RU2786659C2 - Polypeptides binding to adamts5, mmp13, and aggrecan - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, in particular to new hybrid immunoglobulins; it can be used in medicine. The invention discloses polypeptides capable of binding to proteoglycan cartilage aggrecan protein. ADAMTS5, and/or MMP13, as well as structures based on them.

EFFECT: invention can be used in medical practice in the treatment or prevention of joint diseases for prevention of cartilage tissue erosion, in particular in osteoarthritis.

21 cl, 11 dwg, 25 tbl, 14 ex

Description

1. Field to which the invention relates

The present invention relates to polypeptides that bind to Aggrecan, as well as to ADAMTS5 and/or MMP13, more specifically to polypeptides that include or essentially consist of immunoglobulins that bind to Aggrecan, as well as immunoglobulins that bind to ADAMTS5 and/or immunoglobulins, binding to MMP13 (also referred to herein as “ polypeptides of the invention” and “immunoglobulin(s) of the invention” , respectively). , as well as to nucleic acids encoding such immunoglobulins or polypeptides (also referred to in this application as “ nucleic acid(s) of the invention ”; to methods for obtaining such immunoglobulins, polypeptides and constructs; to host cells expressing or capable of expressing such immunoglobulins or polypeptides; to compositions and in particular to pharmaceuticals tic compositions that include such immunoglobulins, polypeptides, constructs, nucleic acids and/or host cells; and to uses of immunoglobulins, polypeptides, constructs, nucleic acids, host cells and/or compositions, in particular for prophylactic and/or therapeutic purposes, such as the prophylactic and/or therapeutic purposes referred to in this application. Other aspects, embodiments, advantages and applications of the invention will become apparent from the description below.

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 disease. It is projected to affect more than 20 percent of the US population by 2025. The disease is not systemic and is usually limited to a few joints. However, the disease can occur in all joints, most commonly in the knees, hips, hands, and spine. OA is characterized by progressive erosion of the articular cartilage (the cartilage that covers the bones), leading to chronic pain and disability. Eventually, the disease leads to complete destruction of the articular cartilage, sclerosis of the underlying bone, formation of osteophytes, etc., all of which lead to loss of movement and pain. Osteoarthritis can be defined as a diverse group of conditions characterized by a combination of joint symptoms, signs due to articular cartilage defects, and changes in adjacent tissues, including bones, tendons, and muscles. The most prominent symptom of OA is pain, which is why patients are the most likely to seek medical attention. OA is not curable, that is, current treatments do not suppress the structural destruction of OA of the joint. Treatment of the disease is limited to treatments that are palliative at best and have little effect on the underlying cause of disease progression.

There is an intense search for osteoarthritis-modifying drugs (DMOADs), which can be defined as drugs that inhibit the progression of structural changes in the disease and ideally also improve symptoms and/or function. DMOADs are likely to be given for long periods in this chronic disease of the aging population, so excellent safety data in the target population with multiple comorbidities and potential drug-drug interactions are needed for this.

Although the onset of the disease may be multifactorial, cartilage destruction appears to be the result of uncontrolled proteolytic destruction of the extracellular matrix (ECM). The most common ECM components of articular cartilage are collagen (primarily collagen II) and proteoglycans, mainly Aggrecan (Kiani et al. 2002 Cell Research 12: 19–32)

Aggrecan is important for the proper functioning of articular cartilage as it provides a hydrated gel structure that gives the cartilage its load-bearing capacity. Aggrecan is a large multimodal molecule (2317 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 attachment of the glycosaminoglycan chain. This extended region contains two domains, one of which is replaced by keratan sulfate chains (KS domain) and the other by chondroitin sulfate chains (CS domain). The CS domain has 100–150 glycosaminoglycan chains (GAGs) attached to it. Aggrecan forms large complexes with hyaluronan, in which 50–100 Aggrecan molecules interact through the G1 domain and binding protein with one hyaluronan molecule. Upon absorption of water (due to the GAG content), these complexes form a reversibly deformable gel that resists compression. The structure, fluid retention and function of articular cartilage are related to the content of Aggrecan in the matrix and the amount of chondroitin sulfate associated with intact core protein. Structurally, OA is characterized by degradation of Aggrecan, gradual release of the G3 and G2 domains (resulting in cartilage “deflation”) and eventually release of the G1 domain and destruction of collagen, with irreversible destruction of the cartilage structure. The most significant Aggrecan cleavage site in the pathogenesis of OA is in the TEGE ³⁷³ ↓ ³⁷⁴ ARGS sequence. This cleavage site is located in the interglobular domain (IGD) of Aggrecan located between the G1 and G2 domains.

Antibodies that recognize neoepitope ³⁷⁴ ARGS led to the discovery of aggrecanase-1, which has been shown to be ADAMTS4, and aggrecanase-2, which is ADAMTS5. Subsequently, other related ADAMTS enzymes, including ADAMTS1, -8, -9, -15 and -20, have been shown to have aggrecanase activity. ADAMTS16 and 18 are also weak aggrecanases. Various data suggest that ADAMTS5 is the main enzyme involved in the pathogenesis of osteoarthritis. In human cartilage explants and chondrocytes, ADAMTS5 knockdown attenuated Aggrecan degradation, suggesting that this enzyme may be involved in human tissues. The expression of the enzyme is enhanced by cytokines such as interleukin-1 and oncostatin-M, which provoke the destruction of Aggrecan in tissues. ADAMTS5-generated Aggrecan fragments have been found in the synovial fluid and serum of patients with OA (Germaschewski et al., 2014, Osteoarthritis Cartilage 22: 690–697). Several pharmaceutical companies are developing DMOAD. Some of these compounds are said to be specific for ADAMTS5, while others also have an effect against other members of ADAMTS or even against matrix metalloproteinases. In particular, these cross-over inhibitions against MMPs are believed to be responsible for musculoskeletal syndrome (MSS), a side effect induced by broad-spectrum inhibitors and including arthralgia, myalgia, joint stiffness, and tendinitis (Santamaria et al., 2015 Biochem J 471: 391-401). These side effects were the reason for stopping further development. The aggrecanase inhibitor AGG-523 from Pfizer was used in a phase I clinical trial for the treatment of OA, but has not been used since. Other small molecule ADAMTS inhibitors have also not progressed further in clinical studies as potential DMOADs (Bondeson et al., 2015 Drug Discovery 10:5–14). The Galapagos/Servier ADAMTS5 inhibitor, GLPG1972, has recently completed phase I trials, but its efficacy has not yet been determined. Indeed, despite a number of recent clinical trials specifically investigating DMOADs, no such treatments have yet been approved (El Bakali et al., Future Medicinal Chemistry (Review) 6:1399). A study of the monoclonal antibody (mAb) CRB0017 from Rottapharm 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). However, there was no comparison with systemic administration, nor was it evaluated for the extent to which the mAb leaks from the synovial space. Another study used systemic administration of mAb 12F4 to mice, which demonstrated both a structural modification of the disease and alleviation of pain-related behavior (Miller et al., 2014 Osteoarthritis Cartilage 22iii, S35). However, a single administration of mAb 12F4 to cynomolgus monkey caused focal hemorrhage, a dose-dependent increase in mean arterial pressure, and cardiac conduction disturbances (more specifically, ST elevation and ventricular arrhythmia on the ECG) indicative of cardiac ischemia, which were maintained up to 8 months after a single dose. (Larkin et al., 2014, Osteoarthritis Cartilage 22iii, S483). Also in this case, side effects stopped further clinical development of mAb 12F4.

Along with ADAMTS enzymes, there is strong evidence that matrix metalloproteinases (MMPs) play a major role in tissue destruction associated with OA. MMPs are a family of zinc-dependent endopeptidases involved in the destruction of the extracellular matrix and tissue remodeling. There are about 28 members of the MMP family, which can be divided into various subgroups, including collagenases, gelatinases, stromelysins, membrane-type MMPs, matrilysins, enamelisins, and others. Collagenases, including MMP1, MMP8, MMP13, and MMP18, are able to degrade trihelix fibrillar collagens into distinct 3/4 and 1/4 fragments. In addition, MMP14 has been shown to cleave fibrillar collagen, and there is evidence that MMP2 is also capable of collagenolysis. MMPs have long been considered attractive therapeutic targets for the treatment of OA. However, as noted above, broad-spectrum MMP inhibitors developed for the treatment of arthritis have not been clinically tested due to painful, joint-numbing side effects, i. MSS.

Therapeutic interventions in the joints have also been difficult due to the difficulty of targeted drug delivery to the articular cartilage. Since articular cartilage is a tissue devoid of blood and lymph vessels, traditional drug delivery routes (oral, intravenous, intramuscular) ultimately depend on the transsynovial transport of drugs from synovial capillaries to cartilage by passive diffusion. This prompted the idea of developing intra-articular (i/a) drug delivery. Although both delivery bypassed the problem of passive diffusion, and/a delivery of therapeutic proteins is limited by their rapid removal from the joint space and, above all, insufficient retention in the cartilage. Notably, the time of drug presence in joint synovial fluid is often less than 24 h (Edwards 2011, Vet J 190: 15–21; Larsen et al., 2008 J Pham Sci 97: 4622–4654). Because of the rapid clearance of most drugs administered by the i/a route, frequent injections will be required to maintain an effective concentration (Owen et al., 1994 Br J Clin Pharmacol 38: 349–355). However, frequent and/a injections are undesirable due to pain and discomfort, which can cause problems with patient compliance, as well as the risk of infection in the joint.

There remains a need for efficient DMOADs.

3. The essence of the invention

The present invention is directed to providing anti-OA polypeptides and constructs with improved prophylactic, therapeutic and/or pharmacological properties, in addition to other useful properties (such as, for example, easier preparation, good stability and/or lower cost of products) compared to amino acid sequences and antibodies of the prior art. In particular, the present invention is directed to providing polypeptides that inhibit ADAMTS5 and/or MMP13 while being retained for an extended period in the joints.

Realizing that osteoarthritis develops unevenly and that the rate of progression of the lesion can be very different - in extreme cases, osteoarthritis can remain stable for decades, while in other patients OA progresses very quickly to complete cartilage destruction in a few months - the authors of the present invention put forward hypothesis (however, without being bound by any theory) that such a variable pattern of disease progression may be due to a heterogeneous pattern of activity of various proteases.

After identifying effective protease inhibitors from various families and cartilage anchoring fragments using creative and innovative screening, characterization and combinatorial strategies, the present inventors developed combinations in which various functional fragments were combined. Two dual specificity polypeptides were constructed comprising cartilage anchoring Aggrecan binding fragments (CAP) and either an ADAMTS5 inhibitor or an MMP13 inhibitor, as well as triple specificity polypeptides comprising an ADAMTS5 inhibitor, an MMP13 inhibitor and CAP binders.

It was demonstrated that the polypeptides of the invention remained effective in various model systems representing various OA states, even when the reference molecules (Wyeth and Pfizer) were not effective.

The present inventors have been able to identify and engineer CAP binders that remain effective even when combined with the other two components. It was also demonstrated that the CAP fragment of the polypeptides of the invention resulted in increased retention in the joint.

It has also been demonstrated that the polypeptides of the invention remain stable in the joint.

Therefore, the polypeptides of the present invention, on the one hand, could be widely useful for patients with OA, while, on the other hand, the burden of the scheme (and/a) administration could be eased. In addition, by combining different fragments in one molecule, the effective dose can be increased.

Accordingly, the present invention provides a polypeptide selected from the group consisting of (a) polypeptides comprising at least 2 immunoglobulin single variable domains (ISVDs), wherein the first ISVD specifically binds to matrix metalloproteinase (MMP) and the second ISVD specifically binds with Aggrecan, and optionally including a third ISVD specifically binding to Aggrecan; (b) polypeptides comprising at least 2 ISVDs, wherein the first ISVD specifically binds to disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and the second ISVD specifically binds to Aggrecan, and optionally comprising a third ISVD that specifically binds to Aggrecan; and (c) polypeptides comprising at least 3 ISVDs, wherein the first ISVD specifically binds to MMP, the second ISVD specifically binds to ADAMTS, and the third ISVD specifically binds to Aggrecan, and optionally including a fourth ISVD that specifically binds to Aggrecan.

In one aspect, the present invention relates to a polypeptide described in this application, in which the first ISVD specifically binds to MMP, the second ISVD specifically binds to ADAMTS, the third ISVD specifically binds to Aggrecan and the fourth ISVD specifically binds to Aggrecan, preferably said MMP is MMP13 .

In one aspect, the present invention provides an ISVD described in this application, where the specified ISVD has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which CDR1, CDR2 and CDR3 have the meaning defined below, and FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention relates to a polypeptide described in this application, where the specified ISVD, specifically binding to MMP13, essentially consists of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively), where (i) (a) CDR1 is SEQ ID NO: 8; and (b) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 8; (ii) (c) CDR2 is SEQ ID NO: 10; and (d) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 10; and (iii) (e) CDR3 is SEQ ID NO: 12; and (f) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 12; preferably, where CDR1 is SEQ ID NO: 8, CDR2 is SEQ ID NO: 10 and CDR3 is SEQ ID NO: 12; even more preferably, said ISVD specifically binding to MMP13 is represented by SEQ ID NO: 2.

In one aspect, the present invention relates to a polypeptide described in this application, where the specified ADAMTS is ADAMTS5, preferably, where the specified ISVD that specifically binds to ADAMTS5, essentially consists of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively), where (i) (a) CDR1 is SEQ ID NO: 14 [GRTVSSYAMG]; and (b) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 14; (ii) (c) CDR2 is SEQ ID NO: 16 [GISRSAERTY]; and (d) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 16; and (iii) (e) CDR3 is SEQ ID NO: 18 [DLDPNRIFSREEYAY]; and (f) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 18; even more preferably, where CDR1 is SEQ ID NO: 14, CDR2 is SEQ ID NO: 16 and CDR3 is SEQ ID NO: 18; and even more preferably, wherein said ADAMTS5-specific ISVD is represented by SEQ ID NO: 3.

In one aspect, the present invention relates to a polypeptide described in this application, where the specified ISVD, which specifically binds to Aggrecan, essentially consists of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively), where (i) (a) CDR1 is SEQ ID NO: 19; and (b) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 19; (ii) (c) CDR2 is SEQ ID NO: 21; and (d) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 21; and (iii) (e) CDR3 is SEQ ID NO: 23; and (f) amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 23; more preferably, where CDR1 is SEQ ID NO: 19, CDR2 is SEQ ID NO: 21 and CDR3 is SEQ ID NO: 23; even more preferably, wherein said Aggrecan-specific ISVD is represented by SEQ ID NO: 4.

In preferred embodiments of all aspects of the invention, an immunoglobulin single variable domain (ISVD) according to the invention preferably consists of or essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions CDR1, CDR2 and CDR3, as indicated in this application above and below. Preferred framework sequences are disclosed, for example, in Table A-2 below, and can be used in the ISVD of the invention. Preferably, the CDRs shown in Table A-2 correspond to corresponding framework regions of the same ISVD design.

In one aspect, the present invention relates to a polypeptide described in this application, where these ISVDs are connected to each other through a linker, preferably the specified linker is selected from the group consisting of SEQ ID NO: 24-40, preferably SEQ ID NO: 28 [9GS] or SEQ ID NO: 35 [35GS].

In one aspect, the present invention provides a polypeptide described herein, wherein said first ISVD specifically binds to MMP13, said second ISVD specifically binds to ADAMTS5, said third ISVD specifically binds to Aggrecan, and said fourth ISVD specifically binds to Aggrecan, preferably represented by SEQ ID NO: 1 or 62.

In one aspect, the present invention provides a polypeptide described herein, wherein said first ISVD specifically binds to MMP13, said second ISVD specifically binds to Aggrecan, and said third ISVD specifically binds to Aggrecan, preferably represented by SEQ ID NO: 5 or 63.

In one aspect, the present invention relates to a polypeptide described in this application, in which the specified first ISVD specifically binds to ADAMTS5, the specified second ISVD specifically binds to Aggrecan, and the specified third ISVD specifically binds to Aggrecan, preferably represented by SEQ ID NO: 6 or 64 .

In one aspect, the present invention relates to a construct that includes or essentially consists of a polypeptide described in this application, which additionally includes one or more other groups, residues, fragments or connecting elements, optionally linked through one or more peptide linkers.

In one aspect, the present invention relates to a nucleic acid encoding a polypeptide described in this application, or a construct described in this application.

In one aspect, the present invention relates to an expression vector comprising the nucleic acid described in this application.

In one aspect, the present invention provides a host or host cell comprising a nucleic acid as described herein or an expression vector as described herein.

In one aspect, the present invention relates to a composition comprising a polypeptide described in this application, a construct described in this application, or a nucleic acid described in this application.

In one aspect, the present invention relates to a method for obtaining a polypeptide described in this application, while this method at least includes the following steps: a) expression, in a suitable host cell, host organism or suitable expression system, the nucleic acid described in this application; optionally followed by b) isolation and/or purification of the polypeptide described in this application.

In one aspect, the present invention relates to a composition described in this application, which is a pharmaceutical composition, preferably said composition further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally includes one or more additional pharmaceutically active polypeptides and/or compounds.

In one aspect, the present invention relates to a composition described in this application, a polypeptide described in this application, or a construct described in this application, for use as a drug.

In one aspect, the present invention relates to a composition described in this application, a polypeptide described in this application, or a construct described in this application, for use in the prevention of a symptom or treatment of arthropathies and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, aggrecanopathy, NASH, chronic periodontitis, and abdominal aortic aneurysms.

In one aspect, the present invention provides a method for preventing a symptom or treating a disease or disorder in an individual, comprising administering a polypeptide described herein, a construct described herein, or a composition described herein, to said individual in an amount effective to treat or prevention of a symptom of arthropathies and chondrodystrophies, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, aggrecanopathy, NASH, chronic periodontitis and abdominal aortic aneurysms.

Other aspects, advantages, uses and uses of the polypeptides and compositions will become apparent from the following disclosure. Several documents are cited in various sections of the description of the present invention. Each of the documents cited above or below in this application (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are incorporated into this application by reference in their entirety. Nothing in this application should be construed as an admission that the invention is not entitled to backdate such disclosure by virtue of prior invention.

4. Description of drawings

Fig. 1: Functional inhibition of the ADAMTS5 molecule by polypeptide 949 ("C010100949", SEQ ID NO: 1) in a FRET-based enzyme activity assay. V _i : rate (process curve) of the inhibited enzymatic reaction; V ₀ : rate of non-inhibited enzymatic reaction. Each point represents the average of technical repeated measurements. 'Whiskers': standard deviation of engineering repeated measurements. This plot is representative of 3 independent experiments.

Fig. 2: Functional inhibition of human, cynomolgus, and rat MMP13 by polypeptide 949 in a FRET-based enzyme activity assay. Left: Inhibition of the cdMMP13 molecule by polypeptide 949 (ALX-1011). Right: Inhibition of activated proMMP13 (949 polypeptide designated C010100949). V _i : rate (process curve) of the inhibited enzymatic reaction, V ₀ : rate of the non-inhibited enzymatic reaction. Cd: catalytic domain; pro: activated pro-MMP13. Each point represents the average of technical repeated measurements. 'Whiskers': standard deviation of engineering repeated measurements. Graphs are representative of at least two independent experiments.

Fig. 3: Efficacy of polypeptide 949 ("NB949") in bovine cartilage explant assay. Efficacy was calculated in comparison to a reference small molecule aggrecanase inhibitor (AGG-523, Wyeth), which completely inhibits IL-1α-stimulated GAG release under these conditions, which was found to be 100% and uninduced cartilage 0%.

Fig. 4: Efficacy of 949 polypeptide in human cartilage explant assay. Efficacy was calculated in comparison to a reference small molecule aggrecanase inhibitor (AGG-523, Wyeth), which completely inhibits IL-1α-stimulated GAG release under these conditions, which was found to be 100% and uninduced cartilage 0%.

Fig. 5: Effect of CAP-mediated anchoring in cartilage of polypeptides in bovine explant assay.

Fig. 6: Polypeptide 949 ("C010100949" or "MAC949") inhibits the release of C2M (Col II cleavage=structure) and C3M (Col III cleavage=associated with symptoms) from co-culture.

Fig. 7: Retention in cartilage: local concentrations of nanobody constructs at various time points in rats.

Fig. 8: Width of significant degeneration of the medial tibial cartilage in different groups.

Fig. 9 : Width of medial tibial cartilage degeneration.

Fig. 10: Analysis of platform gait.

Fig. 11: Serum concentrations (mean concentration in ng/ml) versus time after the first dose (h) of polypeptides in osteoarthritis rats and healthy rats receiving a single intra-articular injection of 400 μg of the nanobody construct per joint (right knee). Points represent individual concentrations in healthy animals; triangles represent individual concentrations in OA animals; and lines represent mean concentrations.

5. Detailed description

There remains a need for safe and effective drugs for the treatment of OA, in particular DMOAD. These medicinal products must meet different and often conflicting requirements, especially when a widely used format is intended. Such a format should preferably be useful to a wide range of patients. The format should preferably be safe and not cause infections due to frequent use. In addition, the format should preferably be convenient for the patient, for example, provide a convenient mode and route of administration, such as systemic administration. For example, it is preferred that such a format is not immediately removed from the bloodstream upon administration. However, the extension of the half-life should preferably not result in off-target activity and side effects or limit efficacy.

The present invention implements at least one of these requirements.

