynomolgus monkey ADAMTS5 (SEQ ID NO: 154), preferably human ADAMTS5, and most preferably SEQ ID NO: 149.

[20] The polypeptide of any one of items 1 to 19, wherein the polypeptide antagonizes an activity of ADAMTS5, such as a protease activity, such as cleavage of aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, preferably cleavage of aggrecan; preferably, antagonizes the aggrecanase activity of ADAMTS5.

[21] The polypeptide of any one of items 1 to 19, wherein the polypeptide blocks binding of ADAMTS5 to aggrecan by at least 20%, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, as determined, for example, by FRET, AlphaLISA, or ELISA.

[22] The polypeptide of any one of items 1 to 20, wherein the polypeptide inhibits the protease activity of ADAMTS5, for example, inhibits proteolysis of a substrate such as aggrecan, versican, brevican, neurocan, decorin, and / or biglycan, preferably aggrecan.

[23] The polypeptide of any one of items 1 to 22, comprising at least two ISVDs, wherein at least one ISVD specifically binds to ADAMTS, preferably ADAMTS5, and is preferably selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18.

[24] The polypeptide according to item 23, wherein at least two ISVDs specifically bind to ADAMTS, preferably ADAMTS5.

[25] A polypeptide comprising two or more ISVDs, each of which individually specifically binds to ADAMTS5, wherein a) at least a "first" ISVD specifically binds to a first antigenic determinant, epitope, portion, domain, subunit, or three-dimensional structure of ADAMTS5; and b) at least a "second" ISVD specifically binds to a second antigenic determinant, epitope, portion, domain, subunit, or three-dimensional structure of ADAMTS5, each different from the first antigenic determinant, epitope, portion, domain, subunit, or three-dimensional structure.

[26] The polypeptide of item 25, wherein the "first" ISVD that specifically binds to ADAMTS5 is selected from the group consisting of SEQ ID NOs: 2, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15, and 18.

[27] The polypeptide of item 25 or 26, wherein the "second" ISVD that specifically binds to ADAMTS5 is SEQ ID NO: 118 or 19.

[28] The polypeptide of any one of Aspects 25 to 27, wherein the polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb), and SEQ ID NO: 128 (clone 131 9D3-093-Alb), or a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO: 126, 127, 128, 130, or 131.

[29] The polypeptide of any one of items 1 to 28, further comprising an ISVD that binds to serum albumin.

[30] The polypeptide of Item 29, wherein the serum albumin-binding ISVD essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SEQ ID NO: 146, CDR2 is SEQ ID NO: 147, and CDR3 is SEQ ID NO: 148. 31. The polypeptide of Item 30, wherein the serum albumin-binding ISVD is selected from the group consisting of ALB8 (SEQ ID NO: 131), ALB23 (SEQ ID NO: 132), ALB129 (SEQ ID NO: 133), ALB132 (SEQ ID NO: 134), ALB11 (SEQ ID NO: 135), ALB11(S112K)-A (SEQ ID NO: 136), ALB82 (SEQ ID NO: 137), ALB82-A (SEQ ID NO: 138), ALB82-AA (SEQ ID NO: 139), ALB82-AAA (SEQ ID NO: 140), ALB82-G (SEQ ID NO: 141), ALB82-GG (SEQ ID NO: 142), ALB82-GGG (SEQ ID NO: 143), ALB92 (SEQ ID NO: 144), and ALB223 (SEQ ID NO: 145).

[32] The polypeptide of item 31, comprising: SEQ ID NO: 129 (clone 577 2F3 SO -Alb), SEQ ID NO: 130 (clone 579 2F3 SO the polypeptide is selected from the group consisting of SEQ ID NO: 120 (clone 4 2A12-Alb), SEQ ID NO: 121 (clone 5 2D7-Alb), SEQ ID NO: 122 (clone 6 2F3-Alb), SEQ ID NO: 123 (clone 69 049-Alb), SEQ ID NO: 124 (clone 70 9D3-Alb), SEQ ID NO: 125 (clone 71 3B2-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb), and SEQ ID NO: 128 (clone 131 9D3-093-Alb).

[33] The polypeptide of any one of items 1 to 32, further comprising at least one ISVD that specifically binds to aggrecan, preferably selected from the group consisting of SEQ ID NO: 156 (Nanobody 00745 PEA114F08) and SEQ ID NO: 157 (Nanobody 00747 PEA604F02).