Based on unconventional screening, characterization and combinatorial strategies, the authors of the present invention unexpectedly found that immunoglobulin single variable domains (ISVD) showed exceptionally good results in experiments in vitro, ex vivo and in vivo.

In addition, the present inventors have succeeded in redesigning ISVDs that are even more superior to comparator drugs in terms of alleviating OA. In addition, the ISVDs of the invention have been shown to be significantly safer than prior art compounds.

In addition, the inventors have demonstrated that by combining various functional components, including MMP13 inhibitors, ADAMTS5 inhibitors and Aggrecan binding components, an even better effect was obtained than with any of the inhibitors.

The present invention is directed to providing combinations of ISVDs that antagonize ADAMTS, in particular ADAMTS5, and/or ISVDs that antagonize MMPs, in particular MMP13, in combination with CAP binders (e.g. Aggrecan binders), with improved prophylactic, therapeutic and/or pharmacological properties, including a safer profile, compared to prior art amino acid sequences and antibodies.

Accordingly, the present invention relates to polypeptides selected from the group consisting of

(a) polypeptides comprising at least 2 immunoglobulin single variable domains (ISVDs), wherein the first ISVD specifically binds to matrix metalloproteinase (MMP) and the second ISVD specifically binds to Aggrecan, and optionally comprising a third ISVD that specifically binds to Aggrecan;

(b) polypeptides comprising at least 2 ISVDs, wherein the first ISVD specifically binds to disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and the second ISVD specifically binds to Aggrecan, and optionally comprising a third ISVD that specifically binds to Aggrecan; and

(c) polypeptides comprising at least 3 ISVDs, wherein the first ISVD specifically binds to MMP, the second ISVD specifically binds to ADAMTS, and the third ISVD specifically binds to Aggrecan, and optionally includes a fourth ISVD that specifically binds to Aggrecan.

Unless otherwise indicated or defined, all terms used have their usual meaning known in the art, which will be understood by the person skilled in the art. For example, reference is made to reference books such as Sambrook et al. (Molecular Cloning: A Laboratory Manual (2nd ^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 (6th Ed.) Mosby/Elsevier, Edinburgh, 2001), ^Roitt et al. (Roitt's Essential Immunology ( ^10th Ed.) Blackwell Publishing, UK, 2001), and Janeway et al. (Immunobiology (6 ^th Ed.) Garland Science Publishing/Churchill Livingstone, New York, 2005), as well as general information known in the art, cited in this application.

Unless otherwise indicated, all methods, steps, procedures, and manipulations that are not described in detail in a specific manner can be and are carried out in a manner known per se , as will be understood by the skilled person. Again, reference is made to the reference books and general information known from the prior art referred to in this application, and to additional reference documents cited therein; and also, for example, 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. (Tumour Biol. 26(1): 31-43, 2005) which describe protein engineering methods such as affinity maturation and other methods to improve the specificity and other desirable properties of proteins such as immunoglobulins.

It should be noted that in the context of this application, the singular forms denoted by the articles "a", "an" and "the" also include the plural, unless the context clearly indicates otherwise. Thus, for example, reference to "reagent" includes one or more of these various reagents, and reference to "method" includes reference to equivalent steps and methods known to those skilled in the art that could be modified or replaced by the methods described. in this application.

Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each 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 embraced by the present invention.

The term "and/or" wherever used in this application includes the meaning of "and", "or" and "all or any other combination of elements associated with the specified term".

The term "about" or "about" in the context of this application means within 20%, preferably within 15%, more preferably within 10% and most preferably within 5% of a given value or range.

Throughout the specification and in the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" are intended to include the specified whole or step or group of integers or steps, but not the exclusion of any other whole or stage or group of wholes or stages. In the context of the present application, the term "including" may be replaced by the term "comprising" or "including", or sometimes the term "having".

The term "sequence" in the context of this application (for example, in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V _HH sequence" or "protein sequence") should generally be understood as including the relevant amino acid sequence, as well as the nucleic acids or nucleotide sequences encoding it, unless the context requires a more limited interpretation.

Amino acid sequences are interpreted to mean a single amino acid or a straight chain of two or more amino acids, depending on the context. Nucleotide sequences are interpreted to mean a straight chain sequence of 3 or more nucleotides.

Amino acids are those L-amino acids that are commonly found in naturally occurring proteins. Amino acid residues will be indicated according to the standard three-letter or single-letter designation of amino acids. See Table A-2 on page 48 of WO 08/020079. Those amino acid sequences that contain D-amino acids are not considered to be covered by this definition. Any amino acid sequence that contains post-translationally modified amino acids can be described as an amino acid sequence that is originally translated using the symbols shown in this Table A-2 with modified positions; for example, hydroxylations or glycosylations, but these modifications need not be directly shown in the amino acid sequence. Any peptide or protein that can be expressed as including sequence-modifying bonds, cross-links and end caps, non-peptidyl bonds, etc., is covered by this definition, as is known in the art.

The terms "protein", "peptide", "protein/peptide" and "polypeptide" are used interchangeably throughout the present disclosure and each has the same meaning for the purposes of the present disclosure. Each term refers to an organic compound formed by a linear chain of two or more amino acids. A compound may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. Those skilled in the art will appreciate that polypeptides generally comprise fewer amino acids than proteins, although there is no recognized point in the art that distinguishes a polypeptide from a protein based on the number of amino acids; that polypeptides can be obtained by chemical synthesis or recombinant methods; and that proteins are typically produced in vitro or in vivo by recombinant methods known in the art. According to the rules, the amide bond in the primary structure of polypeptides is located in the order in which amino acids are written, where the amino-terminus (N-terminus) of the polypeptide is always on the left, while the acid-terminus (C-terminus) is on the right.

A nucleic acid or amino acid sequence is considered to be “(in) (essentially) isolated (form)” – for example, compared to the reaction medium or culture medium from which it was obtained – when it is separated from at least one other component, with which it is normally associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or minor component. In particular, a nucleic acid or amino acid sequence is considered “(substantially) isolated” when purified at least 2-fold, in particular at least 10-fold, more specifically at least 100-fold, and even 1000-fold or more. . A nucleic acid or amino acid that is "in (substantially) isolated form" is preferably substantially homogeneous, as determined using an appropriate method, such as an appropriate chromatographic method, such as polyacrylamide gel electrophoresis.

When a nucleotide sequence or amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or "essentially consist of" another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence is incorporated into the first specified nucleotide sequence or amino acid sequence, respectively, but more typically, this generally means that the first specified nucleotide sequence or amino acid sequence includes in its sequence a section of nucleotides or amino acid residues, respectively, which has the same nucleotide sequence or amino acid sequence, respectively as the last sequence, regardless of how the first specified sequence actually was the image ovan or obtained (for example, this can be using any suitable method described in this application). By way of non-limiting example, when a polypeptide of the invention is indicated to comprise an immunoglobulin single variable domain ("ISVD"), this may mean that said immunoglobulin single variable domain sequence has been incorporated into the sequence of the polypeptide of the invention, but more typically this is generally , means that the polypeptide according to the invention contains in its sequence the sequence of single variable domains of immunoglobulin, regardless of how the specified polypeptide according to the invention was formed or obtained. Also, when a nucleic acid or nucleotide sequence is indicated as including another nucleotide sequence, the first said nucleic acid or nucleotide sequence is preferably such that when it is expressed in an expression product (e.g., a polypeptide), the amino acid sequence encoded by the last nucleotide sequence forms part of said expression product (in other words, the last nucleotide sequence is in the same reading frame as the first specified larger nucleic acid or nucleotide sequence). Also, when a construct of the invention is said to include a polypeptide or ISVD, this may mean that said construct at least includes said polypeptide or ISVD, respectively, but more typically it means that said construct includes groups, residues (e.g., amino acid residues ), fragments, and/or linking elements in addition to said polypeptide or ISVD, regardless of how said polypeptide or ISVD is linked to said groups, residues (e.g., amino acid residues), fragments, and/or linking elements, and regardless of whether how said construct was formed or obtained.

The term "essentially consist of" means that the ISVD used in the invention is either exactly the same as the ISVD of the invention or corresponds to an ISVD of the invention that has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1 –10 amino acid residues and preferably 1-6 amino acid residues, e.g. 1, 2, 3, 4, 5 or 6 amino acid residues added at the amino-terminus, at the carboxy-terminus, or at both the amino-terminus and the carboxy-terminus ISVD site.

For purposes of comparing two or more nucleotide sequences, the percentage of “sequence identity” between the first nucleotide sequence and the second nucleotide sequence can be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [total number of nucleotides in of the first nucleotide sequence] and multiplying 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 of one nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using a known sequence alignment computer algorithm such as NCBI Blast v2.0 using standard settings. Some other methods, computer algorithms and settings for determining the degree of sequence identity are described in, for example, WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768. For purposes of determining the percentage of “sequence identity” between two nucleotide sequences according to the calculation method described above, the nucleotide sequence with the highest number of nucleotides is taken as the “first” nucleotide sequence and the other nucleotide sequence is taken as the “second” nucleotide sequence.

For purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between the first amino acid sequence and the second amino acid sequence (also referred to herein as "amino acid identity") can be calculated by dividing 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] by [total number of amino acid residues in the first amino acid sequence] and multiplying 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 in one amino acid residue (position), i.e. "amino acid difference", as defined in this application. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using a known computer algorithm such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, also using standard settings. Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method described above, the amino acid sequence with the most amino acid residues is taken as the "first" amino acid sequence and the other amino acid sequence is taken as the "second" amino acid sequence.

Also, when determining the degree of sequence identity between two amino acid sequences, one skilled in the art may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical composition. structures and which have little or essentially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example WO 04/037999, GB 335768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected based on the respective teachings in WO 04/037999 as well as WO 98/49185 and other references cited therein.

Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic non-polar or slightly polar residues: Ala, Ser, Thr, Pro and 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; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are the following: Ala to Gly or Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly on Ala or Pro; His to Asn or to Gln; Ile on Leu or on Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met on Leu, on Tyr or on Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

Any amino acid substitutions applicable to the polypeptides described in this application may also be based on the analysis of frequencies of amino acid variation between homologous proteins of different species, developed by Schulz et al. (“Principles of Protein Structure”, Springer-Verlag, 1978), on structure-forming potential analyzes developed by Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45–149, 1978), and on pattern analysis hydrophobicity in proteins 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, 1986), all of which are incorporated herein by reference in their entirety. Information about the primary, secondary and tertiary structure of nanobodies is presented in the description of the present invention and in the general level of technology indicated above. In addition, for this purpose, the crystal structure of the V _HH domain of the llama, for example, is presented in Desmyter et al. (Nature Structural Biology, 3: 803, 1996), Spinelli et al. (Natural Structural Biology, 3: 752–757, 1996) and Decanniere et al. (Structure, 7 (4): 361, 1999). Further information on certain amino acid residues that form the V _H /V _L interface in conventional V _H domains and potential camel-derived substitutions at these positions can be found in the prior art referenced above.

Amino acid sequences and nucleic acid sequences are referred to as "exactly the same" if they have 100% sequence identity (as defined in this application) throughout their length.

When comparing two amino acid sequences, the term “amino acid(s) difference” refers to an insertion, deletion, or substitution of one amino acid residue at a specific position in the first sequence compared to the second sequence; it should be understood that 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(s)” refers to the insertion, deletion, or substitution of a single amino acid residue at a specific position in the CDR sequence defined in (b), (d) or (f), compared to the CDR sequence, respectively (a), (c) or (e); it should be understood that the CDR sequence (b), (d) and (f) may contain one, two, three, four or a maximum of five such amino acid differences compared to the CDR sequence, respectively (a), (c) or (e) .

An “amino acid difference(s)” can be any one, two, three, four or up to five substitutions, deletions or insertions, or any combination thereof, that either enhances the properties of the ISVD of the invention, i.e. an ADAMTS5-binding, MMP13-binding, and/or Aggrecan-binding agent of the invention, such as a polypeptide of the invention, or which at least slightly impairs the desired properties or the balance or combination of desirable properties of the ISVDs of the invention, i.e. an ADAMTS5-binding, MMP13-binding and/or Aggrecan-binding agent of the invention, such as a polypeptide(s) of the invention comprising an Aggrecan-binding and MMP13-binding and/or ADAMTS5-binding component. In this regard, the resulting polypeptide(s) of the invention should at least bind Aggrecan and MMP13 and/or ADAMTS5 with the same, nearly the same, or higher affinity compared to a polypeptide comprising one or more CDR sequences without one, two , three, four or a maximum of five substitutions, deletions or insertions. Affinity can be measured by any suitable method known in the art, but is preferably measured by the method described in the Examples section.

In this regard, the amino acid sequence of the CDR according to (b), (d) and/or (f), as specified in this application, may be an amino acid sequence that is derived from the amino acid sequence according to (a), (c ) and/or (e), respectively, by affinity maturation using one or more affinity maturation methods known per se or described in the examples. For example, and depending on the host organism used to express the polypeptide of the invention, such deletions and/or substitutions can be calculated to remove one or more sites for post-translational modification (such as one or more glycosylation sites), which should be known to those skilled in the art (see Examples).

“Nanobody family,” “V _HH family,” or “family,” as used herein, refers to a group of VHH _nanobodies and/or sequences that are the same length ( i.e. , they have the same number of amino acids in their sequence) and in which the amino acid sequences between position 8 and position 106 (according to Kabat numbering) have an amino acid sequence identity of 89% or more.

The terms "epitope" and "antigenic determinant", which may be used interchangeably, refer to the portion of a macromolecule, such as a polypeptide or protein, that is recognized by antigen-binding molecules such as immunoglobulins, conventional antibodies, immunoglobulin single variable domains, and/or polypeptides of the invention. , and more specifically the antigen-binding site of these molecules. Epitopes define the minimum binding site for an immunoglobulin and thus represent the target of immunoglobulin specificity.

The portion 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 referred to as a "paratope".

An amino acid sequence (such as an immunoglobulin single variable domain, an antibody, a polypeptide of the invention, or typically an antigen-binding protein or polypeptide or fragment thereof) that can “bind to” or “specifically bind to” that “has affinity for” and/or which “has specificity for” a particular epitope, antigen or protein (or at least one part, fragment or epitope thereof) is referred to as “against” or “directed against” the specified epitope, antigen or protein, or is a “binding molecule with respect to such an epitope, antigen, or protein, or is referred to as an “anti”-epitope, “anti”-antigen, or “anti”-protein ( e.g., “anti”-Aggrecan, “anti”-MMP13 and/or “anti” ”–ADAMTS5).

Affinity refers to the strength or stability of a molecular interaction. Affinity is usually represented as K_D, or the dissociation constant, which is represented in units of mol/liter (or M). Affinity can also be expressed as an association constant, K_A, which is equal to 1/K_D and which is represented in units (mol/liter)^-one (or M^-one). In the present description, the stability of the interaction between two molecules can basically be expressed as K_D the significance of their interaction; a qualified specialist will understand that based on the ratio K_A=1/K_D, which determines the strength of molecular interaction by its K_D value, you can also use the corresponding K_A meaning. K_D-meaning characterizes the strength of the molecular interaction also from a thermodynamic point of view, since it is associated with a change in the free energy (DG) of binding by the well-known relation DG=RT.ln(K_D) (equivalent to DG=–RT.ln(K_A)), where R=gas constant, T=absolute temperature and ln means natural logarithm.

K _D for biological interactions that are considered significant (eg, specific) typically range from ^10–12 M (0.001 nM) to ^10–5 M (10,000 nM). The stronger the interaction, the lower its K _D .

K _D can also be expressed as the ratio of the dissociation rate constant of the complex, denoted as k _off , to the rate of its association, denoted as k _on (thus, K _D =k _off /k _on and K _A =k _on /k _off ). The dissociation rate k _off is expressed in units of sec ^-1 (where sec means second (SI unit)). The association rate k _on is expressed in units of M– ¹ sec– ¹ . The association rate can vary from 10 ² M ^-1 s ^-1 to about 10 ⁷ M ^-1 s ^-1 , approaching the diffusion-limited association rate constant for bimolecular interactions. The dissociation rate is related to the half-life of this molecular interaction by the ratio t _1/2 =ln(2)/k _off . The dissociation rate can vary from 10 ^-6 sec ^-1 (close to an irreversible complex with t _1/2 for several days) to 1 sec ^-1 (t _1/2 = 0.69 sec).

The specific binding of an antigen-binding protein, such as ISVD, to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, binding saturation assays and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and competitive sandwich assays, and their various variants known per se in the art; as well as other methods specified in this application.

The affinity of a molecular interaction between two molecules can be measured by a variety of methods known per se, such as the well-known surface plasmon resonance (SPR) biosensor method (see, for example, Oberet al. 2001, Intern. Immunology 13: 1551–1559), where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule in flow mode to obtain k_on, k_offvalues and, therefore, K_D (or K_A) values. This can for example be carried out using the well known BIACORE® devices (Pharmacia Biosensor AB, Uppsala, Sweden). Exclusion Kinetic Analysis (KINEXA®) (Drakeet al. 2004 Analytical Biochemistry 328: 35–43) measures binding events in solution without labeling binding partners and is based on the kinetic exclusion of complex dissociation. Solution affinity assays can also be performed using the GYROLAB® Immunoassay System, which provides an automated bioassay platform and rapid sample turnaround (Fraleyet al. 2013, Bioanalysis 5: 1765–74), or ELISA.

Also, it should be clear to the skilled artisan that the measured K _D may correspond to the apparent K _D if the method of measurement affects the true binding affinity of potential molecules in any way, for example, through artifacts associated with applying a single molecule to the biosensor. Also, apparent K _D can be measured if one molecule contains more than one recognition site for another molecule. In such a situation, the measured affinity can be affected by the avidity of the interaction between two such molecules. In particular, accurate measurement of K _D can be very labor intensive, and as a result often apparent K _D values are determined to estimate the binding strength of two molecules. It should be noted that, provided that all measurements are made in a consistent manner (for example, keeping the analysis conditions unchanged), apparent K _D values can be used as an approximation to the true K _D , and therefore, in this document, K _D and apparent K _D should be regarded as equally important or relevant.

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 an ISVD or a polypeptide of the invention) 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 methods for measuring antigen–antigen binding. a binding molecule (such as a polypeptide or ISVD of the invention) and an appropriate antigen. Typically, antigen-binding proteins (such as ISVDs and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (K _D ) of ^10–5 to ^10–12 mol/liter or less, and preferably ^10–7 to 10 ^-12 mol/liter or less, and more preferably 10 ^-8 to 10 ^-12 mol/liter ( i.e. with an association constant (K _A ) of 10 ⁵ to 10 ¹² liter/mol or more, and preferably from 10 ⁷ to 10 ¹² liter/mol or more, and more preferably 10 ⁸ to 10 ¹² liter/mol). Any K _D value greater than 10 ^-4 mol/liter (or any K _A value less than 10 ⁴ liter/mol) is generally considered to be indicative of non-specific binding. Preferably, the monovalent ISVD 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 between 10 and 5 pM or less. See also paragraph n) on pages 53-56 of WO 08/020079.

An ISVD and/or a polypeptide is said to be "specific for" a (first) target or antigen compared to another (second) target or antigen when it binds to the first antigen with an affinity (as described above, and appropriately expressed as a K _D value, K _A value, K _off rate and/or K _on rate) which is at least 10 times greater, such as at least 100 times and preferably at least 1000 times or more, than the affinity with which ISVD and /or the polypeptide binds to the second target or antigen. For example, the ISVD and/or polypeptide may bind to a first target or antigen with a K _D value that is at least 10 times smaller, such as at least 100 times smaller, and preferably at least 1000 times smaller, or even more less than the K _D with which said ISVD and/or polypeptide binds to the second target or antigen. Preferably, when the ISVD and/or polypeptide is "specific for" the first target or antigen as compared to the second target or antigen, it is directed against (as defined in this application) said first target or antigen, but not directed against said second target or antigen.

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, binding saturation assays and/or competitive binding assays such as radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and their various options known from the prior art; as well as other methods specified in this application.

The preferred approach that can be used to assess affinity is the 2-step ELISA procedure (Enzyme-linked immunosorbent assay) by Friguet et al. 1985 (J. Immunol. Methods 77: 305-19). This method establishes a measurement of the binding equilibrium in the solution phase and avoids possible artifacts associated with the adsorption of one of the molecules on the substrate, such as plastic. As the skilled artisan will appreciate, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining the dissociation constant will be understood by those skilled in the art and, for example, include the methods indicated on pages 53-56 of WO 08/020079.

Finally, it should be noted that in many situations the skilled artisan may find it convenient to determine binding affinity relative to some reference molecule. For example, to assess the strength of binding between molecules A and B, one can, for example, use a reference molecule C, known to bind to B and appropriately labeled with a fluorophore or chromophore group or other chemical group such as biotin, so that detection can be easily carried out in ELISA or FACS (fluorescence-activated cell sorting) or other format (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 according to a given concentration or amount of B. This results in an IC ₅₀ value corresponding to the concentration of A at which the signal measured for C in the absence of A is halved. Provided that K _{D ref} , K _D of the reference molecule, and the total concentration c _ref of the reference molecule are known, the apparent K _D for the A–B interaction can be obtained using the following formula: K _D =IC ₅₀ /(1+c _ref / _KDref ). It should be noted that if c _ref << K _{D ref} , K _D ≈ IC ₅₀ . Provided that the measurement of IC ₅₀ is carried out in a uniform manner (for example, by maintaining a fixed c _ref ) for binders that are compared, the difference in strength or stability of molecular interaction can be estimated by comparing IC ₅₀ and this indicator is considered equivalent to K _D or apparent K _D everywhere in the text of this description.

The half-maximal inhibitory concentration (IC ₅₀ ) can also be an indication of the effectiveness of a compound in inhibiting a biological or biochemical function, such as a pharmacological effect. This quantitative indicator indicates how much of a polypeptide or ISVD (for example, a nanobody) is necessary to inhibit half of a given biological process (or process component, i.e. enzyme, cell, cell receptor, chemotaxis, anaplasia, metastases, invasiveness, etc.) . In other words, this is the half-maximal (50%) inhibitory concentration (IC) of the substance (50% IC, or IC ₅₀ ). IC ₅₀ values can be calculated for a given antagonist, such as a polypeptide or ISVD (eg, nanobody) of the invention, by determining the concentration required to inhibit half the maximum biological response of the agonist. The K _D of a drug can be determined by plotting a dose-response curve and examining the effect of different concentrations of an antagonist, such as a polypeptide or ISVD (eg, nanobody) of the invention, on reversing agonist activity.

The term "half maximum effective concentration" (EC ₅₀ ) refers to the concentration of a compound that induces a response halfway between baseline and maximum after a specified exposure time. In the context of the present application, it is used as an indicator of the effectiveness of the polypeptide, ISVD (for example, nanobodies). The EC ₅₀ value, obtained from the dose-response curve with gradual increase in dose, is the concentration of the compound at which 50% of its maximum effect is observed. The concentration is preferably expressed in molar units.

In biological systems, small changes in ligand concentration usually result in rapid changes in response according to a sigmoidal function. The inflection point at which the enhancement of the response with increasing ligand concentration begins to slow down is the EC ₅₀ value. This can be determined mathematically by deriving the best fitting line. In most cases, it is convenient to evaluate based on a schedule. In the case where EC ₅₀ is indicated in the Examples section, the experiments were designed to reflect K _D as accurately as possible. In other words, EC ₅₀ values can then be considered as K _D values. The term "average K _D " refers to the average K _D obtained in at least 1, but preferably more than 1, eg at least 2 experiments. The term "average" refers to the mathematical term "mean" (the sum of the data divided by the number of elements in the data).

This is also related to the value of IC_fifty, which is a criterion for evaluating a compound, its inhibitory action (50% inhibition). For competitive binding assays and functional IC antagonist assays_fifty is the most typical outcome of the dose-response curve. For agonist/stimulant assays, the most typical outcome measure is EC_fifty.

The inhibition constant (Ki) is a measure of how strong an inhibitor is; this is the concentration required to provide half-maximal inhibition. Unlike IC ₅₀ , which may vary depending on experimental conditions, Ki is an absolute value and is often reported as a drug inhibition constant. The inhibition constant Ki can be calculated using the Cheng–Prusoff equation:

in which [L] represents a fixed concentration of the ligand.

The term "activity" of a polypeptide and/or ISVD according to the invention in the context of the present application means a function of the amount of the polypeptide and/or ISVD according to the invention, necessary for the manifestation of its specific effect. This refers to the ability of said polypeptide and/or ISVD of the invention to modulate and/or partially or completely inhibit MMP13 activity and/or modulate and/or partially or completely inhibit ADAMTS5 activity.

In particular, this may refer to the ability of the specified polypeptide to reduce or even completely inhibit the activity of MMP13 defined in this application. Thus, it may refer to the ability of said polypeptide to inhibit proteolysis such as protease activity, endopeptidase activities, substrate binding such as, for example, Aggrecan, Collagen II, Collagen I, Collagen III, Collagen IV, Collagen IX, Collagen X, Collagen XIV and gelatin. The effectiveness can be measured using any suitable assay known in the art or described in this application.

In particular, this may refer to the ability of said polypeptide and/or ISVD to reduce or even completely inhibit the activity of ADAMTS5 as defined herein and/or the activity of MMP13 as defined herein. The effectiveness can be measured using any suitable assay known in the art or described in this application. In the context of the present application, "aggrecanase activity" is defined as the proteolytic cleavage of Aggrecan.

The "potency" of a polypeptide of the invention determines the maximum strength of its effect, as such, at saturating concentrations of the polypeptide. Efficacy means the maximum response achieved by a polypeptide of the invention. This refers to the ability of a polypeptide to provide a desired (therapeutic) effect.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide binds to ADAMTS5 with a K _D value between 1E- ⁰⁷ M and 1E ^-13 M, such as between 1E ^-08 M and 1E ^-12 M, preferably at most 1E ^-07 M, preferably less than 1E ^-08 M or 1E ^-09 M, or even less than 1E ^-10 M, such as 5E ^-11 M, 4E ^-11 M, 3E ^-11 M, 2E ^-11 M, 1, 7E ^-11 M, 1E ^-11 M or even 5E ^-12 M, 4E ^-12 M, 3E ^-12 M, 1E ^-12 M, for example, as determined by the Gyrolab or KinExA method.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide binds to MMP13 with a K _D value between 1E- ⁰⁷ M and 1E ^-13 M, such as between 1E ^-08 M and 1E ^-12 M, preferably at most 1E ^-07 M, preferably less than 1E ^-08 M or 1E ^-09 M, or even less than 1E ^-10 M, such as 5E ^-11 M, 4E ^-11 M, 3E ^-11 M, 2E ^-11 M, 1, 7E ^-11 M, 1E ^-11 M or even 5E ^-12 M, 4E ^-12 M, 3E ^-12 M, 1E ^-12 M, for example, as determined by the Gyrolab or KinExA method.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide binds to Aggrecan with a K _D value between 1E- ⁰⁷ M and 1E ^-13 M, such as between 1E ^-08 M and 1E ^-12 M, preferably at most 1E ^-07 M, preferably less than 1E ^-08 M or 1E ^-09 M, or even less than 1E ^-10 M, such as 5E ^-11 M, 4E ^-11 M, 3E ^-11 M, 2E ^-11 M, 1, 7E ^-11 M, 1E ^-11 M or even 5E ^-12 M, 4E ^-12 M, 3E ^-12 M, 1E ^-12 M, for example, as determined by the Gyrolab or KinExA method.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide binds to ADAMTS5 with a dissociation rate of less than 5E ^-04 sec ^-1 such as, for example, less than 1E ^-04 sec ^-1 or 5E ^-05 sec ^{- 1} , or even less than 1E ^-05 sec ^-1 , for example, as determined by the SPR method.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide binds to MMP13 with a dissociation rate of less than 5E ^-04 sec ^-1 such as less than 1E ^-04 sec ^-1 or 5E ^-05 sec ^-1 , or even less than 1E ^-05 sec ^-1 , for example, as determined by the SPR method.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide binds to Aggrecan with a dissociation rate of less than 5E ^-04 sec ^-1 , such as less than 1E ^-04 sec ^-1 or 5E ^-05 sec ^-1 , or even less than 1E ^-05 sec ^-1 , for example, as determined by the SPR method.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide modulates the activity of ADAMTS5 and/or the activity of MMP13 with an EC ₅₀ value between 1E ^-07 M and 1E ^-12 M, such as between 1E ^-08 M and 1E ^-11 M, for example, as determined in an ELISA binding assay (to determine ADAMTS5 activity), or, for example, in a competitive ELISA, competitive TIMP-2 ELISA, fluorogenic peptide assay, fluorogenic collagen assay, or collagenolytic assay (to determine MMP13 activity).

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide inhibits the activity of ADAMTS5 and/or MMP13 with an IC ₅₀ value between 1E ^-07 M and 1E ^-12 M, such as between 1E ^-08 M and 1E ^-11 M , e.g., as determined in a human protein FRET assay or human protein AlphaLISA assay (to determine ADAMTS5 activity) or, e.g., in a competitive ELISA, a competitive TIMP-2 ELISA, a fluorogenic peptide assay, a fluorogenic collagen assay, or a collagenolytic assay (to determine MMP13 activity).

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide inhibits the enzymatic activity of ADAMTS5 and/or MMP13 with an IC ₅₀ value of maximum 1E ^-07 M, preferably 1E ^-08 M, 5E ^-09 M, or 4E ^-9 M , 3E ^–9 M, 2E ^–9 M, such as 1E ^–9 M.

An amino acid sequence, such as an ISVD or a polypeptide, is listed as “cross-reactive” to two different antigens or antigenic determinants (such as , for example, ADAMTS5 from different mammalian species, e.g., human ADAMTS5, bovine ADAMTS5, rat ADAMTS5, guinea pig ADAMTS5, ADAMTS5 mouse or cynomolgus ADAMTS5, or such as , for example, MMP13 from different mammalian species, such as human MMP13, dog MMP13, bovine MMP13, rat MMP13, porcine MMP13, mouse MMP13, rabbit MMP13, cynomolgus monkey MMP13 and/or rhesus monkey MMP13 , or such as, for example, Aggrecan from different mammalian species, for example, Aggrecan of a human, Aggrecan of a dog, Aggrecan of a bull, Aggrecan of a rat, Aggrecan of a pig, Aggrecan of a mouse, Aggrecan of a rabbit, Aggrecan of a cynomolgus macaque and/or Aggrecan of a rhesus macaque), if it is is specific for (as defined in this application) these various antigens or antigenic determinants. It should be understood that an ISVD or polypeptide may be considered cross-reactive, although the binding affinity for two different antigens may differ, for example by factors of 2, 5, 10, 50, 100 or more, provided that it is specific for (as defined in this application) of these different antigens or antigenic determinants.

ADAMTS5 is also known as ADAMTS11, ADMP-2 or Aggrecanase-2. Relevant information about the ADAMTS5 structure can be found, for example, by UniProt access numbers, as shown in Table B-1 below (see Table B).

Table B–1 Protein, access number Gene organism Q9UNA0 ADAMTS5 H. sapiens Q9TT92 ADAMTS5 B. taurus Q6TY19 ADAMTS5 R.norvegicus H0VFP0 ADAMTS5 Cavia Porcellus Q9R001 ADAMTS5 M.musculus F6Z3S6 ADAMTS5 M. mulatta

"Human ADAMTS5" refers to ADAMTS5 comprising the amino acid sequence of SEQ ID NO: 67. In one aspect, the polypeptide of the invention specifically binds to ADAMTS5 from Human sapiens, Mus musculus, Cavia Porcellus, Bos taurus, Macaca mulatta and/or Rattus norvegicus, preferably ADAMTS5 human, preferably SEQ ID NO: 67.

MMP13 is also known as CLG3 or Collagenase 3, MANDP1, MMP-13, Matrix metallopeptidase 13 or MDST. Relevant information about the structure of MMP13 can be found, for example, by UniProt access numbers, as shown in Table B-2 below (see Table B).

Table B–2 Protein, access number Gene organism NP_002418.1 MMP13 H. sapiens XP_001154361.1 MMP13 P.troglodytes XP_001098996.1 MMP13 M. mulatta XP_536598.3 MMP13 C. lupus NP_776814.1 MMP13 B. taurus NP_032633.1 Mmp13 M.musculus NP_598214.1 Mmp13 R.norvegicus XP_003640635.1 MMP13 G. gallus

"Human MMP13" refers to an MMP13 comprising the amino acid sequence of SEQ ID NO: 66. In one aspect, a polypeptide of the invention specifically binds to MMP13 from Human sapiens, Mus musculus, Canis lupus, Bos taurus, Macaca mulatta, Rattus norvegicus, Gallus gallus and/ or P. troglodytes , preferably human MMP13, preferably SEQ ID NO: 66.

Aggrecan is also known as aggrecan 1, ACAN, AGC1, AGCAN, CSPGCP, MSK16, SEDK, cartilage-specific proteoglycan core protein (CSPCP), or chondroitin sulfate proteoglycan 1 (CSPG1). Aggrecan in humans is encoded by the ACAN gene, which is located on chromosome Chr 15: q26.1. Relevant information about Aggrecan's structure can be found, for example, through UniProt access numbers, as shown in Table B-3 below (see Table B).

Table B–3 Protein, access number Gene organism P16112 ACAN H. sapiens XP_003952775.2 ACAN P.troglodytes XP_002804990.1 ACAN M. mulatta Q28343 ACAN C. lupus P13608 ACAN B. taurus Q61282 ACAN M.musculus P07897 ACAN R.norvegicus Q29011 ACAN S. scrofa G1U677–1 ACAN O. cuniculus NP_990286.2 ACAN G. gallus

"Human aggrecan" refers to an aggrecan comprising the amino acid sequence of SEQ ID NO: 68. In one aspect, a polypeptide of the invention specifically binds to Aggrecan from Human sapiens, Mus musculus, Bos taurus, Macaca mulatta, Pan troglodytes, Gallus gallus, Canis lupus, Sus scrofa, Oryctolagus cuniculus and/or Rattus norvegicus, preferably human Aggrecan, preferably SEQ ID NO: 68.

The terms "(cross)-block", "(cross)-block", "(cross)-block", "competitive binding", "(cross)-compete", "(cross)-compete" and "(cross)-compete" “competitive” are used interchangeably herein as referring to the ability of an immunoglobulin, antibody, ISVD, polypeptide, or other binding agent to prevent other immunoglobulins, antibodies, ISVDs, polypeptides, or binding agents from binding to a given target. The degree to which an immunoglobulin, antibody, ISVD, polypeptide, or other binding agent is able to interfere with the binding of another agent to a target, and, accordingly, whether this can be called cross-blocking in accordance with the invention, can be determined using competitive binding assays, which are conventional in the art, such as, for example, screening purified ISVDs against phage-exposed ISVDs in a competitive ELISA. Particularly suitable quantitative cross-blocking assays include ELISA.

Other methods for determining whether an immunoglobulin, antibody, ISVD, polypeptide, or other binding agent directed against a target, (cross)-blocks, is able to (cross)-block, competitively binds, or is (cross)-competitive, as defined herein application may include a "sandwich analysis" based on SPR, such as, for example, 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).

"ADAMTS5 activities" and "ADAMTS5 activities" (these terms are used interchangeably in this application) include, but are not limited to, enzymatic activities such as proteolysis, for example, protease activity (also called proteinase or peptidase activity) and endopeptidase activities, with one side, and activities via exosites, such as, for example, recognition and/or binding of a substrate, for example, via a disintegrin-like domain, a central thrombospondin type I-like (TS) repeat, a cysteine-rich domain, a spacer region, and/or additional TS motives. ADAMTS5 activities include binding and/or proteolysis of substrates such as hyaluronan-binding extracellular chondroitin sulfate proteoglycan (CSPG) proteins such as Aggrecan, Versican, Brevican, Neurocan, Decorin and Viglican. In the context of the present application, proteolysis is the cleavage of proteins into smaller polypeptides or amino acids by hydrolysis of peptide bonds that link amino acids together in a polypeptide chain.

"MMP13 activities" and "MMP13 activities" (these terms are used interchangeably in this application) include, but are not limited to, proteolysis such as protease activity (also referred to as proteinase or peptidase activity) and endopeptidase activities on the one hand, and binding to substrate, for example through the Hemopexin-like domain and the peptidoglycan-binding domain. MMP13 activities include binding and/or proteolysis of substrates such as Aggrecan, Collagen II, Collagen I, Collagen III, Collagen IV, Collagen IX, Collagen X, Collagen XIV and Gelatin. In the context of the present application, proteolysis is the cleavage of proteins into smaller polypeptides or amino acids by hydrolysis of peptide bonds that link amino acids together in a polypeptide chain.

In the context of the present invention, “modulating” or “modulating” generally means a change in ADAMTS5 and/or MMP13 activity measured using an appropriatein vitro, cellular or in vivo assay (such as assays referred to in this application). In particular, "modulating" or "modulate" can mean either a decrease or inhibition of activity, or alternatively an increase in ADAMTS5 and/or MMP13 activity as measured using an appropriate in vitro, cellular or in vivo analysis (for example, such as the analyzes specified in this application), 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 and/or MMP13 in the same assay under the same conditions, but without the presence of ISVD or a polypeptide of the invention.

Accordingly, the present invention relates to a polypeptide described in this application, where the specified polypeptide modulates the activity of ADAMTS5 and/or MMP13, preferably inhibits the activity of ADAMTS5 and/or MMP13.

Accordingly, the present invention relates to a polypeptide described in this application, where the specified polypeptide inhibits the protease activity of ADAMTS5, for example inhibits the proteolysis of a substrate such as Aggrecan, Versican, Brevican, Neurocan, Decorin, and/or Viglican.

Accordingly, the present invention provides a polypeptide as described herein, wherein said polypeptide blocks the binding of ADAMTS5 to a substrate such as Aggrecan, Versican, Brevican, Neurocan, Decorin and/or Viglican, where said substrate is preferably Aggrecan.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide blocks the binding of ADAMTS5 to Aggrecan by at least 20%, for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, for example, as determined by ELISA.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide antagonizes or inhibits the activity of ADAMTS5, such as (i) protease activity, preferably cleavage of Aggrecan, Versican, Brevican, Neurocan, Decorin and/or Viglican, preferably cleavage of Aggrecan; preferably antagonizes ADAMTS5 aggrecanase activity; (ii) binding of a substrate to ADAMTS5, such as an ADAMTS5 exosite, eg, a disintegrin-like domain, a central thrombospondin type I-like (TS) repeat, a cysteine-rich domain, a spacer region, or an additional TS motif.

Accordingly, the present invention relates to a polypeptide described in this application, where the specified polypeptide inhibits the protease activity of MMP13, for example inhibits the proteolysis of a substrate such as Aggrecan, Collagen II, Collagen I, Collagen III, Collagen IV, Collagen IX, Collagen X, Collagen XIV and/or gelatin.

Accordingly, the present invention relates to a polypeptide described in this application, where the specified polypeptide blocks the binding of MMP13 to a substrate such as Aggrecan, Collagen II, Collagen I, Collagen III, Collagen IV, Collagen IX, Collagen X, Collagen XIV and/or Gelatin where said Collagen is preferably Collagen II.

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide blocks the binding of MMP13 to Collagen and/or Aggrecan by at least 20%, for example, at least 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, or even more, for example, as determined by competitive ELISA assays (see Howes et al. 2014 J. Biol. Chem. 289:24091-24101).

In one aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide antagonizes or inhibits the activity of MMP13, such as (i) protease activity, preferably the cleavage of Aggrecan and/or Collagen, where the specified Collagen is preferably Collagen II; (ii) Collagen binding to the hemopexin-like domain.

In one aspect, the present invention relates to a polypeptide described in this application, where the specified polypeptide inhibits the protease activity of ADAMTS5 and/or MMP13, preferably at least 5%, for example 10%, 20%, 30%, 40%, 50% or even greater, such as at least 60%, 70%, 80%, 90%, 95%, or even greater, as determined by any suitable method known in the art, such as competitive assays, or as described in the Examples section.

Although ADAM, ADAMTS and MMP share an Aggrecan binding site that is very similar in both sequence and overall shape, e.g. the ADAMTS4 and ADAMTS5 catalytic domains share a high degree of sequence similarity, the present inventors were able to identify ISVDs that were target-specific, as demonstrated in the examples. Target specificity could also help to avoid or at least limit musculoskeletal syndrome, which is a side effect caused by broad spectrum inhibitors.

In one aspect, the invention relates to an ADAMTS5 binding agent, such as an ISVD and a polypeptide of the invention, wherein said ADAMTS5 binding agent does not bind to ADAMTS4, ADAMTS1, ADAMTS15, MMP1 and/or MMP14 (membrane type). Preferably, the present invention relates to a polypeptide as defined herein, wherein said ADAMTS5-binding ISVD does not bind to ADAMTS4, MMP1, or MMP14.

In one aspect, the invention provides an MMP13 binding agent, such as an ISVD and a polypeptide of the invention, wherein said MMP13 binding agent does not bind to MMP1 and/or MMP14 (membrane type). Preferably, the present invention provides a polypeptide as defined herein, wherein said MMP13-binding ISVD does not bind to MMP1 or MMP14.

Unless otherwise indicated, the terms “immunoglobulin” and “immunoglobulin sequence”—whether used herein as referring to a heavy chain-only antibody or to a conventional 4-chain antibody—are used as generic terms, including both full-length antibody , and its individual chains, as well as all their parts, domains or fragments (including, but not limited to, antigen-binding domains or fragments, such as V _HH domains or V _H /V _L domains, respectively).

The term "domain" (polypeptide or protein) in the context of the present application refers to a folded protein structure that has the ability to maintain its tertiary structure independently of the rest of the protein. Typically, domains are responsible for certain functional properties of proteins, and in many cases can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and/or domain.

The term “immunoglobulin domain” as used herein refers to the globular region of an antibody chain (such as , for example, a conventional 4-chain or heavy chain-only antibody chain) or a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are characterized in that they retain the immunoglobulin-specific folding of antibody molecules, which consists of a bilayer sandwich of about seven antiparallel beta chains arranged in two beta sheets, optionally stabilized by a conserved disulfide bond.

The term "immunoglobulin variable domain" in the context of this application means an immunoglobulin domain essentially consisting of four "framework regions", which are referred to in the art and hereinafter as "framework region 1" or "FR1"; as “wireframe 2” or “FR2”; as “wireframe 3” or “FR3”; and as “frame region 4” or “FR4”, respectively; wherein said 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”; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be specified as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. This is the variable domain(s) of an immunoglobulin that confers antigen specificity to an antibody because it contains an antigen-binding site.