[34] The polypeptide of any one of items 1 to 32, comprising at least two ISVDs that specifically bind to aggrecan.

[35] The polypeptide according to Item 34, wherein the at least two ISVDs that specifically bind to aggrecan may be the same or different.

[36] The polypeptide of item 34 or 35, wherein at least two ISVDs that specifically bind to aggrecan are independently selected from the group consisting of SEQ ID NOs: 156 and 157.

[37] The polypeptide according to any one of Aspects 33 to 36, wherein the ISVD that specifically binds to aggrecan specifically binds to human aggrecan [SEQ ID NO: 155].

[38] The polypeptide according to any one of Items 33 to 37, wherein the ISVD that specifically binds to aggrecan specifically binds to dog aggrecan, bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan; rabbit aggrecan; cynomolgus monkey aggrecan, and / or rhesus monkey aggrecan.

[39] The polypeptide according to any one of items 33 to 38, wherein the ISVD that specifically binds to aggrecan preferably binds to cartilaginous tissue such as cartilage and / or meniscus.

[40] The polypeptide of any one of items 1 to 39, wherein the polypeptide has stability at 37°C in synovial fluid (SF) for at least 7 days, such as 14 days, 21 days, 1 month, 2 months, or even 3 months.

[41] The polypeptide of any one of items 23 to 40, wherein at least two ISVDs are linked to each other directly or via a linker.

[42] The polypeptide according to Item 41, wherein the linker is selected from the group consisting of SEQ ID NOs: 158 to 174, preferably SEQ ID NO: 169.

[43] The polypeptide of any one of items 1 to 42, further comprising a C-terminal extension.

[44] The polypeptide of item 43, wherein the C-terminal extension is a C-terminal extension (X)n, where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[45] The polypeptide of any one of paragraphs 1 to 44, wherein the polypeptide has at least 80%, 90%, 95%, or 100% sequence identity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 116, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130.

[46] A method for treating and / or preventing a disease or disorder in an individual, for example one in which ADAMTS5 activity is implicated, the method comprising administering to the individual a polypeptide according to any one of items 1 to 45 in an amount effective to treat or prevent a symptom of the disease or disorder.

[47] The method of item 46, wherein the disease or disorder is selected from the group consisting of arthropathy and chondrodystrophy, arthritis such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tear or avulsion, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans, and aggrecanopathy.

[48] ​​The polypeptide according to any one of items 1 to 45, for use as a pharmaceutical.

[49] The polypeptide of any one of paragraphs 1 to 45 for use in the treatment or prevention of ADAMTS5-related diseases, such as arthropathy and chondrodystrophy, arthritis such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or avulsions, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated discs, lumbar degenerative disc disease, degenerative joint disease, and symptoms of relapsing polychondritis, osteochondritis dissecans, and aggrecanopathy.

[50] The polypeptide of any one of Items 1 to 45, wherein the polypeptide cross-blocks the binding to ADAMTS5 of at least one polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18 and / or is cross-blocked in binding to ADAMTS5 by at least a polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18.

[51] A polypeptide that cross-blocks binding to ADAMTS5 by a polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18, and / or is cross-blocked in binding to ADAMTS5 by at least a polypeptide represented by any one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, and 18, wherein the polypeptide comprises at least one VH, VL, dAb, or immunoglobulin single variable domain (ISVD) that specifically binds to ADAMTS5, and wherein the binding to ADAMTS5 modulates the activity of ADAMTS5.

Claims

1. A polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that binds to A disintegrin and metalloproteinase with thrombospondin 5 motifs (ADAMTS5), wherein the ISVD that binds to ADAMTS5 comprises three complementarity determining regions (CDR1, CDR2, and CDR3), wherein CDR1 has the sequence of SEQ ID NO:21, CDR2 has the sequence of SEQ ID NO:37, and CDR3 has the sequence of SEQ ID NO:

55.

2. The polypeptide of claim 1 , wherein the ISVD comprises four framework regions (FR1, FR2, FR3, and FR4).

3. 3. The polypeptide of claim 2, wherein FR1 has the amino acid sequence: DVQLVESGGGVVQPGGSLRLSCAAS, FR2 has the amino acid sequence of SEQ ID NO: 88, FR3 has the amino acid sequence: YVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAA, and FR4 has the amino acid sequence of SEQ ID NO:

114.