The term “immunoglobulin single variable domain” (abbreviated as “ISVD” or “ISV”), and used interchangeably with “single variable domain”, means molecules where an antigen binding site is present on, and formed by, a single immunoglobulin domain. This distinguishes single immunoglobulin variable domains from "traditional" immunoglobulins or fragments thereof, where two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ) interact to form an antigen binding site. In the latter case, the complementarity determining regions (CDRs) of both V _H and V _L will contribute to the antigen binding site, ie. a total of 6 CDRs will be involved in the formation of the antigen binding site.

As defined above, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD, or IgE molecule; known in the art) or Fab fragment, F(ab')2 fragment, Fv fragment, such as a disulfide-linked Fv or scFv fragment, or a diabody (all known in the art) derived from such a conventional 4-chain antibody would not normally be considered a single immunoglobulin variable domain because, in these cases, binding to the appropriate antigen epitope would normally does not occur through a single (single) immunoglobulin domain, but through a pair of (associated) immunoglobulin domains, such as the light and heavy chain variable domains, ie. via the V _H –V _L pair of immunoglobulin domains that co-bind to the epitope of the corresponding antigen.

In contrast, ISVDs are able to specifically bind to an antigen epitope without pairing with an additional immunoglobulin variable domain. The ISVD binding site is formed by a single V _HH , V _H or V _L domain. Therefore, an ISVD antigen-binding site is formed by no more than three CDRs.

Thus, a single variable domain may be a light chain variable domain sequence ( eg, V _L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence ( eg, V _H sequence or V _HH sequence) or a suitable fragment thereof; provided that it is capable of forming a single antigen-binding unit ( i.e. , a functional antigen-binding unit that essentially consists of a single variable domain, such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

In one embodiment, the ISVDs are heavy chain variable domain sequences ( eg, V _H sequence); more specifically, ISVDs can be heavy chain variable domain sequences that are derived from a conventional four chain antibody, or heavy chain variable domain sequences that are derived from a heavy chain only antibody.

For example, an ISVD may be a (single)-domain antibody (or an amino acid that is suitable for use as a (single)-domain antibody), a "dAb" or sdAb (or an amino acid that is suitable for use as a dAb), or Nanobody (defined in this application and including, but not limited to, VHH); other single variable domains, or any suitable fragment of any of the above.

In particular, the ISVD may be a Nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobody® and Nanoclone® are registered trademarks of Ablynx N.V.]. With regard to the general description of nanobodies, reference is made to the following further description, as well as to the prior art cited in this application, for example, WO 08/020079 (p. 16).

“V _HH domains”, also known as VHH, V _H H domains, VHH antibody fragments and VHH antibodies, were originally described as the antigen-binding (variable) domain of an immunoglobulin “antibody with only heavy chains” ( i.e. “an antibody lacking a lung chains"; Hamers-Casterman et al. 1993 Nature 363: 446-448). The term “V _HH domain” was chosen to distinguish these variable domains from the heavy chain variable domains that are present in traditional 4-chain antibodies (which are referred to in this application as “V _H domains” or “VH domains”), and from light chain variable domains that are present in traditional 4-chain antibodies (which are referred to in this application as "V _L domains" or "VL domains"). A more detailed description of VHH and Nanotel can be found in the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as in the following patent applications, which are listed as prior art: WO 94/04678, WO 95/04079 and WO 96/34103 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 Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 Algonomics NV and Ablynx NV; WO 01/90190 National Research Council of Canada; WO 03/025020 (= EP 1433793) Institute of Antibodies, as well as 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 Ablynx NV and other published patent applications Ablynx NV You can also refer to other documents of the prior art referred to in these applications, and in particular to the list reference documents on pages 41-43 of international application WO 06/040153, this list and reference documents are incorporated into this application by reference. As described in these references, Nanobodies (particularly VHH sequences and partially humanized Nanobodies) may in particular be characterized by the presence of one or more "Unique Residues" in one or more framework sequences. A more detailed description of Nanobodies, including humanization and/or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or “fusions of Nanobodies”, multivalent constructs (including some non-limiting examples of linker sequences) and various modifications to increase the half-life of Nanobodies and their preparations can be found, for example, in WO 08/101985 and WO 08/142164. Also a general description of Nanobodies can be found in the prior art cited in this application, for example, described in WO 08/020079 (p. 16).

In particular, the framework sequences present in Aggrecan, ADAMTS5 and/or MMP13 binding agents of the invention, such as ISVD and/or polypeptides of the invention, may contain one or more unique residues (for example, as described in WO 08/020079 (Tables A -3-A-8)), thus the aggrecan, ADAMTS5 and/or MMP13 binding agent of the invention is a Nanobody. Some preferred, but non-limiting examples (suitable combinations) of such framework sequences will be apparent from the further disclosure provided in this application (see, for example, Table A-2). Typically, Nanobodies (particularly V _HH sequences and partially humanized Nanobodies) may in particular be characterized by the presence of one or more “ Unique Residues ” in one or more framework sequences (as, for example, described in more detail in WO 08/020079, page 61 line 24 - page 98 line 3). As used herein, the term "represented" in the context of any SEQ ID NO is equivalent to "includes or consists of" the specified SEQ ID NO, and preferably is equivalent to "consists of" the specified SEQ ID NO.

More specifically, the invention provides Aggrecan, ADAMTS5 and/or MMP13 binding agents comprising at least one immunoglobulin single variable domain which is an amino acid sequence with the (general) structure:

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and which:

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, 3 or 4 (see Table A-1), where in order to determining the degree of amino acid identity, amino acid residues that form CDR sequences are not taken into account. In this regard, reference is also made to Table A-2, which lists framework sequences 1 (SEQ ID NO: 7), framework sequences 2 (SEQ ID NO: 9, 15 and 20), framework sequences 3 (SEQ ID NO: 11, 17 and 22) and framework sequences 4 (SEQ ID NO: 13) of immunoglobulin single variable domains of SEQ ID NO: 2, 3 and 4; or

ii) framework sequence combinations as shown in Table A-2;

and in which:

iii) preferably one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from Unique residues, such as those listed in Table A-3 - Table A-8 of WO 08/020079.

Aggrecan, ADAMTS5 and/or MMP13 binding agents of the invention, such as ISVDs and/or polypeptides of the invention, may also contain the specific mutations/amino acid residues described in the following co-pending US Provisional Applications, which are all entitled “Improved immunoglobulin variable domains ”: US 61/994552 filed May 16, 2014; US 61/014015 filed June 18, 2014; US 62/040167 filed August 21, 2014; and US 62/047560 filed September 8, 2014 (all owned by Ablynx N.V.).

In particular, Aggrecan, ADAMTS5 and/or MMP13 binding agents of the invention, such as ISVDs and/or polypeptides of the invention, may respectively 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 with K or Q at position 110; or (v) V at position 11 and L at position 89; or any suitable combination of (i) - (v).

As also described in these co-pending US Provisional Applications, when Aggrecan, ADAMTS5 and/or MMP13 binding agents of the invention, such as ISVD and/or polypeptides of the invention, contain mutations according to one of the above (i) - ( v) (or a suitable combination of these):

- the amino acid residue at position 11 is preferably selected from L, V or K (and most preferably is V); and/or

- the amino acid residue at position 14 is preferably respectively selected from A or P; and/or

- the amino acid residue at position 41 is preferably respectively selected from A or P; and/or

- the amino acid residue at position 89 is preferably respectively selected from T, V or L; and/or

- the amino acid residue at position 108 is preferably respectively selected from Q or L; and/or

- the amino acid residue at position 110 is preferably respectively selected from T, K or Q; and/or

- the amino acid residue at position 112 is preferably selected from S, K or Q, respectively.

As described in these co-pending US Provisional Applications, these mutations are effective in preventing or reducing the binding of so-called "preexisting antibodies" to immunoglobulins and compounds of the invention. For this purpose, Aggrecan, ADAMTS5 and/or MMP13 binding agents of the invention, such as ISVD and/or polypeptides of the invention, may also contain (optionally in combination with the indicated mutations) a C-terminal extension (X)n (where n is from 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 a (preferably natural) amino acid residue which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)), see for example US Provisional Applications and also WO 12/175741. In particular, an Aggrecan, ADAMTS5 and/or MMP13 binding agent of the invention, such as an ISVD and/or a polypeptide of the invention, may contain such a C-terminal extension when it forms or includes a C-terminal region of a protein, polypeptide or other compound. designs (see, for example, said US Provisional Applications and also WO 12/175741).

The Aggrecan, ADAMTS5 and/or MMP13 binding agent of the invention may be an immunoglobulin, such as an immunoglobulin single variable domain, obtained by any suitable method and from any suitable source, and may, for example, be natural V _HH sequences ( i.e. , from camelid species) or synthetic or semi-synthetic amino acid sequences, including, but not limited to, "humanized" (as defined in this application) Nanobodies or VHH sequences, "camelized" (as defined in this application) immunoglobulin sequences (and, in particular , camelized sequences of heavy chain variable domains), as well as Nanobodies, which are obtained by methods such as affinity maturation (for example, based on synthetic, randomized or natural sequences of immunoglobulins), CDR-grafting, masking of surface residues, pooling of fragments derived from various immunoglobulin sequences, PCR assembly using overlapping primers, and similar methods for constructing immunoglobulin sequences well known to those skilled in the art; or using any suitable combination of any of the above, as described later in this application. Also, when an immunoglobulin comprises a V _HH sequence, said immunoglobulin may be suitably humanized, as described hereinafter, to provide one or more additional (partially or fully) humanized immunoglobulins of the invention. Similarly, when an immunoglobulin comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), said immunoglobulin may optionally be further humanized in an appropriate manner, also as described herein, also to provide one or more additional (partially or fully) humanized immunoglobulins according to invention.

"Domain Antibodies", also known as "Dab", "Domain Antibodies" and "dAb" (the terms "Domain Antibodies" and "dAb" are used as trademarks by 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, as well as, for example, WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. Domain antibodies essentially correspond to VH or VL domains of non-camelid mammals, in particular human 4-chain antibodies. For binding to an epitope as a single antigen-binding domain, i.e. without mating with a V _L or V _H domain, respectively, specific selection is needed based on such antigen-binding properties, eg using human single V _H or V _L domain sequence libraries. Domain antibodies have, like V _HH s, a molecular weight of about 13 to about 16 kDa and, if formed from fully human sequences, do not require humanization, eg, for human therapeutic use.

It should also be noted that, although less preferred in the context of the present invention because they are non-mammalian, single variable domains can be derived from certain shark species (e.g. so-called “IgNAR domains”, see e.g. WO 05/18629 ).

The present invention relates in particular to ISVDs, wherein said ISVDs are selected from the group consisting of VHHs, humanized VHHs, and camelized VHs.

The amino acid residues of the VHH domain are numbered according to the common numbering for V _H domains according to Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) applicable to VHH domains from camelids, as shown , for example, in FIG. 2 in Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999), as is known in the art. Alternative methods for numbering amino acid residues of V _H domains, which can also be applied similarly to VHH domains, are known in the art. However, in the present specification, claims, and drawings, Kabat numbering applies to VHH domains as described above, unless otherwise indicated.

It should be noted that - as is well known in the art for V _H domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., in of the actual sequence, one or more positions according to the Kabat numbering may be unoccupied, or the actual sequence may contain more amino acid residues than the number that is possible according to the Kabat numbering). This means that, in general, the numbering according to Kabat 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 the VH domain and VHH domain should typically be in the range of 110 to 120, often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

With regard to CDRs, as is well known in the art, there are many conventional methods for defining and describing VH or VHH fragment CDRs, such as the Kabat definition (which is based on sequence variability and is the most widely used) and the Chothia definition (which is based on the localization structural loop regions). See, for example, the website http://www.bioinf.org.uk/abs/. For purposes of this specification and claims, CDRs are most preferably determined based on the Abm definition (which is based on the Oxford Molecular “AbM” Antibody Modeling Program), as this is considered to be an optimal compromise between the Kabat and Chothia definitions (see http://www.bioinf. org.uk/abs/). In the context of this application, FR1 includes amino acid residues at positions 1-25, CDR1 includes amino acid residues at positions 26-35, FR2 includes amino acids at positions 36-49, CDR2 includes amino acid residues at positions 50-58, FR3 includes amino acid residues at positions 59-94, CDR3 includes amino acid residues at positions 95-102, and FR4 includes amino acid residues at positions 103-113.

In the context of the present invention, the term “immunoglobulin single variable domain” or “single variable domain” includes polypeptides that are derived from a non-human source, preferably camelid, preferably camelid heavy chain antibody only. They can be humanized as described in this application. In addition, the term includes polypeptides derived from non-camelid sources, such as mice or humans, that have been "camelized" as described in this application.

Therefore, ISVDs such as Domain Antibodies and Nanobodies (including VHH domains) can be humanized. In particular, humanized ISVDs such as Nanobodies (including VHH domains) may be ISVDs as defined herein in general but in which at least one amino acid residue (and in particular at least one from framework residues) which is and/or which corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions can be determined by comparing the framework sequence of a native V _HH sequence with the corresponding framework sequence of one or more closely related human V _H sequences, whereupon one or more potentially useful humanizing substitutions (or combinations thereof) so determined can be introduced into said V _HH sequence (by any method known per se, as described hereinafter) and the resulting humanized V _HH sequences can be tested for target affinity, stability, ease and level of expression, and/or other desired properties. In this way, with a limited degree of trial and error, a person skilled in the art will be able to determine other suitable humanizing substitutions (or suitable combinations thereof) based on the disclosure presented in this application. Also, based on the foregoing, the (framework regions) of an ISVD such as a Nanotbody (including VHH domains) may be partially humanized or fully humanized.

Another particularly preferred class of ISVDs of the invention includes ISVDs with an amino acid sequence that matches the amino acid sequence of the natural V _H domain, but which has been "camelized", ie. by replacing one or more amino acid residues in the amino acid sequence of the native V _H domain from a conventional 4-chain antibody with one or more amino acid residues that are present at the corresponding position(s) in the V _HH domain of the heavy chain-only antibody. This can be done in a manner known per se, which should be clear to the skilled person, for example, based on the description presented in this application. Such “camelizing” substitutions are preferably introduced at amino acid positions that form and/or are present at the V _H –V _L interface and/or at positions of so-called Camelid-derived unique residues as defined herein (see also e.g. WO 94/04678 and Davies and Riechmann (1994 and 1996)). Preferably, the V _H sequence that is used as a starting material or starting point for the formation or construction of camelized immunoglobulin single variable domains is preferably a V _H sequence from a mammal, more preferably a human V _H sequence, such as a V _H 3 sequence. However, it should be noted that such camelized immunoglobulin single variable domains of the invention can be prepared by any suitable method known per se, and thus are not strictly limited to polypeptides that are prepared using a polypeptide that includes a natural V _H domain as a starting point. 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)

For example, again as described hereinafter, both “humanization” and “camelization” can be accomplished by providing a nucleotide sequence that encodes a natural V _HH domain or V _H domain, respectively, and then substituting in a manner known per se, one or more codons in said nucleotide sequence such 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 ISVDs of the invention. Alternatively, based on the amino acid sequence of the natural V _HH domain or V _H domain, respectively, the amino acid sequence of the desired humanized or camelized ISVDs of the invention, respectively, can be designed and then synthesized de novo using peptide synthesis techniques known per se. Also, based on the amino acid sequence or nucleotide sequence of a natural V _HH domain or V _H domain, respectively, a nucleotide sequence encoding the desired humanized or camelized ISVDs of the invention, respectively, can be designed and then synthesized de novo using nucleic acid synthesis methods known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se to provide the desired ISVDs of the invention.

ISVDs such as Domain Antibodies and Nanobodies (including VHH domains and humanized VHH domains) can also be affinity matured by making one or more changes to the amino acid sequence of one or more CDRs, where such changes result in improved affinity of the resulting ISVD for its corresponding antigen compared to the corresponding parent molecule. The affinity matured ISVD molecules of the invention can be prepared by methods known in the art, for example as described by 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).

A method for constructing/selecting and/or obtaining a polypeptide based on an ISVD, such as V _H , V _L , V _HH , Domain antibody or Nanotelo, is also referred to in this application as “formatting” said ISVD; and an ISVD that is part of a polypeptide is listed as "formatted" or "in the format" of said polypeptide. Examples of ways in which an ISVD format can be specified and examples of such formats will be understood by those skilled in the art based on the disclosure provided in this application; and such a single immunoglobulin variable domain in a predetermined format constitutes another aspect of the invention.

Preferred CDRs are shown in Table A-2.

In particular, the present invention relates to a polypeptide described in this application, where the specified ISVD, which specifically binds to MMP13, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein

(i) CDR1 is SEQ ID NO: 8; and amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 8;

(ii) CDR2 is SEQ ID NO: 10; and amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 10; and

(iii) CDR3 is SEQ ID NO: 12; and amino acid sequences that are 1, 2, or 3 amino acid different from SEQ ID NO: 12.

In particular, the present invention relates to a polypeptide described in this application, where the specified ISVD, which specifically binds to MMP13, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is SEQ ID NO: 8, CDR2 is SEQ ID NO: 10, and CDR3 is SEQ ID NO: 12.

In particular, the present invention relates to an ISVD described in this application, where the specified ISVD, specifically binding to ADAMTS5, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein

(i) CDR1 is SEQ ID NO: 14 [GRTVSSYAMG]; and amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 14;

(ii) CDR2 is SEQ ID NO: 16 [GISRSAERTY]; and amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 16; and

(iii) CDR3 is SEQ ID NO: 18 [DLDPNRIFSREEYAY]; and amino acid sequences that are 1, 2, or 3 amino acid different from SEQ ID NO: 18.

In particular, the present invention relates to an ISVD described in this application, where the specified ISVD, specifically binding to ADAMTS5, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is SEQ ID NO: 14, CDR2 is SEQ ID NO: 16, and CDR3 is SEQ ID NO: 18.

In particular, the present invention relates to the ISVD described in this application, where the specified ISVD, specifically binding to Aggrecan, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein

(i) CDR1 is SEQ ID NO: 19; and amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 19;

(ii) CDR2 is SEQ ID NO: 21; and amino acid sequences that have 1, 2 or 3 amino acid differences from SEQ ID NO: 21; and

(iii) CDR3 is SEQ ID NO: 23; and amino acid sequences that are 1, 2 or 3 amino acid different from SEQ ID NO: 23.

In particular, the present invention relates to the ISVD described in this application, where the specified ISVD, specifically binding to Aggrecan, essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is SEQ ID NO: 19, CDR2 is SEQ ID NO: 21, and CDR3 is SEQ ID NO: 23.

In particular, the present invention relates to a polypeptide described in this application, where the specified ISVD that specifically binds to MMP13 is SEQ ID NO: 2.

In particular, the present invention relates to a polypeptide described in this application, where the specified ISVD that specifically binds to ADAMTS5 is SEQ ID NO: 3.

In particular, the present invention relates to a polypeptide described in this application, where the specified ISVD, specifically binding to Aggrecan, is SEQ ID NO: 4.

In yet another preferred embodiment, the Aggrecan, ADAMTS5 and/or MMP13 binding agent of the invention comprises at least two CDR1 sequences, at least two CDR2 sequences, and at least two CDR3 sequences, each independently selected from the following table:

CDR1 CDR2 CDR3 SEQ ID NO: 8(CDR1a) SEQ ID NO: 10(CDR2a) SEQ ID NO: 12(CDR3a) SEQ ID NO: 14(CDR1b) SEQ ID NO: 16(CDR2b) SEQ ID NO: 18(CDR3b) SEQ ID NO: 19(CDR1c) SEQ ID NO: 21(CDR2c) SEQ ID NO: 23(CDR3c)

In the above Aggrecan, ADAMTS5 and/or MMP13 binding agent, the sequence order is preferably CDR1a-CDR2a-CDR3a-Linker-CDR1b-CDR2b-CDR3b, where the Linker is a polypeptide having a length of more than 5 amino acids which is suitable for first row binding CDR (CDR1a-CDR2a-CDR3a) with a second row (CDR1b-CDR2b-CDR3b). Preferably, the linker is selected from Table C below, and most preferably is a linker with an amino acid sequence according to SEQ ID NO: 35. In a more preferred embodiment, Aggrecan, ADAMTS5 and/or MMP13 binding agent includes all nine CDR sequences from the table below. above, where the CDR sequences and linker polypeptides are in the following order: CDR1a-CDR2a-CDR3a-Linker1-CDR1b-CDR2b-CDR3b-Linker2-CDR1c-CDR2c-CDR3c, where Linker1 and Linker2 are each a polypeptide of at least 5 amino acids, and wherein the Linker1 and Linker2 polypeptide sequences are identical or not identical to each other. Preferably, Linker1 and Linker2 are each independently selected from Table C below, and most preferably, at least one (preferably both) of the linkers has/have an amino acid sequence according to SEQ ID NO: 35.

In yet another preferred embodiment, the Aggrecan, ADAMTS5 and/or MMP13 binding agent of the invention preferably comprises at least the CDR sequences listed in the following table:

SEQ ID NO: 8 (CDR1a) SEQ ID NO: 10(CDR2a) SEQ ID NO: 12(CDR3a) SEQ ID NO: 14(CDR1b) SEQ ID NO: 16(CDR2b) SEQ ID NO: 18(CDR3b) SEQ ID NO: 19(CDR1c) SEQ ID NO: 21(CDR2c) SEQ ID NO: 23(CDR3c) SEQ ID NO: 19(CDR1d) SEQ ID NO: 21(CDR2d) SEQ ID NO: 23(CDR3d)

In the above embodiment, the order of the CDR sequences may be CDR1a-CDR2a-CDR3a-Linker1-CDR1b-CDR2b-CDR3b-Linker2-CDR1c-CDR2c-CDR3c-Linker3-CDR1d-CDR2d-CDR3d; where Linker1, Linker2 and Linker3 each is a polypeptide of at least 5 amino acids, and where the polypeptide sequences of Linker1, Linker2 and Linker3 are identical to each other or not identical to each other. Preferably, Linker1, Linker2 and Linker3 are independently selected from Table C below, and most preferably at least one (preferably all three) of the linkers has/have an amino acid sequence according to SEQ ID NO: 35. It is understood that in the preferred embodiments presented in the context of the two tables above, the CDR sequences may be linked to each other via framework sequences as described herein, and preferably the framework sequences disclosed in Table A-2 may be used in this connection.

It should be understood that, without limitation, the immunoglobulin single variable domains of the present invention can be used as a "building block" to produce a polypeptide, which optionally contains one or more additional immunoglobulin single variable domains that can serve as a building block.

There is a need in the art for an effective therapy for joint cartilage disorders such as osteoarthritis. Even when administered intra-articularly, the retention time of most drugs for the treatment of affected cartilage is insufficient. Without wishing to be bound by a particular theory, the present inventors hypothesized that the efficacy of a therapeutic drug, such as a construct, polypeptide, and ISVD of the invention, could be modulated by combining the therapeutic drug with a moiety that could "anchor" the drug in the joint and, therefore, increase drug retention time, but which should not interfere with the efficacy of said therapeutic drug (this group is also referred to in this application as "cartilage anchoring protein" or "CAP"). This concept of anchoring could modulate not only drug efficacy but also functional specificity for the diseased joint by reducing toxicity and side effects, thus expanding the range of possible beneficial drugs.

It is contemplated that the format of the molecule for clinical use includes one or two building blocks, such as ISVDs, binding MMP13 and/or ADAMTS5, and one or more building blocks, such as ISVDs, with such a confining mode of action, and possibly other components. The present invention demonstrates that such formats support both MMP13 and/or ADAMTS5 binding and therapeutic effect, eg inhibitory activity, as well as retention properties. One or more building blocks, such as ISVD, with a confining mode of action can be any building block that has a confining effect ("CAP building block") in diseases in which MMP13 and/or ADAMTS5 are involved, such as arthritic disease, osteoarthritis, spondyloepimetaphyseal dysplasia, lumbar disc degeneration, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, aggrecanopathy.

A "CAP building block" is used to guide, anchor and/or retain another, e.g. therapeutic, building blocks, such as ISVDs, binding to MMP13 and/or ADAMTS5 at a desired site, e.g., a joint, where said other, e.g., therapeutic the agent, the building block, must exhibit its effect, such as binding and/or inhibition of MMP13 and/or ADAMTS5.

Again, without wishing to be bound by any theory, the present inventors also hypothesized that Aggrecan binding agents such as ISVD(s) that bind Aggrecan could potentially function as such an anchor, although Aggrecan is highly glycosylated and degraded by various disorders affecting the cartilage in the joints. In addition, given the high costs and laborious testing in various animal models that are required before a drug can be approved for clinical use, such Aggrecan binding agents should preferably have wide cross-reactivity, for example, Aggrecan binding agents should bind to Aggrekan from various species.

Using various original methods of immunization, screening and characterization, the authors of the present invention were able to identify various Aggrecan-binding agents with excellent properties of selectivity, stability and specificity, which allowed for prolonged retention and activity in the joint.

In one aspect, the present invention provides a method for reducing and/or inhibiting efflux of a composition, polypeptide, or construct from a joint, wherein said method comprises administering a pharmaceutically active amount of at least one polypeptide of the invention, construct of the invention, or composition of the invention. with the invention to a subject in need of it.

In the present invention, the term "reducing and/or inhibiting outflow" means reducing and/or inhibiting the outward flow of a composition, polypeptide or construct from a joint. Preferably, efflux is reduced and/or inhibited by at least 10%, such as at least 20%, 30%, 40%, or 50% or even more, such as at least 60%, 70%, 80% , 90% or even 100% compared to the efflux of the above composition, polypeptide or construct from the joint under the same conditions, but without the presence of Aggrecan-binding agent according to the invention, for example ISVD(s), binding Aggrecan.

In addition to diseases in which MMP13 and/or ADAMTS5 are involved, such as arthritic disease, osteoarthritis, spondyloepimetaphyseal dysplasia, lumbar disc degeneration, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, and aggrecanopathy, it is believed that the Aggrecan binding agent of the invention can also be use in various other diseases that affect cartilage, such as arthropathies and chondrodystrophies, arthritic disease (such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment), achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar degeneration disc, degenerative joint disease and relapsing polychondritis (generally referred to in this application as "Aggrecan-associated diseases").

Specified CAP building block, for example, ISVD(s), binding Aggrecan, preferably binds to cartilage tissue, such as cartilage and/or meniscus. In a preferred aspect, the CAP building block is cross-reactive to other species and specifically binds to one or more of human Aggrecan (SEQ ID NO: 68), dog Aggrecan, bovine Aggrecan, rat Aggrecan; Aggrecan pig; Aggrekan mouse, Aggrekan rabbit; Aggrecan cynomolgus monkey and/or Aggrecan rhesus monkey. Relevant information about Aggrecan's structure can be found, for example, from access numbers (UniProt) shown in Table B-3 above.

A preferred CAP building block is an ISVD binding Aggrecan, preferably human Aggrecan, preferably represented by SEQ ID NO: 68, as shown in Table B.

The present invention thus relates to a polypeptide or construct according to the invention further comprising at least one CAP building block.

The present invention thus relates to a polypeptide or construct according to the invention further comprising at least one ISVD specifically binding to Aggrecan, preferably said ISVD is represented by SEQ ID NO: 4.

In one aspect, the present invention relates to a polypeptide described in this application, comprising at least 2 ISVDs that specifically bind to Aggrecan.

In one aspect, the present invention relates to a polypeptide described in this application, comprising at least 2 Aggrecan specific binding ISVDs, wherein said at least 2 Aggrecan specific binding ISVDs may be the same or different from each other.

In one aspect, the present invention relates to a polypeptide described in this application, comprising at least 2 ISVDs that specifically bind to Aggrecan, where each of these at least 2 ISVDs that specifically bind to Aggrecan is represented by SEQ ID NO: 4.

In one aspect, the present invention provides a polypeptide described herein comprising an Aggrecan-specific ISVD, wherein said Aggrecan-specific ISVD specifically binds to human Aggrecan [SEQ ID NO: 68].

In one aspect, the present invention relates to a polypeptide described in this application, where the specified ISVD, which specifically binds to Aggrecan, specifically binds to human Aggrecan (SEQ ID NO: 68), dog Aggrecan, bovine Aggrecan, rat Aggrecan, porcine Aggrecan, mouse Aggrecan , rabbit aggrecan, cynomolgus macaque aggrecan and/or rhesus monkey aggrecan.

In one aspect, the present invention relates to a polypeptide described in this application, where the specified ISVD, which specifically binds to Aggrecan, preferentially binds to cartilage tissue, such as cartilage and/or meniscus.

It should be understood that the ISVD, polypeptide and construct of the invention are preferably stable. The stability of a polypeptide, construct or ISVD of the invention can be measured using routine assays known to those skilled in the art. Exemplary assays include, but are not limited to, assays that measure the activity of a specified polypeptide, construct, or ISVD, followed by incubation in synovial fluid for a desired period of time, after which the activity is measured again.

In one aspect, the present invention provides an ISVD, polypeptide or construct of the invention with stability for at least 7 days, such as at least 14 days, 21 days, 1 month, 2 months or even 3 months in synovial fluid (SF ) at 37°C.

The desired activity of a therapeutic building block, such as an ISVD, that binds MMP13 and/or ADAMTS5 in a multivalent polypeptide or construct of the invention can be measured using routine assays known to those skilled in the art. Representative assays include (without limitation) the GAG release assays detailed in the Examples section.

The polypeptide of the invention (also referred to herein as the "Nanobody construct") is selected from the group consisting of

(a) polypeptides comprising at least 2 immunoglobulin single variable domains (ISVDs) comprising a first ISVD specifically binding to Aggrecan and a second ISVD specifically binding to matrix metalloproteinase (MMP);

(b) polypeptides comprising at least 2 ISVDs, including a first ISVD specifically binding to Aggrecan and a second ISVD specifically binding to disintegrin A and thrombospondin metalloproteinase (ADAMTS); and

(c) polypeptides comprising at least 3 ISVDs, including a first ISVD that specifically binds to Aggrecan, a second ISVD that specifically binds to ADAMTS, and a third ISVD that specifically binds to MMP.

In a polypeptide of the invention, the ISVDs may be directly linked or linked via a linker. Even more preferably, the polypeptide of the invention includes a C-terminal extension. As will be detailed below, C-terminal extension essentially prevents/removes the binding of pre-existing antibodies/factors in most samples from human subjects/patients. The C-terminal extension is present C-terminally from the last amino acid residue (usually a serine residue) of the last (most C-terminal) ISVD.

As described in more detail below, ISVDs may be derived from the V _HH , V _H , or V _L domain, however, ISVDs are chosen such that they do not form complementary pairs from the V _H and V _L domains in the polypeptides of the invention. The nanobody, V _HH , and humanized V _HH are unusual in that they are derived from natural camelid antibodies that do not contain light chains, and indeed these domains are unable to associate with camelid light chains to form complementary V _HH and V _L pairs. Thus, the polypeptides of the present invention do not include complementary ISVDs and/or do not form complementary ISVD pairs, such as, for example, complementary V _H /V _L pairs.

Generally, polypeptides or constructs that include or essentially consist of one building block, one ISVD, or one Nanobody will be referred to as "univalent" polypeptides and "univalent constructs", respectively. Polypeptides or constructs that include two or more building blocks (such as, for example, ISVDs) will also be referred to as "multivalent" polypeptides or constructs, and building blocks/ISVDs present in such polypeptides or constructs will also be referred to below as being in a "multivalent format". For example, a "bivalent" polypeptide may include two ISVDs optionally linked via a linker sequence, while a "trivalent" polypeptide may include three ISVDs optionally linked via two linker sequences; while a "tetravalent" polypeptide may include four ISVDs, optionally linked via three linker sequences, and so on.

In a multivalent polypeptide, 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 part(s) or epitope(s) or against different parts or epitopes ) or, alternatively, may be directed against different antigens or antigenic determinants; or may include any suitable combination of the above, such as directed against Aggrekan. Polypeptides and constructs that contain at least two building blocks (such as, for example, ISVD), in which at least one building block is directed against the first antigen (for example, Aggrecan) and at least one building block is directed against the second antigen ( i.e. , other than Aggrecan, e.g. directed against ADAMTS5) will also be referred to as “multispecific” polypeptides and constructs, and building blocks (such as, for example, ISVD) present in such polypeptides and constructs will also be referred to below as in a “multi-specific format”. Thus, for example, a “bispecific” polypeptide of the invention is a polypeptide that includes at least one ISVD directed against a first antigen ( e.g., Aggrecan) and at least one more ISVD directed against a second antigen ( i.e. , other than Aggrecan, e.g. directed against ADAMTS5), while a “trispecific” polypeptide of the invention is a polypeptide that includes at least one ISVD directed against the first antigen ( e.g. Aggrecan), at least one more ISVD directed against a second antigen ( ie , other than Aggrecan, eg directed against ADAMTS5), and at least one more ISVD directed against a third antigen ( ie , different from both Aggrecan and ADAMTS5, eg directed against MMP); etc.

In one aspect, the present invention relates to a polypeptide comprising at least 2 ISVD, where at least one ISVD specifically binds to MMP, preferably MMP13, more preferably said one ISVD is represented by the amino acid sequence of SEQ ID NO: 2.

In one aspect, the present invention relates to a polypeptide comprising at least 2 ISVDs, wherein at least one ISVD specifically binds to ADAMTS, preferably ADAMTS5, more preferably said one ISVD is represented by the amino acid sequence of SEQ ID NO: 3.

In one aspect, the present invention relates to a polypeptide comprising at least 2 ISVDs, wherein at least one ISVD specifically binds to Aggrecan, more preferably said one ISVD is represented by the amino acid sequence of SEQ ID NO: 4.

"Multiparatopic" polypeptides and "multiparatopic" constructs, such as, for example, "biparatopic" polypeptides or constructs and "triparatopic" polypeptides or constructs, comprise or essentially consist of two or more building blocks, each having a different paratope.

One or more ISVDs of the invention may be used as a building block in such a polypeptide or construct to produce a monovalent, multivalent, or multiparatopic polypeptide or construct of the invention, respectively, as described herein.

The present invention thus also relates to a polypeptide or construct that is a multivalent polypeptide or a multivalent construct, respectively, for example, a divalent or trivalent polypeptide or construct comprising or essentially consisting of two or more ISVDs of the invention (with respect to multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also Conrath et al . 23221 and WO 2010/115998).

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 Nanobody directed against Aggrecan, and a second ISVD, such as Nanobody directed against a second antigen, such as, for example, ADAMTS5 or MMP13, in which said first and second ISVDs, such as Nanobodies, may optionally be linked via a linker sequence (as defined herein); while the 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, for example, Aggrecan, and a third ISVD, such as a Nanobody directed against a third antigen, such as for example MMP13, wherein said first, second and third ISVDs, such as Nanobodies, may optionally be linked via one or more, and in particular two, linker sequences.

The invention also relates to a multivalent polypeptide that includes or (essentially) consists of at least one ISVD (or suitable fragments thereof) that binds ADAMTS5, preferably human ADAMTS5, and one additional ISVD, such as an ISVD that binds Aggrecan.

Particularly preferred bivalent bispecific polypeptides or constructs and tetravalent trispecific polypeptides or constructs according to the invention are those shown in the examples described in this application and in Table A-1 (e.g., SEQ ID NOS: 1, 5, 6, 62, 63 or 64).

The two or more ISVDs present in a multivalent polypeptide or construct of the invention may consist of a light chain variable domain sequence (eg, a V _L sequence) or a heavy chain variable domain sequence ( eg, a V _H sequence); they may consist of a heavy chain variable domain sequence that is derived from a conventional four chain antibody, or a heavy chain variable domain sequence that is derived from a heavy chain-only antibody. In a preferred aspect, they consist of a Domain antibody (or an amino acid that is suitable for use as a domain antibody), a single domain antibody (or an amino acid that is suitable for use as a single domain antibody), a "dAb" (or an amino acid that is is suitable for use as a dAb), from Nanotel® (including but not limited to V _HH ), from a humanized V _HH sequence, from a camelized V _H sequence; or from a V _HH sequence that is obtained by affinity maturation. The two or more single variable domains of an immunoglobulin may consist of a partially or fully humanized Nanobody or a partially or fully humanized VHH.

In a particularly preferred aspect, a polypeptide or construct of the invention comprises or essentially consists of four or more ISVDs, of which at least two ISVDs are directed against Aggrecan. It should be understood that the at least two ISVDs directed against Aggrecan may be the same or different from each other, may be directed against the same epitope or different epitopes of Aggrecan, may belong to the same epitope group or to different epitope groups and/or can bind to the same or different Aggrecan domains.

Relative affinities may depend on the location of the ISVDs in the polypeptide. It should be understood that the order of the ISVDs in the polypeptide of the invention (orientation) can be chosen as desired by one skilled in the art. The location of the individual ISVDs, and whether or not the polypeptide includes a linker, is a matter of design choice. Certain orientations, with or without linkers, may provide preferred binding characteristics over other orientations. For example, the order of the first ISVD (eg ISVD 1) and the second ISVD (eg ISVD 2) in a polypeptide of the invention may be N-terminal to C-terminal: (i) ISVD 1 (eg nanobody 1) - [linker ] – ISVD 2 (for example, nanobody 2) – [C-terminal extension]; or (ii) ISVD 2 (eg nanobody 2) - [linker] - ISVD 1 (eg nanobody 1) - [C-terminal extension]; (where the fragments in square brackets, i.e. linker and C-terminal extension, are optional). All orientations are covered by the invention. Polypeptides that contain an orientation of ISVDs that provide the desired binding characteristics can be easily identified using routine screening, for example, as illustrated in the Examples section.

In the preferred order, the Aggrecan-binding ISVD is located on the C-terminal side of the polypeptide. A particularly preferred order from N-terminus to C-terminus is: ISVD binding ADAMTS5 - [linker] - ISVD binding Aggrecan - [C-terminal extension] or ISVD binding MMP13 - [linker] - ISVD binding Aggrecan - [C-terminal extension] where the fragments in square brackets are optional. Another particularly preferred order from N-terminus to C-terminus is: ADAMTS5 binding ISVD - [linker] - Aggrecan binding ISVD - [linker] - Aggrecan binding ISVD - [C-terminal extension] or Aggrecan binding ISVD MMP13 - [linker] - ISVD binding Aggrecan - [linker] - ISVD binding Aggrecan - [C-terminal extension], where fragments in square brackets are optional. For example, the preferred order from N-terminus to C-terminus is: ISVD binding MMP13 - [linker] - ISVD binding ADAMTS5 - [linker] - ISVD binding Aggrecan - [C-terminal extension], where the fragments are in square brackets are optional. For example, a particularly preferred order from N-terminus to C-terminus is: ISVD linking MMP13 - [linker] - ISVD linking ADAMTS5 - [linker] - ISVD linking Aggrecan - [linker] - ISVD linking Aggrecan - [C –terminal extension], where the fragments in square brackets are optional.

In another aspect, the invention relates to a polypeptide described in this application, where the specified polypeptide has at least 80%, 90%, 95% or 100% sequence identity with any of SEQ ID NO: 1, 5, 6, 62, 63 or 64.

In one aspect, the present invention relates to a polypeptide described in this application, which is selected from the group consisting of SEQ ID NO: 1 (ALX-1011), SEQ ID NO: 5 (MMP13-CAP-CAP), and SEQ ID NO: 6 (ATS5-CAP-CAP), SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64.

In a specific aspect of the invention, a construct or polypeptide of the invention may contain a moiety conferring a property such as a longer half-life compared to a corresponding construct or polypeptide of the invention without said moiety. Some preferred, but non-limiting examples of such constructs and polypeptides of the invention will be apparent to those skilled in the art based on the further disclosure provided herein, and include, for example, ISVDs or polypeptides of the invention that have been chemically modified to increase their half-life (e.g., by pegylation); polypeptides or constructs of the invention that include at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that include at least one ISVD of the invention that is linked to at least one fragment (and in particular at least one amino acid sequence) that increases the half-life of the amino acid sequence of the invention. Examples of constructs of the invention and polypeptides of the invention comprising such half-life increasing fragments or ISVDs will be apparent to those skilled in the art based on the further disclosure provided herein; and, for example, include, without limitation, polypeptides 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 one or more binding elements that can bind to serum proteins (such as, for example, domain antibodies, immunoglobulin single variable domains that are suitable for use as a domain antibody, single domain antibodies, ISVDs that are suitable for use as a single domain antibody, dAbs, ISVD , which are suitable for use as dAbs, or Nanobodies, which can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin, see further description and referenced therein links); polypeptides in which the amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable portion or fragment thereof; or polypeptides in which one or more single variable domains of an immunoglobulin of the invention are suitably linked to one or more small proteins or peptides that can bind to serum proteins, such as, for example, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489, WO2008/068280, WO2009/127691 and PCT/EP2011/051559.

In one aspect, the present invention provides a polypeptide and a construct of the invention, wherein said construct or said polypeptide further comprises a whey protein-binding moiety or whey protein. Preferably, said serum protein-binding moiety binds to serum albumin, such as human serum albumin.

In one aspect, the present invention relates to a polypeptide described in this application, including ISVD, which binds to serum albumin.

In general, constructs or polypeptides of the invention with extended half-life preferably have a half-life that is at least 1.5 fold, preferably at least 2 fold, such as at least 5 fold, such as at least 10 fold or greater than 20 times the half-life of the corresponding constructs or polypeptides of the inventionperse,those. without a fragment providing a longer half-life. For example, constructs or polypeptides of the invention with an extended half-life may have a half-lifeeg, in a human 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 respective constructs or polypeptides according to the invention per se, those. without a fragment providing a longer half-life.

In a preferred 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 greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours or even more than 24, 48 or 72 hours compared to the corresponding constructs or polypeptides of the inventionperse,those. without a group providing a longer half-life.

In another preferred aspect of the invention, such constructs and polypeptides of the invention exhibit a human serum half-life of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, constructs or polypeptides of the invention may have a 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 to 18 days or more) or more than 14 days (e.g., about 14-16 days). 19 days).

In a particularly preferred aspect of the invention, the invention provides a construct of the invention and a polypeptide of the invention comprising, in addition to one or more Aggrecan-binding building blocks and one or more ADAMTS5 and/or MMP13-binding building blocks, at least one building block , which binds to serum albumin, such as ISVD, which binds to serum albumin, such as human serum albumin, as described in this application. Preferably, said serum albumin-binding ISVD comprises or essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), wherein CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR. Preferably said ISVD binding to human 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).