4. 2. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO: 129 or 130 or has at least 95% sequence identity with SEQ ID NO: 129 or 130.

5. The polypeptide is K D is 1E -08 The polypeptide of any one of claims 1 to 4, which binds to ADAMTS5 with less than M.

6. The polypeptide is an IC 50 is up to 1E -09 The polypeptide according to any one of claims 1 to 4, which inhibits the enzymatic activity of ADAMTS5 in M.

7. The polypeptide is 50 is 1E -07 M to 1E -12 The polypeptide according to any one of claims 1 to 4, which regulates ADAMTS5 during M.

8. The polypeptide has an off-rate of 1E -04 s -1 The polypeptide of any one of claims 1 to 4, which binds to ADAMTS5 with less than 1 kJ / s.

9. The polypeptide according to any one of claims 1 to 4, wherein the ADAMTS5 is human ADAMTS5 having the sequence of SEQ ID NO:

149.

10. The polypeptide of any one of claims 1 to 4, wherein the polypeptide antagonizes the activity of ADAMTS5.

11. The polypeptide of any one of claims 1 to 4, wherein the polypeptide inhibits binding of ADAMTS5 to aggrecan by at least 20%.

12. The polypeptide of any one of claims 1 to 4, wherein the polypeptide inhibits the protease activity of ADAMTS5.

13. The polypeptide of any one of claims 1 to 4, further comprising a second ISVD.

14. The polypeptide of claim 13, wherein the second ISVD specifically binds to ADAMTS5.

15. The polypeptide of any one of claims 1 to 4, further comprising an ISVD that binds to serum albumin.

16. 16. The polypeptide of claim 15, wherein the ISVD that binds to serum albumin comprises four framework regions and three complementarity determining regions (CDR1, CDR2, and CDR3), wherein CDR1 has the sequence of SEQ ID NO: 146, CDR2 has the sequence of SEQ ID NO: 147, and CDR3 has the sequence of SEQ ID NO:

148.

17. 16. The polypeptide of claim 15, wherein the serum albumin-binding ISVD is selected from the group consisting of ALB8 (SEQ ID NO:131), ALB23 (SEQ ID NO:132), ALB129 (SEQ ID NO:133), ALB132 (SEQ ID NO:134), ALB11 (SEQ ID NO:135), ALB11(S112K)-A (SEQ ID NO:136), ALB82 (SEQ ID NO:137), ALB82-A (SEQ ID NO:138), ALB82-AA (SEQ ID NO:139), ALB82-AAA (SEQ ID NO:140), ALB82-G (SEQ ID NO:141), ALB82-GG (SEQ ID NO:142), ALB82-GGG (SEQ ID NO:143), ALB92 (SEQ ID NO:144), and ALB223 (SEQ ID NO:145).

18. below: SEQ ID NO: 129 (clone 577 2F3 SO -Alb), SEQ ID NO: 130 (clone 579 2F3 SO -093-Alb), SEQ ID NO: 122 (clone 6 2F3-Alb), and SEQ ID NO: 126 (clone 129 2F3-093-Alb) 15. The polypeptide of claim 14, selected from the group consisting of:

19. 14. The polypeptide of claim 13, wherein an ISVD that binds to ADAMTS5 is linked to a second ISVD directly or via a linker.

20. 20. The polypeptide of claim 19, wherein the linker is selected from the group consisting of SEQ ID NOs: 158-174.

21. 5. The polypeptide of any one of claims 1 to 4, wherein the polypeptide has at least 90% sequence identity to any one of SEQ ID NOs: 2, 129 or 130.

22. 22. The polypeptide of any one of claims 1 to 21 for use in the treatment of a disease or disorder in which ADAMTS5 activity is involved, selected from arthropathy, chondrodystrophy, arthritis, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans and aggrecanopathy.

23. 23. The polypeptide of claim 22, wherein the arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic lacerations and abrasions.

24. A pharmaceutical composition for treating a disease or condition associated with ADAMTS5 activity, comprising the polypeptide of any one of claims 1 to 21, wherein the disease or condition is selected from arthropathy, chondrodystrophy, arthritis, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, and aggrecanopathy.

25. 25. The pharmaceutical composition of claim 24, wherein the arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic lacerations and abrasions.

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