In one embodiment, the present invention provides a construct of the invention, such as a polypeptide comprising a whey protein binding moiety, wherein said whey protein binding moiety is a non-antibody polypeptide.

In one aspect, the present invention relates to a construct described in this application, including at least one ISVD or polypeptide and one or more other groups, residues, fragments or connecting elements. One or more other groups, residues, fragments or binding elements are preferably selected from the group consisting of a polyethylene glycol molecule, whey proteins or fragments thereof, binding elements that can bind to whey proteins, an Fc moiety, and small proteins or peptides that can bind to serum proteins, other amino acid residues, markers, or other functional moieties, such as toxins, labels, radiochemicals, etc.

In one embodiment, as set forth below, the present invention relates to a construct of the invention, such as a polypeptide comprising a moiety conferring a half-life extension property, wherein said moiety is PEG. Therefore, the present invention also relates to the construction or polypeptide according to the invention, including PEG.

Additional amino acid residues may or may not alter, modify or otherwise affect other (biological) properties of the polypeptide of the invention and may or may not add additional functionality to the polypeptide of the invention. For example, such amino acid residues:

a) may include an N-terminal Met residue, for example as a result of expression in a heterologous host cell or host organism;

b) can form a signal sequence or leader sequence that directs the secretion of the polypeptide from the host cell during synthesis (for example, to obtain a pre-, pro- or pre-pro 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 those described later in this application. Typically, such a leader sequence will be linked to the N-terminus of the polypeptide, although the invention in its broadest sense is not limited to this;

c) may form a "tag", eg an amino acid sequence or residue, which allows or facilitates purification of the polypeptide, eg using affinity methods directed against said sequence or residue. The specified sequence or residue can then be removed (for example, by chemical or enzymatic cleavage) to obtain a polypeptide (for this purpose, the label may optionally be linked to the amino acid sequence or polypeptide sequence through a cleavable linker sequence or contain a cleavable motif). Some preferred, but non-limiting, examples of such residues are several histidine residues, glutathione residues, and a myc-tag such as AAAEQKLISEEDLNGAA;

d) there may be one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups will be obvious to the skilled person and include, but are not limited to, the amino acid residues and functional groups specified in this application for derivatives of the polypeptides of the invention.

Also contemplated by the present invention are constructs comprising a polypeptide and/or ISVD of the invention, which further include other functional moieties, eg, toxins, tags, radiochemicals, etc.

Other groups, residues, fragments or connecting elements may, for example, be chemical groups, residues, fragments, which themselves may or may not be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to one or more ISVDs or polypeptides of the invention in such a manner as to provide a “derivative” of the polypeptide or construct of the invention.

Accordingly, the 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 ( eg, enzymatic) modification, of the constructs and/or polypeptides of the invention and/or one or more amino acid residues that form the polypeptide of the invention.

Examples of such modifications, as well as examples of amino acid residues in polypeptide sequences that can be modified in this way ( i.e. either in the protein backbone, but preferably in the side chain), methods and procedures that can be used to introduce such modifications, and the potential applications and benefits of such modifications will be apparent to the skilled artisan (see also Zangi et al. , Nat Biotechnol 31(10):898-907, 2013).

For example, such modification may include the introduction ( for example, by covalent linkage or any other suitable method) of one or more (functional) groups, residues or fragments in or on the polypeptide according to the invention, in particular, one or more functional groups, residues or fragments, which confer one or more desired properties or functionalities on a construct and/or polypeptide of the invention. Examples of such functional groups will be apparent to those skilled in the art.

For example, such modification may include the introduction ( for example, by covalent linkage or any other suitable method) of one or more functional fragments that increase the half-life, solubility and/or absorption of the construct or polypeptide of the invention, which reduce the immunogenicity and/or toxicity of the construct, or a polypeptide of the invention that eliminates or reduces any undesirable side effects of the construct or polypeptide of the invention and/or that confer other beneficial properties of the construct or polypeptide of the invention and/or reduce undesirable properties thereof; or any combination of two or more of the above. Examples of functional moieties and methods for their administration will be apparent to the skilled artisan and will generally include all of the functional moieties and methods described in the prior art cited herein above, as well as functional moieties and methods known per se for modifying pharmaceutical proteins and, in particular, for the modification of antibodies or antibody fragments (including ^ScFv and single domain antibodies), for example, see Remington (Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA, 1980). Such functional fragments, for example, can be linked directly (eg covalently) to the polypeptide of the invention, or optionally via a suitable linker or spacer, as will also be apparent to those skilled in the art.

One specific example is a derivative of a polypeptide or construct of the invention, where the polypeptide or construct of the invention has been chemically modified to increase its half-life (eg, by pegylation). This is one of the most widely used methods to increase the half-life and/or decrease the immunogenicity of pharmaceutical proteins and involves the attachment of a suitable pharmacologically acceptable polymer such as poly(ethylene glycol) (PEG) or its derivatives (such as methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFvs); 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 protein pegylation reagents are also commercially available, for example from Nektar Therapeutics, USA.

Preferably, site-directed PEGylation is used, in particular via a cysteine residue (see, for example, Yang et al. (Protein Engineering 16: 761-770, 2003). For example, PEG can be attached to a cysteine residue that is naturally present for this purpose. in a polypeptide of the invention, a construct or polypeptide of the invention may be modified so that one or more cysteine residues for PEG attachment are suitably introduced, or an amino acid sequence comprising one or more cysteine residues for PEG attachment may be fused to N- and/or C - the end of the construct or polypeptide of the invention - in all cases using protein engineering techniques known per se to those skilled in the art.

Preferably, for the constructs or polypeptides of the invention, PEGs with a molecular weight greater than 5000, such as greater than 10,000 and less than 200,000, such as less than 100,000, are used; for example, in the range of 20000–80000.

Another, usually less preferred, modification involves N-linked or O-linked glycosylation, usually as part of a co-translational and/or post-translational modification, depending on the host cell used to express the polypeptide of the invention.

Another modification may include the introduction of one or more detectable labels or other signal-generating groups or fragments, depending on the intended use of the polypeptide or construct of the invention. Suitable labels and methods for attaching, using and detecting them will be understood by those skilled in the art and, for example, include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phtaldehyde and fluorescamine and fluorescent metals such as ¹⁵² Eu or other lanthanide metals), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, acridinium aromatic ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogues) , radioisotopes (such as 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. metal cations such as ^99m Tc, ¹²³ I, ¹¹¹ In, ¹³¹ I, ⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga and ⁶⁸ Ga, or other metals or metal cations which are particularly suitable for use in in vivo, in vitro or in situ diagnostics and imaging such as ( ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr and ⁵⁶ Fe)), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels will be apparent to those skilled in the art and include, for example, fragments that can be detected using NMR or EPR spectroscopy.

Such labeled polypeptides and constructs of the invention, for example, can be used 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 diagnostics and in vivo imaging, depending on the choice of a particular label.

As the skilled person will appreciate, another modification may include the introduction of a chelating group, for example to chelate one of the metals or metal cations mentioned above. Suitable chelating groups, for example, include, without limitation, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Another modification may include the introduction of a functional fragment that is one part of a specific binding pair, such as a biotin-(strept)avidin binding pair. Such a functional moiety can be used to link a polypeptide of the invention to another protein, polypeptide, or chemical that is linked to the other half of the binding pair, i.e. through bonding pair formation. For example, a construct or polypeptide of the 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 invention can be used as a reporter, for example in a diagnostic system where the detectable signal-emitting agent is conjugated to avidin or streptavidin. Such binding pairs, for example, can also be used to link a construct or polypeptide of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposome compositions described by Cao and Suresh (Journal of Drug Targeting 8: 257, 2000). Such binding pairs can also be used to bind a therapeutically active agent to a polypeptide of the invention.

Other potential chemical and enzymatic modifications will be apparent to those skilled in the art. Such modifications may also be introduced for research purposes (eg, to investigate the function-activity relationship). For example, see Lundblad and Bradshaw (Biotechnol. Appl. Biochem. 26: 143-151, 1997).

Preferably the constructs, polypeptides and/or derivatives are those that bind to Aggrecan and ADAMTS5 and/or MMP13 with affinity (appropriately measured and/or expressed as K _D -value (real or apparent), K _A -value (real or apparent ), k _on is the rate or rate of association and/or k _off or the rate of dissociation, or alternatively as an IC ₅₀ value, as described later in this application), which is as defined in this application (for example, as defined for polypeptides according to invention).

Such constructs and/or polypeptides of the invention and derivatives thereof may also be in substantially isolated form (as defined herein).

In one aspect, the present invention provides a construct of the invention that comprises or essentially consists of an ISVD of the invention or a polypeptide of the invention, and that further comprises one or more other groups, residues, fragments, or linking elements that are optionally linked via one or more peptide linkers.

In one aspect, the present invention relates to a construct of the invention wherein one or more other groups, residues, fragments or binding elements are selected from the group consisting of a polyethylene glycol molecule, whey proteins or fragments thereof, binding elements that can bind to whey proteins, Fc portions and small proteins or peptides that can bind to serum proteins.

In the constructs of the invention, such as the polypeptides of the invention, two or more building blocks, such as ISVDs, and optionally one or more other groups, drugs, agents, residues, fragments, or linking elements can be directly linked to each other ( as, for example, 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. Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be apparent to those skilled in the art and generally can be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing constructs, proteins or polypeptides intended for pharmaceutical use.

For example, a polypeptide of the invention, for example, may be a trivalent trispecific polypeptide comprising one building block, such as an ISVD that binds Aggrecan, an ISVD that binds ADAMTS5, and potentially another building block, such as a third ISVD that binds MMP13, wherein: the first, second and third building blocks, such as the ISVD, may optionally be linked via one or more and in particular 2 linker sequences. Also, the present invention provides a construct or polypeptide of the invention comprising a first ISVD binding Aggrecan and possibly a second ISVD binding Aggrecan and/or possibly a third ISVD binding ADAMTS5 and/or possibly a fourth ISVD binding MMP13, wherein said first ISVD and/or said second ISVD and/or possibly said third ISVD and/or possibly said fourth ISVD are linked via linkers, in particular 3 linkers.

Some particularly preferred linkers include those that are used in the art to link antibody fragments or antibody domains. These include the linkers described in the general art above, as well as, for example, linkers that are used in the art to construct diabodies or ScFv fragments (in this regard, however, it should be noted that while in diabodies and in ScFv fragments, the linker sequence used must have a length, degree of flexibility, and other properties that allow the respective V _H and V _L domains to combine to form a complete antigen-binding site, there are no particular restrictions as to the length or flexibility of the linker used in the polypeptide of the invention, since each ISVD, such as Nanobodies, itself forms a complete antigen-binding site as such)

For example, the linker may be a suitable amino acid sequence, and in particular amino acid sequences of 1-50, preferably 1-30, eg 1-10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, e.g. of the (gly _x ser _y ) ₂ type, such as (e.g. (gly ₄ ser) ₃ or (gly ₃ ser ₂ ) ₃ , as described in WO 99/42077, and GS30, GS15, GS9, and GS7 linkers as described in the Ablynx applications cited above (see, for example, WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of natural antibodies composed only of heavy chains, or similar sequences (such as those described in WO 94/04678) Preferred linkers are shown in Table C.

Some particularly preferred linkers are GS9 (see also SEQ ID NO: 84 in WO 06/122825) and GS35, as well as polyalanine (such as AAA) and the GS30 linker (see also SEQ ID NO: 85 in WO 06/122825 ).

Other suitable linkers generally include organic compounds or polymers, in particular, which are suitable for use in proteins for pharmaceutical use. For example, poly(ethylene glycol) moieties are used to bind antibody domains, see eg WO 04/081026.

It is within the scope of the present invention that the length, degree of flexibility and/or other properties of the linker(s) used (although they are not critical, as is usually the case for linkers used in ScFv fragments) may have some effect on the properties of the final construct of the invention, such as a polypeptide of the invention, including, but not limited to, affinity, specificity, or avidity for a chemokine or one or more other antigens. Based on the disclosure presented in this application, one skilled in the art will be able to determine the optimal linker(s) for use in a particular construct of the invention, such as a polypeptide of the invention, optionally after a few limited routine experiments.

For example, in multivalent polypeptides of the invention that include building blocks, ISVDs, or Nanobodies directed against Aggrecan and another target such as, for example, ADAMTS5 and/or MMP13, the length and flexibility of the linker is preferably such that each building block, such as An ISVD of the invention present in a polypeptide will bind to its cognate target, eg, an antigenic determinant on each of the targets. Also, based on the disclosure presented in this application, one skilled in the art will be able to determine the optimal linker(s) for use in a particular construct of the invention, such as a polypeptide of the invention, optionally after a few limited routine experiments.

It is also within the scope of the present invention that the linker(s) used impart one or more other useful properties or functionalities to the constructs of the invention, such as the polypeptides of the invention, and/or provide one or more sites for derivatization and/or attachment of functional groups. (for example, as described in this application for derivatives of ISVDs according to the invention). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, while linkers that form or contain small epitopes or tags can be used for detection, identification and/or purification purposes. Again, based on the disclosure presented herein, one skilled in the art will be able to determine the optimal linkers for use in a particular polypeptide of the invention, optionally after a few limited routine experiments.

Of course, when two or more linkers are used in constructs such as the polypeptides of the invention, these linkers may be the same or different from each other. Again, based on the disclosure presented herein, one skilled in the art will be able to determine the optimal linkers for use in a particular construct or polypeptide of the invention, optionally after a few limited routine experiments.

Typically, to allow for easier expression and production, a construct of the invention, such as a polypeptide of the invention, may be a linear polypeptide. However, the invention in its broadest sense is not limited to this. For example, when a construct of the invention, such as a polypeptide of the invention, includes three or more building blocks, ISVD or Nanotel, they can be connected using a linker with three or more "arms", where each "arm" is associated with a building block, ISVD or Nanobody to provide a "star" design. It is also possible, although generally less preferred, to use annular structures.

Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein said ISVDs are directly linked to one another or linked through a linker.

Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein a first ISVD and/or a second ISVD and/or possibly a serum albumin-binding ISVD are linked via a linker.

Accordingly, the present invention provides a construct of the invention, such as a polypeptide of the invention, wherein said linker is selected from the group consisting of linkers 9GS, 35GS, 3A, 5GS, 7GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, poly- A, 8GS, 40GS, G1 hinge, 9GS-G1 hinge, llama top long hinge and G3 hinge, such as those shown in Table C (SEQ ID NOs: 28, 35, 24-27, 29-34 and 36 –40).

Accordingly, the present invention 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: 62-64, 1, 5 and 6.

The invention also relates to methods for obtaining constructs, polypeptides, ISVDs, nucleic acids, host cells and compositions described in this application.

The multivalent polypeptides of the invention can generally be prepared by a process that includes at least the step of linking, in an appropriate manner, an ISVD and/or a monovalent polypeptide of the invention to one or more other ISVDs, optionally via one or more suitable linkers, to provide a multivalent polypeptide of the invention. . The polypeptides of the invention can also be produced by a method that typically includes at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and isolating the expressed polypeptide of the invention. This can be done in a manner known per se, which should be understood by the skilled person, for example, based on the methods and procedures described later in this application.

The method for obtaining multivalent polypeptides according to the invention may include at least the steps of linking two or more ISVD according to the invention and, for example, one or more linkers together in a suitable manner. The ISVDs of the invention (and linkers) can be linked by any method known in the art and as described later in this application. Preferred methods include linking nucleic acid sequences that encode the ISVDs of the invention (and linkers) to generate a genetic construct that expresses a multivalent polypeptide. Methods for linking amino acids or nucleic acids will be apparent to those skilled in the art, in which case reference can also be made to reference books such as Sambrook et al. and Ausubel et al. above, as well as the examples below.

Accordingly, the present invention also relates to the use of the ISVD of the invention to produce a multivalent polypeptide of the invention. A method for producing a multivalent polypeptide will include linking an ISVD of the invention to at least one additional ISVD of the invention, optionally via one or more linkers. The ISVD of the invention is then used as a binding domain or building block to provide and/or obtain a multivalent polypeptide comprising 2 (eg, in a divalent polypeptide), 3 (eg, in a trivalent polypeptide), 4 (e.g., in a tetravalent) or more (eg, in a multivalent polypeptide) building blocks. In this regard, an ISVD of the invention can be used as a binding domain or binding element to provide and/or obtain a multivalent, such as divalent, trivalent or tetravalent, polypeptide of the invention comprising 2, 3, 4 or more building blocks.

Accordingly, the present invention also relates to the use of an ISVD polypeptide of the invention (as described in this application) to obtain a multivalent polypeptide. A method for producing a multivalent polypeptide will include linking an ISVD of the invention to at least one other ISVD of the invention, optionally via one or more linkers.

The polypeptides and nucleic acids of the invention can be obtained in a manner known per se, as will be understood by the skilled person from the description below. For example, the polypeptides of the invention can be prepared by any method known per se for antibody production, and in particular for antibody fragment production (including, but not limited to, (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for obtaining polypeptides and nucleic acids include the methods and procedures described in this application.

The method for producing a polypeptide of the invention may include the following steps: expression, in a suitable host cell or organism (also referred to in this application as the “ host of the invention ”), or in another suitable nucleic acid expression system that encodes the specified polypeptide of the invention (also referred to in this application as " nucleic acid according to the invention "); optionally followed by isolation and/or purification of the polypeptide of the invention thus obtained.

In particular, such a method may include the following steps: culturing and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one polypeptide of the invention; optionally followed by isolation and/or purification of the polypeptide of the invention thus obtained.

Accordingly, the present invention also relates to a nucleic acid or nucleotide sequence that encodes a polypeptide, ISVD, or construct of the invention (also referred to as " nucleic acid of the invention ").

The nucleic acid of the invention may be in the form of single or double stranded DNA or RNA. In accordance with one embodiment of the invention, the nucleic acid of the invention is in substantially isolated form, as defined in this application. The nucleic acid of the invention may also be in the form of a vector, may be present in a vector and/or be part of a vector, eg an expression vector such as, for example, a plasmid, cosmid or YAC, which may also be in a substantially isolated form. Accordingly, the present invention also relates to an expression vector comprising the nucleic acid or nucleotide sequence of the invention.

Nucleic acids of the invention can be made or obtained by a method known per se, based on the information on the polypeptides of the invention presented in this application, and/or they can be isolated from a suitable natural source. Also, as will be understood by the skilled person, in order to obtain a nucleic acid of the invention, also several nucleotide sequences, for example, at least two nucleic acids encoding an ISVD of the invention, and, for example, nucleic acids encoding one or more linkers, can be linked together in a suitable way. Methods for producing nucleic acids of the invention will be apparent to those skilled in the art and may, for example, include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more natural and/or synthetic sequences (or two or more parts thereof), introducing mutations that result in the expression of a truncated expression product; introducing one or more restriction sites (eg, to create cassettes and/or regions that can be easily cleaved and/or ligated using suitable restriction enzymes), and/or introducing mutations by PCR using one or more "mismatched" primers. These and other methods will be apparent to the skilled artisan, in which case reference may again be made to reference books such as Sambrook et al. and Ausubel et al. above, as well as the examples below.

In a preferred, but non-limiting embodiment, the genetic construct of the invention comprises

a) at least one nucleic acid according to the invention;

b) operably linked to one or more regulatory elements such as a promoter and optionally a suitable terminator; and optionally also

c) one or more additional elements of genetic constructs known per se;

where the terms "regulatory element", "promoter", "terminator" and "operably linked" have their usual meaning in the art.

The genetic constructs of the invention can generally be obtained by linking in an appropriate manner the nucleotide sequence(s) of the invention to one or more of the other elements described above, for example using the methods described in general reference books such as Sambrook et al. and Ausubel et al. above.

The nucleic acids of the invention and/or the genetic constructs of the invention can be used to transform a host cell or host organism, i. for expression and/or production of a polypeptide of the invention. Suitable hosts or host cells will be apparent to the skilled artisan, and may, 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 all other host cells. or (non-human) hosts known per se to express and produce antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments), as will be understood by the skilled artisan. See also the prior art reference above and also, for example, WO 94/29457; W096/34103; WO 99/42077; Franken 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 other reference documents specified in this application. In addition, the polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be apparent to those skilled in the art. Suitable methods for transforming a host or host cell of the invention will be apparent to those skilled in the art and may depend on the intended host cell/host organism and the genetic construct being used. See also reference books and patent applications cited above. A transformed host cell (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) constitute other aspects of the present invention. Accordingly, the present invention relates to a host or host cell comprising a nucleic acid according to the invention or an expression vector according to the invention. Preferably, these host cells or host organisms are those that express, or (at least) are capable of expressing ( e.g., under suitable conditions), a polypeptide of the invention (and in the case of a host organism: in at least one of its cells , part, tissue or organ). The invention also includes further generations, progeny and/or daughter products of the host cell or host organism of the invention, which, for example, can be obtained by cell division or sexual or asexual reproduction.

In order to obtain/attain expression of the polypeptides of the invention, the transformed host cell or transformed host organism can generally be stored, maintained and/or cultured under conditions under which the (desired) polypeptide of the invention is expressed/produced. Suitable conditions will be apparent to those skilled in the art and generally depend on the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the reference books and patent applications cited above in the paragraphs disclosing the genetic constructs of the invention.

The polypeptide of the invention can then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism has been cultured using protein isolation and/or purification methods known per se, such as, (preparative) chromatography and/or electrophoresis, differential precipitation methods, affinity methods (e.g. using a specific cleavable amino acid sequence fused to a polypeptide of the invention) and/or preparative immunological methods (i.e. using antibodies against the polypeptide to be isolated)

In one aspect, the invention relates to a method for producing a construct, polypeptide or ISVD according to the invention, comprising at least the following steps: (a) expressing, in a suitable host cell or host organism, or other suitable expression system, a nucleic acid sequence in accordance with the invention; optionally followed by (b) isolation and/or purification of the construct, polypeptide or ISVD in accordance with the invention.

In one aspect, the invention relates to a composition comprising a construct, polypeptide, ISVD or nucleic acid in accordance with the invention.

As stated above, there is still a need for safe and effective drugs for the treatment of OA. First, the present inventors have identified very effective cartilage anchoring proteins, i.e. Aggrecan-binding ISVDs that are used as building blocks to construct molecules that also bind to ADAMTS5 and/or MMP13. The resulting molecules have an increased retention period in the subject's body and can be administered systemically while maintaining activity. The present inventors then demonstrated that the combination of both ADAMTS5 inhibitors and MMP13 inhibitors was more effective in alleviating OA than inhibiting either target alone. In addition, the polypeptides and constructs of the invention have also shown significantly improved efficacy compared to prior art compounds.

The present invention thus provides compositions, constructs and/or polypeptides with improved prophylactic, therapeutic and/or pharmacological properties, including a safer profile compared to prior art amino acid sequences and antibodies.

In one aspect, the present invention relates to a method for treating or preventing diseases or disorders in an individual, for example, which are associated with ADAMTS5 and/or MMP13 activity, the method comprising administering to said individual a composition, polypeptide and/or construct in accordance with the invention in an amount effective for the treatment or prevention (symptom) of said disease or disorder.

In one aspect, the present invention relates to a composition according to the invention, a polypeptide according to the invention and/or a construct according to the invention for use as a drug.

In another aspect, the invention relates to the use of a composition, polypeptide and/or construct according to the invention for the preparation of a pharmaceutical composition for the prevention and/or treatment of at least ADAMTS5 and/or an MMP13-associated disease such as OA; and/or for use in one or more of the treatments described herein.

The invention also relates to the use of a composition, polypeptide and/or construct according to the invention for the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder, which can be prevented and/or treated by modulating the activity of ADAMTS, preferably by inhibiting the activity ADAMTS5.

The invention also relates to the use of a composition, polypeptide and/or construct according to the invention for 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 MMP activity, preferably by inhibiting MMP13 activity .

The invention also relates to the use of an ISVD, a polypeptide, a composite and/or a construct according to the invention for 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 to a patient an ISVD, a polypeptide, compositions and/or structures according to the invention.

The invention also relates to an ISVD, a composition, a polypeptide and/or a construct of the invention, or a pharmaceutical composition comprising them, for use in the prevention and/or treatment of at least an ADAMTS5-associated disease and/or an MMP13-associated disease.

It is contemplated that the ADAMTS5 binding agents of the invention can be used in various diseases affecting cartilage such as arthropathies and chondrodystrophies, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration, degenerative joint disease and relapsing polychondritis, osteochondritis dissecans and aggrecanopathy, and non-alcoholic steatohepatitis (NASH) (collectively referred to herein as "ADAMTS5-associated diseases"), preferably OA.

It is contemplated that the MMP13-binding agents of the invention can be used in various diseases affecting cartilage such as arthropathies and chondrodystrophies, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, aggrecanopathy, chronic periodontitis, and abdominal aortic aneurysm (collectively referred to herein as "MMP13-associated diseases"), preferably OA.

In one aspect, the present invention relates to a composition, ISVD, polypeptide and/or construct according to the invention for use in the treatment or prevention of a symptom of ADAMTS5-associated disease and/or MMP13-associated disease, such as, for example, arthropathies and chondrodystrophy, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, aggrecanopathy, NASH, chronic periodontitis and an aneurysm of the abdominal aorta, preferably OA.

In one aspect, the present invention relates to a method for the prevention or treatment of arthropathies and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration , degenerative joint disease, relapsing polychondritis, NASH, chronic periodontitis and abdominal aortic aneurysm, preferably OA, wherein said method comprises administering to a subject in need thereof a pharmaceutically active amount of at least a composition, an immunoglobulin, a polypeptide and/or a construct according to the invention .

In one aspect, the present invention relates to the use of an ISVD, a polypeptide, composition and/or construct according to the invention for the preparation of a pharmaceutical composition for the treatment or prevention of a disease or disorder such as arthropathy and chondrodystrophy, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, aggrecanopathy, NASH, chronic periodontitis and abdominal aortic aneurysm, preferably OA.

It is also expected that by binding to Aggrecan, ISVD, the constructs and/or polypeptides of the invention may reduce or inhibit the activity of a member of the serine protease family, cathepsins, matrix metalloproteinases (MMPs other than MMP13), such as for example MMP20, but also ADAMTS4 (Aggrecanase– 1) and/or ADAMTS11 associated with the destruction of Aggrekan.

In the context of the present invention, the term "prevention and/or treatment" includes not only the prevention and/or treatment of a disease, but also generally includes preventing the onset of a disease, slowing or modifying the course of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and/or alleviation of one or more symptoms associated with the disease, reduction of the severity and/or duration of the disease and/or any symptoms associated with it, and/or prevention of further increase in the severity of the disease and/or any associated symptoms, prevention, reduction or reversal of any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

The regimen of administration should be determined by the attending physician and clinical factors. As is well known in medicine, the dosage for any patient depends on many factors, including size, body weight, body surface area, age of the patient, the particular compound administered, the activity of the polypeptide used (including antibodies), the time and route of administration, general health, and combination with other therapies or treatments. The proteinaceous pharmaceutically active agent may be present in amounts between 1 g and 100 mg/kg body weight per dose; however, doses below or above this exemplary range are also contemplated. If continuous infusion is used, the amount may be in the range of 1 pg to 100 mg per kilogram of body weight per minute.

An ISVD, polypeptide or construct of the invention can be used at a concentration of, for example, 0.01, 0.1, 0.5, 1, 2, 5, 10, 20, or 50 pg/mL to inhibit and/or neutralize ADAMTS5 activity and/ or MMP13 by at least about 50%, preferably 75%, more preferably 90%, 95%, or up to 99%, and most preferably about 100% (substantially all), as determined by methods well known in the art.

Typically, a treatment regimen will include the administration of one or more polypeptides and/or constructs of the invention, or one or more compositions comprising them, in one or more pharmaceutically effective amounts or doses. Specific amounts or doses to be administered may be determined by the clinician, again based on the factors listed above. Useful doses of the compositions, constructs and/or polypeptides of the invention can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses from mice and other animals to humans are known in the art; for example, see US 4938949.

Generally, depending on the particular disease, disorder or condition being treated, the activity of the particular polypeptide and/or construct of the invention being used, the particular route of administration, and the particular pharmaceutical formulation or composition being used, the clinician will be able to determine an appropriate administration schedule.

The number of compositions, constructs and/or polypeptides of the invention required for use in treatment will vary not only depending on the particular composition, polypeptide and/or construct, but also on the route of administration, the nature of the condition being treated, and the age and condition of the patient. , and ultimately this will be determined by the attending physician or clinician. Also, the dosage of the compositions, constructs and/or polypeptides of the invention varies depending on the target cell, tissue or organ.

The desired dose may conveniently be provided as a single dose or, less preferably, as divided doses administered at appropriate intervals, eg, as two, three, four or more sub-doses per day. The sub-dose itself can be further divided, for example, into several separate freely distributed administrations.

The administration schedule may include long-term daily treatment. By "long term" is meant at least two weeks, and preferably several weeks, months or years. Necessary modifications within this dosage range can be determined by one of ordinary skill in the art using only routine experimentation in light of the present disclosure. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the attending physician in case of any complications. The compositions, polypeptides and constructs have been shown to be extremely stable and remain effective for extended periods of time.

Typically, a composition, polypeptide and/or construct of the invention may be used in the method described above. However, the scope of the invention covers the use of two or more compositions, polypeptides and/or constructs according to the invention in combination, such as, for example, the combination of SEQ ID NOS: 5 and 6, SEQ ID NOS: 63 and 64, SEQ ID NOS: 5 and 64 or SEQ ID NO:s 63 and 6.

The compositions, polypeptides and/or constructs of the invention may be used in combination with one or more other pharmaceutically active compounds or active substances, i. as a combination treatment regimen, which may or may not result in a synergistic effect.

The pharmaceutical composition may also include at least one other active substance, for example, one or more other antibodies or antigen-binding fragments thereof, peptides, proteins, nucleic acids, organic and inorganic molecules.

Again, the clinician will be able to select such other compounds or actives as well as an appropriate combination treatment regimen based on the factors listed above and his expert judgment.

In particular, the compositions, polypeptides and/or constructs of the invention can be used in combination with other pharmaceutically active compounds or active substances that are intended or can be used for the prevention and/or treatment of diseases, disorders and conditions listed in this application, as a result which may or may not achieve a synergistic effect. Examples of such compounds and active substances, as well as routes, methods, and pharmaceutical formulations or compositions for their administration, will be apparent to the clinician.

When two or more substances or active principles, such as for example (a composition comprising) a polypeptide comprising an ISVD inhibiting ADAMTS5 and another polypeptide comprising an ISVD inhibiting MMP13, are used as part of a combined treatment regimen such as, for example, the combination of SEQ ID NOs: 5 and 6, SEQ ID NOs: 63 and 64, SEQ ID NOs: 5 and 64, or SEQ ID NOs: 63 and 6, they can be administered by the same route of administration or by different routes of administration, substantially simultaneously or at different time (eg, substantially simultaneously, sequentially, or intermittently). When the substances or active principles can be administered simultaneously by the same route of administration, they can be administered as different pharmaceutical formulations or compositions, or as part of a combined pharmaceutical formulation or composition, as will be understood by the skilled person.

Also, when two or more active substances or active principles are used as part of a combined treatment regimen, such as, for example (a composition comprising) a polypeptide comprising an ISVD inhibiting ADAMTS5 and another polypeptide comprising an ISVD inhibiting MMP13, each of the substances or active principles can be entered in the same amount and in accordance with the same scheme of administration, which are used in the introduction of the connection or the active principle as such, and such a combined introduction may or may not lead to a synergistic effect. However, when the combined use of two or more active substances or active principles leads to a synergistic effect, then it will be possible to reduce the amount of one, several or all substances or active principles to be administered, while achieving the desired therapeutic effect. This may, for example, be useful in preventing, limiting or reducing any undesirable side effects associated with the use of one or more substances or active principles when used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used in accordance with the invention can be determined and/or tracked in any way known per se for the disease, disorder or condition, as will be understood by the clinician. The clinician will also be able, if necessary and on a case-by-case basis, to change or modify a particular treatment regimen in order to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect, on the one hand hand, and the prevention, limitation or reduction of unwanted side effects, on the other hand.

In general, the treatment regimen should be followed until the desired therapeutic effect is achieved and/or until the desired therapeutic effect is to be maintained. Again, this can be determined by the attending physician.

Thus, in yet another aspect, the invention relates to a pharmaceutical composition that contains at least one construct according to the invention or at least one polypeptide according to the invention and at least one suitable carrier, diluent or excipient ( i.e. suitable for pharmaceutical use ) and, optionally, one or more additional active substances. In a specific aspect, the invention relates to a pharmaceutical composition which comprises at least one composition, construct or polypeptide according to the invention, preferably at least one of SEQ ID NOs: 1 and 62, or a combination of SEQ ID NOs: 5 and 6, SEQ ID NOs: 63 and 64, SEQ ID NOs: 5 and 64, or SEQ ID NOs: 63 and 6, and at least one suitable carrier, diluent, or excipient ( i.e. , suitable for pharmaceutical use), and optionally one or more additional active substances.

The subject to be treated may be any warm-blooded animal, but in particular a mammal and in particular a human. In veterinary applications, the subject to be treated includes any animal raised commercially or kept as a pet. As will be understood by a person skilled in the art, the subject to be treated will, in particular, be a human suffering from or at risk of diseases, disorders and conditions specified in this application. Therefore, in a preferred embodiment of the invention, pharmaceutical compositions comprising a polypeptide of the invention are for use in treatment or diagnosis. Preferably, the pharmaceutical compositions are for use in medicine, but they can also be used in veterinary medicine.

Again, in such a pharmaceutical composition, one or more compositions, polypeptides and/or constructs according to the invention, or a nucleotide encoding them and/or a pharmaceutical composition comprising them, can also be suitably combined with one or more other active substances, such as specified in this application.

The invention also relates to a composition (such as, without limitation, a pharmaceutical composition or formulation as further described in this application) for use either in vitro (for example, in an in vitro assay or cell assay) or in vivo (for example, in a single cell or a multicellular organism, and in particular for a mammal and, more specifically, a human, such as a human who is at risk of developing or suffering from the disease, disorder or condition of the present invention).

It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless expressly stated otherwise.

Generally, for pharmaceutical use, compositions, constructs, polypeptides and/or ISVDs of the invention may be formulated as a pharmaceutical preparation or composition comprising at least one construct, polypeptide and/or ISVD of the invention and at least one pharmaceutically acceptable carrier, a diluent or excipient and/or adjuvant; and optionally one or more pharmaceutically active polypeptides and/or compounds. As non-limiting examples, such a composition may be in a form suitable for oral administration, for parenteral administration (for example, via intra-articular, intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by means of a skin patch , implant, suppository, etc., with parenteral administration being preferred. Such suitable administration forms, which may be solid, semi-solid or liquid, depending on the mode of administration, as well as methods and carriers for use in their preparation, will be understood by a person skilled in the art and are further described in this application. Such a pharmaceutical preparation or composition will be referred to generically as a "pharmaceutical composition".

As examples of excipients, disintegrators, binders, fillers and lubricants may be mentioned. Examples of disintegrants include agar-agar, algins, calcium carbonate, cellulose, colloidal silicon dioxide, gums, magnesium aluminum silicate, methylcellulose, and starch. Examples of binders include microcrystalline cellulose, hydroxymethylcellulose, hydroxypropylcellulose, and polyvinylpyrrolidone. Examples of excipients include calcium carbonate, calcium phosphate, tribasic calcium sulfate, calcium carboxymethylcellulose, cellulose, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrins, maltose, sorbitol, starch, sucrose, sugar, and xylitol. Examples of lubricants include agar, ethyl oleate, ethyl laurate, glycerin, glyceryl palmitostearate, hydrogenated vegetable oil, magnesium oxide, stearates, mannitol, poloxamer, glycols, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc. Conventional stabilizers, preservatives, wetting agents and emulsifiers, texture-improving agents, flavoring agents, osmotic salts, buffering agents, solubilizers, diluents, emollients, coloring and masking agents, and antioxidants are considered as pharmaceutical adjuvants.

Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, water, alcohols, polyols, glycerin, vegetable oils, etc.

In general, the constructs, polypeptides and/or ISVDs of the invention may be formulated and administered by any suitable method known per se. See for example the prior 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 reference books such as Remington's Pharmaceutical Sciences, 18th ^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, for example, pp. 252–255).

In a specific aspect, the invention relates to a pharmaceutical composition which comprises at least a composition, construct, polypeptide, ISVD or nucleic acid according to the invention, and which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant and, optionally includes one or more additional pharmaceutically active polypeptides and/or constructs.

Compositions, constructs, polypeptides and/or ISVDs of the invention may be formulated and administered by any known method for conventional antibodies and antibody fragments (including ScFvs and diabodies) and other pharmaceutically active proteins. Such compositions and methods for their preparation will be understood by those skilled in the art and, for example, include preparations preferred for parenteral administration (e.g., intraarticular, intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial, intrathecal, intranasal, or intrabronchial administration), but also for topical ( i.e. transdermal or intradermal) administration.

Formulations for parenteral administration may, for example, be sterile solutions, suspensions, dispersions or emulsions which are suitable for infusion or injection. Suitable carriers or diluents for such preparations, for example, include, without limitation, those listed on page 143 of WO 08/020079. Generally, aqueous solutions or suspensions will be preferred.

Compositions, constructs, polypeptides and/or ISVDs of the invention can also be administered using gene therapy delivery methods, see, for example, US Pat. No. 5,399,346, which is incorporated by reference with respect to gene therapy delivery methods. Using a gene therapy delivery method, primary cells transfected with a gene encoding a construct, polypeptide and/or ISVD of the invention can be further transfected with tissue-specific promoters to target specific organs, tissues, transplants, tumors, or cells and can be further transfected with signaling and stabilizing sequences for subcellularly localized expression.

In accordance with other aspects of the invention, the compositions, constructs and/or polypeptide of the invention may be used in additional in vivo and in vitro applications. For example, compositions, constructs and/or polypeptides of the invention can be used for diagnostic purposes, for example in assays designed to detect and/or quantify the presence of ADAMTS5 and/or MMP13 and/or to purify ADAMTS5 and/or MMP13. Compositions, polypeptides and/or constructs can also be tested in animal models of specific diseases and in tests to evaluate toxicity, safety and dosage.

Finally, the invention relates to a kit comprising at least one composition, polypeptide or construct of the invention, at least one nucleic acid sequence encoding said components, a vector or vector system of the invention, and/or a host cell of the invention. It is contemplated that the kit may be provided in various forms, such as a diagnostic kit.

Hereinafter the invention will be further described using the following non-limiting preferred aspects, examples and drawings.

The entire contents of all references (including references to the literature, issued patents, published patent applications and pending patent applications) cited in this application are expressly incorporated into this application by reference, in particular with regard to the fundamental references indicated above.

The sequences are disclosed in the main body of the description and in a separate sequence listing in accordance with WIPO ST.25. SEQ ID with a specific number should be the same in the body of the description and in a separate sequence listing. By way of example, SEQ ID NO: 1 would define the same sequence in both the main body of the description and the separate sequence listing. In the event of a discrepancy between a sequence definition in the main text of the description and a separate sequence listing (if, for example, SEQ ID no: 1 in the main text of the description erroneously matches SEQ ID no: 2 in a separate sequence listing), a reference to a specific sequence in the application, in particular to specific embodiments should be understood as a reference to a sequence in the main body of the application and not to a separate sequence listing. In other words, the inconsistency between the definition/designation of a sequence in the main part of the description and in a separate sequence listing should be eliminated by correcting the separate sequence listing in accordance with the sequences and their designations disclosed in the main part of the application, which includes a description, examples, drawings and claims .

6. EXAMPLES

Without being bound by a particular theory, the inventors of the present invention hypothesized that inhibition of both ADAMTS5 and MMP13 could potentially be more effective because

(1) inhibition of a wider spectrum of OA-inducing and maintaining proteases could occur;

(2) it would be possible to target a wider range of patients, for example, without the need to separate patients into different groups; and

(3) a wider range of developing diseases could be treated.

For the convenience of patients, inhibitors are preferably retained and active in the joints for extended periods of time.

Accordingly, the inventors set out to isolate and characterize ISVDs that specifically bind MMP13, ISVDs that specifically bind ADAMTS5, and ISVDs that specifically bind Aggrecan. Subsequently, ISVDs were combined in various formats and tested in various in vitro, ex vivo and in vivo models.

Example 1 MMP13 ISVD

1.1 anti-MMP13 ISVD 62C02

After screening more than 10E7 clones, MMP13-specific ISVD 62C02 was identified in an assay using fluorogenic peptides, collagenolytic assays, and fluorogenic collagen assays.

Briefly, the scheme of the fluorogenic peptide assays of human, cynomolgus, rat, dog and bovine MMP13 as well as the fluorogenic peptide assays of human MMP1 and MMP14 is as follows. Activated MMP was incubated with the fluorogenic peptide substrate Mca-PLGL-Dpa-AR-NH2 (R&D Systems # ES001) and a 1/5 dilution of periplasmic extract or a dilution series of purified Nanotel/positive control (total volume=20 µl in 50 mM Tris assay buffer, pH 7.5, 100 mM NaCl, 10 mM CaCl2, 0.01% Tween 20) for 2 hours at 37°C. A linear increase in fluorescence (v0– between 15 and 45 min incubation) was used as an indicator of enzymatic activity, and % inhibition was calculated using the formula 100–100 (v0 in the presence of test Nanobody/v0 in the presence of negative control Nanobody (Cablys)).

Brief description of the collagenolytic assay procedure: 250 ng/ml human collagen II for immunization (Chondrex # 20052) was incubated with 5 nM activated MMP13 in 100 µl assay buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 10 mM CaCl2, 0.01% Twin-20). After 1.5 h incubation at 35° C., the reaction was neutralized with EDTA (10 μl of a 30 mM stock solution). The MMP13-cleaved collagen was further digested with elastase for 20 min at 38°C to avoid re-annealing the destroyed Collagen II (10 µl 1/3 diluted stock solution provided in the Collagen type II detection kit (Chondrex #6009)). Collagen remaining intact was determined by ELISA (reagents provided in Collagen Type II Detection Kit (Chondrex #6009))

The collagen fluorogenic assay scheme is essentially as follows: 100 µg/ml DQ™ Bovine Skin Type I Collagen (fluorescein conjugate; Molecular Probes #D-12060 lot 1149062) was incubated with 10 nM activated MMP13 and a dilution series of purified Nanobody/positive control for 2 h at 37°C in 40 µl of assay buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM CaCl2, 0.01% Tween-20). A linear increase in fluorescence (v0– between 15 and 45 min incubation) was used as an indicator of enzymatic activity, and % inhibition was calculated using the formula 100–100 (v0 in the presence of test Nanobody/v0 in the presence of negative control Nanobody (Cablys)).

To further characterize the 62CO2 ISVD, this ISVD was recloned into the pAX129 vector, transformed into E. coli, and expressed and purified according to standard protocols (eg, Maussang et al. 2013 J Biol Chem 288: 29562–72). This ISVD was then subjected to various in vitro functional assays. TIMP-2, which is a non-selective MMP inhibitor, was used as a positive control in these assays. The small molecule drug MSC2392891A was used as a positive control. An overview of activities in enzyme assays is shown in Table 1.1

Table 1.1 IC50 [nM] clone fluorogenic peptide assay, human fluorogenic peptide assay, rat fluorogenic peptide assay, dog fluorogenic peptide assay, bovine fluorogenic peptide assay, cynomolgus macaque collagenolytic analysis, human fluorogenic collagen analysis, human TIMP-2 0.5 0.4 0.9 1.1 0.4 0.4 2.4 62C02 1.4 1.1 1.0 3.1 1.4 1.4 8.3 MSC2392891A 3.7 0.7 0.7 8.5 3.0 4.7 partial inhibition

In summary, ISVD 62C02 outperformed the comparator MSC2392891A in all assays.

1.2 anti-MMP13 ISVD 62C02 is selective

Fluorogenic peptide assays for MMP1 and MMP14 were used to determine the selectivity of ISVD 62C02 for MMP13. MMP1 and MMP14 are two closely related members of the MMP family. TIMP-2 was used as a positive control in these assays. The same analysis scheme was used as described in Example 1.1.

ISVD 62C02 was shown to be highly selective, showing no inhibition of MMP1 or MMP14 (data not shown).

Example 2 ADAMTS5 ISVD

2.1 anti-ADAMTS5 ISVD 02F03

Also in this case, more than 10E7 clones were screened to identify ISVD 02F03, which specifically binds to ADAMTS5. ISVD 02F03 was further characterized for inhibition of ADAMTS5-mediated Aggrecan cleavage in FRET and AlphaLISA assays.

Briefly, ISVD 02F03 periplasmic extract was tested for binding to recombinant human ADAMTS5 in an ELISA binding assay. It was then confirmed that ISVD 02F03 was able to prevent ADAMTS5-mediated cleavage of Aggrecan in a FRET-based human ADAMTS5 enzymatic assay. After FRET analysis, an AlphaLISA (Perkin Elmer, Waltham, MA, US) assay of human ADAMTS5 was performed with a biotinylated 43mer Aggrecan oligopeptide as a substrate. ADAMTS5 cleavage of this substrate releases a biotinylated ARGSV neoepitope product and can be detected using streptavidin-AlphaScreen donor beads and anti-α-neoepitope ("ARGSV") antibody captured on anti-mouse IgG-coated acceptor beads AlphaLISA, which generates a luminescent AlphaScreen signal under laser excitation. To determine the ability of ISVD 02F03 to prevent ADAMTS5-mediated substrate cleavage, signal reduction was analyzed as a function of ISVD 02F03 concentration and IC ₅₀ values were calculated.

The low molecular weight MSC2310852A, which inhibits both ADAMTS4 activity and ADAMTS5 activity, was used as a positive control.

The results are presented in Table 2.1.1.

Table 2.1.1
Activity (IC ₅₀ ) and % inhibition of human ADAMTS5 for ISVD 02F03 and control compounds in FRET and AlphaLISA enzyme assays Connection ID fret,
human alphalisa,
human IC50
[M] Inhibition, % IC50
[M] Inhibition, % mAb 12F4 H4L0 1.0E–09 75 6.5E–11 100 02F03 1.8E–09 89 7.5E–11 100 MSC2310852A 6.0E–08 100 1.4E–07 99

Comparable to the divalent mAb 12F4, ISVD 02F03 showed better activity than the low molecular weight MSC2310852A.

In addition, ISVD 02F03 was further evaluated for its ability to block cartilage destruction in an ex vivo assay in which the substrate is presented in a state closer to the physiological state compared to biochemical assays. Briefly, bovine cartilage explant chips (4 mm in diameter) were prepared fresh from bovine knee joints and incubated in 96-well plates in the presence of IL-1α to induce cartilage destruction. Glycosaminoglycan (GAG) release in the supernatant after 5 days of incubation (37°C, 7.5% CO2) was determined as an indicator of cartilage/Aggrecan degradation using the metachromatic dye 1,9-dimethylmethylene blue (emission at 633 nm). Chondroitin sulfate was included as a standard for analysis. Efficacy was determined using IL-1α-induced controls without compound (0%) and in the presence of MSC2310852A (100% effect).

The results are presented in Table 2.1.2.

Table 2.1.2
IC ₅₀ value for ISVD 02F03 in bovine explant assay ID IC50[M] mAb 12F4 H4L0 3.2E–06 02F03 1.4E–08

In this ex vivo assay, ISVD 02F03 showed higher efficacy than divalent mAb 12F4, ie. IC ₅₀ is about 50 times smaller.

Cross-species cross-reactivity was initially assessed using SPR dissociation rate analysis on a Biacore T100 device. The polypeptides were tested for binding to human ADAMTS5, cynomolgus ("cyno"), guinea pig, mouse and bovine. For this, recombinant ADAMTS5 was immobilized on a CM5 chip via amine coupling using EDC and NHS. Purified ISVD 02F03 was injected for 2 minutes at a concentration of 100 nM and allowed to dissociate for 15 minutes at a flow rate of 45 μl/min. The dissociation rate for each individual ADAMTS5 was determined by fitting a 1:1 interaction model (Langmuir model) to individual dissociation curves using the BIA Evaluation program. As a control, dissociation rates on human ADAMTS5 were determined in each experiment.

The results are presented in Table 2.1.3.

Table 2.1.3
Review of ISVD 02F03 Interspecies Cross-Reactivity Data Nanobody ID Dissociation rate determined by the SPR method, kd (1/sec) on ADAMTS-5 Experiment 1 Experiment 2 Experiment 3 Man Cyno The guinea pig Man Mouse Man Bull 02F03 3.3E–05 2.2E–05 3.4E–05 6.5E–05 2.9E–04 9.7E–05 4.3E–05

ISVD 02F03 showed comparable dissociation rates (cross-reactivity) with human ADAMTS5, cynomolgus monkey (cyno), guinea pig and bovine.

2.2 Selectivity ISVD 02F03

To confirm the selectivity of ISVD 02F03 for ADAMTS5, inhibition of the enzymatic activity of MMP1, MMP14 and ADAMTS4 was assessed in FRET-based assays and in the AlphaLISA assay of human ADAMTS4 using the respective enzymes.

Activated human MMP1 or MMP14 was incubated for 30 minutes at room temperature with 10 μl of the ISVD 02F03 dilution series. After incubation, 20 µl of respectively 5 µM or 2.5 µM fluorogenic peptide substrate (Mca-PLGL-Dpa-AR-NH2 Fluorogenic MMP Substrate (R&D Systems cat #ES001)) was added. The ability of ISVD 02F03 to prevent MMP1- and MMP14-mediated cleavage was monitored every minute for 2 hours at 37°C on a Tecan Infinite M1000 plate reader.

While natural inhibitors of TIMP2 and TIMP3 inhibited MMP1 and MMP14 activity, ISVD 02F03 showed no inhibition (data not shown).

To evaluate inhibition of human ADAMTS4, an assay similar to the AlphaLISA assay of human ADAMTS5 was performed essentially as described in Example 2.1, but using human ADAMTS4 (R&D Systems, Minneapolis, US; cat # 4307-AD). To determine the ability of ISVD 02F03 to prevent human ADAMTS4-mediated substrate degradation, signal reduction was analyzed as a function of ISVD 02F03 concentration and IC ₅₀ values were calculated.

While the small molecule MSC2310852A inhibited the activity of human ADAMTS4, ISVD 02F03 or mAb 12F4 showed no inhibitory activity (data not shown). The mAb 12F4 (H4L0) monoclonal antibody has been described as selective for ADAMTS4 in WO 2011/002968.

Example 3 Aggrecan ISVD

3.1 anti-Aggrecan ISVD 114F08

Aggrecan-specific ISVD 114F08 was isolated and characterized after a large-scale screening. Llamas were immunized with recombinant human Aggrecan (G1-IGD-G2 domains, R&D Systems # 1220-PG) and showed specific and high serum titers. However, only a very small fraction of the isolated Nanobodies met the two requirements, such as binding to the G1 domain of Aggrecan and demonstrating broad interspecies cross-reactivity. After arduous attempts, various members of the family were identified showing substantially similar characteristics (see Table 31A - Table 3.1C sequence variability in CDRs). As a result, ISVD 114F08 ("C0101PMP114F08") was selected for further characterization.

Domain mapping and interspecies cross-reactivity data are presented in Table 3.1.1.

Table 3.1.1 Periplasmic extract, ELISA. OD 450 nM Mapping Clone G1-IGD-G2
human G1-IGD-G2
Cyno G1-IGD-G2
rats G1-IGD-G2
dogs G1–IGD–G2 bull G1 114F08 2.38 2.32 2.05 1.90 1.18

Following initial screening, initial ELISA binding, rate of dissociation, and interspecies cross-reactivity, ISVD 114F08 was further characterized.

Sequence variability in the CDRs of ISVD 114F08 family members is presented in Tables 3.1A, 3.1B and 3.1C below. The CDR amino acid sequences of clone 114F08 were used as a reference against which the CDRs of all other family members were compared (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

Table 3.1A 114F08 CDR1* Numbering according to the Kabat system 26 27 28 29 thirty 31 32 33 34 35 Absolute numbering one 2 3 4 five 6 7 eight nine 10 Wild type sequence G S T F I I N V V R Mutations S M * Up to 2 CDR1 mutations in one clone

Table 3.1B 114F08 CDR2* Numbering according to the Kabat system fifty 51 52 53 54 55 56 57 58 Absolute numbering one 2 3 4 five 6 7 eight nine Wild type sequence T I S S G G N A N Mutations A R T T D * Up to 5 CDR2 mutations in one clone

Table 3.1C 114F08 CDR3* Numbering according to the Kabat system 95 96 97 98 99 100 100a 100b 100c 100d 100e 100f 100g Absolute numbering one 2 3 4 five 6 7 eight nine 10 eleven 12 13 Wild type sequence P T T H Y G G V Y Y G P Y Mutations . . . R . . . D . . . . . * Up to 2 CDR3 mutations in one clone

3.2 Hold in bovine cartilage ex vivo

Since there is no established assay to evaluate cartilage retention, the present inventors developed an ex vivo cartilage retention assay using bovine cartilage.

The ability of polypeptides comprising ISVD 114F08 to be retained in cartilage for an extended period of time after a relatively short exposure of the polypeptide to cartilage was determined (as would be expected with intra-articular injection). The assay was typically performed with 4 cartilage discs per Nanotel sample; 2 disks were analyzed immediately after incubation with the polypeptide (t ₀ ) to determine the initial amount of bound polypeptide; 2 disks were analyzed after washing (t _{1–5 days} ). The degree of retention was determined as the relative amount of the polypeptide determined at t _{1–5 days} and t _0. This amount was determined by visual control of Western blots using a score from 0 to 6, where 0 means no retention and 6 means complete retention. Simulation construct C0101030 ("30"), consisting of 2 inactive ALB26 ISVDs, showed no binding.

The results are shown in Table 3.2.

Table 3.2. Target Design Description Retention in cartilage* G1 118 ALB26-114F08-114F08 6.0 G1 54 114F08-ALB26 5.0 Imitation thirty ALB26–ALB26 0.0

Table 3.2: Cartilage retention of anti-Aggrecan ISVD 114F08 in ALB26 format. *Scores are mean scores from 1-4 independentex vivo analyzes retention in bovine cartilage on a scale of 0 to 6: 0 means no retention and 6 means complete retention.

The polypeptides comprising ISVD 114F08 were found to be very well retained (scores 5–6) in cartilage. This included designs including both one and two ISVD 114F08 tie pieces.

3.3 Binding characteristics - ELISA

Based on data retention in bovine cartilageex vivo, ISVD 114F08 was further characterized by ELISA on the recombinant G1-IGD-G2 region from human, cynomolgus, rat, dog and bovine Aggrecan to determine its cross-species cross-reactivity (see also Example 3.1) and on recombinant human Neurocan and Brevican to determine its selectivity. Certain ECs_fifty values are presented in Table 3.3.

Table 3.3
Characterization of anti-Aggrecan ISVD 114F08 in ALB26 format by ELISA EC50(M) Design Description Man Cyno Rat Dog Bull Neurokan Brevican 54 114F08-ALB26 6.0E–09 4.4E–09 7.6E–09 3.0E–09 5.6E–09 No binding No binding 118 ALB26-114F08-114F08 1.1E–10 7.6E–11 1.9E–10 2.4E–10 3.7E–10 No binding No binding Imitation ALB26–ALB26 No binding No binding No binding No binding No binding No binding No binding

3.4 Tissue specificity

It has been demonstrated above that the polypeptides of the invention bind specifically to Aggrecan in vitro. and with cartilageex vivo. In addition, these polypeptides should also bind preferentially to the cartilage of the joint, not binding or to a lesser extent binding to other tissues in the joint.

Binding of polypeptides comprising ISVD 114F08 to synovial membrane, tendon and epimysium was evaluated using the same assay scheme as for the ex vivo cartilage binding assay. Polypeptide isolation and Western blot analysis were performed after rapid tissue washing (30 min) after overnight incubation with polypeptides.

The results are presented in Table 3.4.

Table 3.4 Design Description Cartilage synovial membrane Tendon Epimysium 054 114F08-ALB26 6 one one one 118 ALB26-114F08-114F08 6 one one one Imitation ALB26–ALB26 one one one 0

Table 3.4: Tissue specificity. Binding of anti-Aggrecan ISVD 114F08 in ALB26 format to articular cartilage, synovial membrane, tendon, and epimysium. Scores on a scale of 0 to 6, where 0 means no binding and 6 means maximum observed binding.

The results show that polypeptides comprising either one or two ISVD 114F08 binding elements show preferential binding to cartilage over other tissues present in the joint.

3.5 Stability of Nanotel in bovine synovial fluid

For various reasons, including convenience and safety for the patient, it is preferred that the polypeptides remain stable for longer periods of time in the synovial layer.

Accordingly, the stability of polypeptides comprising ISVD 114F08 in synovial fluid (SF) was assessed by incubating the polypeptides in bovine SF for up to 7 days at 37 ^° C.

The results are presented in Table 3.5.

Table 3.5 Design Description Stability in SF bull, 37 ^o C 054 114F08-ALB26 > 7 days 118 ALB26-114F08-114F08 > 7 days Imitation ALB26–ALB26 > 7 days

Table 3.5: Stability of polypeptides comprising ISVD 114F08 in bovine SF.

No destruction of any of the structures was observed.

Example 4 Polypeptides

Polypeptide 754 was constructed by genetic linking of anti-MMP13 ISVD 62C02 and two anti-Aggrecan ISVD 114F08 using 35GS linkers (M-C-C). The 754-9 polypeptide was similar to the 754 polypeptide, but in this case 9GS linkers were used. The resulting amino acid sequences are shown in Table A-1 as SEQ ID NOs: 5 and 63, respectively.

The 954 polypeptide was constructed by genetic linking of anti-ADAMTS5 ISVD 2F03 and two anti-Aggrecan ISVD 114F08 using 35GS linkers (A-C-C). The 954-9 polypeptide (see "973") was similar to the 954 polypeptide, but in this case 9GS linkers were used. The resulting amino acid sequences are shown in Table A-1 as SEQ ID NOs: 6 and 64, respectively.

The 949 polypeptide was constructed by genetic linking of anti-MMP13 ISVD 62C02, anti-ADAMTS5 ISVD 2F03, and two anti-Aggrecan ISVD 114F08 using 35GS linkers (M-A-C-C). The 949-9 polypeptide was similar to the 949 polypeptide, but in this case 9GS linkers were used. The resulting amino acid sequences are shown in Table A-1 as SEQ ID NOs: 1 and 62, respectively.

Example 5 Affinity of polypeptides for ADAMTS5, MMP13 and Aggrecan from different species

5.1 Affinity of 949 polypeptide for human, cynomolgus and rat ADAMTS5

The affinity of the 949 polypeptide for human, cynomolgus and rat ADAMTS5 was determined in solution using the KinExA method. A fixed concentration of 20 pM (final concentration) of 949 polypeptide was equilibrated over 24 hours using a 16-point dose-response curve with a 1/2.2 serial dilution of human, cynomolgus or rat ADAMTS5, each time starting at 20 nM, with the last the point is a control point (= without ADAMTS5). All dilutions were made in sample buffer (PBS+0.1% BSA+0.02% NaN ₃ ). To test for Aggrecan interference, recombinant Aggrecan G1-IGD-G2 was added to this pre-incubation in selected experiments at a concentration of 2 nM (= over 100x its K _D for polypeptide 949, see below).

Then, preincubated mixtures (ADAMTS5 DRC+20 pM polypeptide 949 ± Aggrecan G1–IGD–G2) were injected through a KinExA autosampler into a column with human ADAMTS5-conjugated poly(methyl methacrylate) (PMMA) beads to capture free Nanotel constructs on the beads. Captured Nanobody constructs were determined using the Alexa Fluor (AF) 647 instrument labeled anti-Nanobody recognizing the 949 polypeptide. The percentage of free Nanobody construct was plotted as a function of ADAMTS5 concentration and KinExA Pro v3.6.5 was used to determine K _D .

The results presented in Table 5.1 show that the affinity of the 949 polypeptide for human ADAMTS5 is 32.69 pM in the absence of G1-IGD-G2 and 26.56 pM in the presence of Aggrecan. Based on the fact that the CIs overlap with the K _D values, the presence of Aggrecan did not affect the affinity of the 949 polypeptide for ADAMTS5. The cynomolgus and rat ADAMTS5 affinities were 24.35 pM and 25.17 pM, respectively, demonstrating the binding cross-reactivity of the 949 polypeptide for both species, since both K _D values corresponded to human CI K _D.

Table 5.1: Affinity of 949 polypeptide for human ADAMTS5 in the absence or presence of Aggrecan G1-IGD-G2, and for cynomolgus monkey and rat ADAMTS5

Table 5.1 N Mean value K _D (pM) 95% CI for K _D (pM) Human - Aggrekan 3 32.69 16.35–65.38 Human + Aggrekan 3 26.56 16.38–58.46 Javanese macaque one 24.35 9.25–66.27 Rat one 25.17

5.2 Functional inhibition of human, cynomolgus and rat ADAMTS5 by 949 polypeptide

Functional inhibition of human, cynomolgus and rat ADAMTS5 by C010100949 was investigated using FRET-based kinetic analysis. This assay was performed using FRET assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl2.2H ₂ 0.5% glycerol, 0.1% CHAPS in MilliQ). A fixed concentration of 10 nM (final concentration) ADAMTS5 from the respective species was mixed and equilibrated using an 11-point (12 ^{-n =} control) DRC 1/1.7 serial dilution of the 949 polypeptide starting at 50 nM (final concentration). Then 30 μM (final concentration) of the ADAMTS5 species-specific fluorogenic substrate was added. This substrate, Aggrecan peptide labeled with quencher (2,4-dinitrophenyl, Dnp) and fluorochrome (aminobenzoic acid, Abz) was enzymatically cleaved with ADAMTS5 and fluorescence (excitation wavelength 340 nm/emission wavelength 430 nm) was kinetically measured every minute for 2 hours using the Tecan F200 microplate reader. The resulting process curves (fluorescent signal vs. reaction time) were analyzed using the GraphPad Prism software by superimposing the linear part of each process curve with a straight line (Y=slope * X+Y-cut-off segment on the axis). Obtained slopes or reaction rates (v _i ) were normalized to the mean slope of all controls (uninhibited reaction rate, v ₀ ), plotted as a function of 949 polypeptide concentration, and fitted using a non-symmetrical 5-parameter logistic curve (5PL) fit to obtaining inhibition curves.

The results are presented in Fig. 1 which is a representative graphical illustration of the results. The results show dose dependent and complete inhibition of ADAMTS5 enzymatic activity from all species tested by the 949 ("C010100949") polypeptide, demonstrating that they were all fully functionally inhibited by the Nanotel construct.

5.3 Affinity of 949 polypeptide for human, cynomolgus and rat MMP13

Functional activities as well as enzyme inhibition constants (K _I ) of polypeptide 949 against MMP13 from different species were examined using FRET-based kinetic analysis. All dilutions were made in assay buffer (50 mM Tris (pH 7.5), 100 mM NaCl, 10 mM CaCl ₂ .2H ₂ 0, 0.01% Tween20). 11-point DRC ( ^12th = control) 1/3 serial dilution of the 949 polypeptide, starting at 9 μM (final concentration), was balanced with a constant concentration of 0.2 nM of the catalytic domain (cd) of MMP-13 or 0.2–20 nM activated pro-MMP13 (nominal concentration) of each species. Then, a constant concentration of 22 μM (final concentration) of fluorogenic MMP substrate was added to all wells. This collagen-based substrate contains the typical MMP cleavage sequence Proline-Leucine-Glycine-Leucine (PLGL) and is labeled with fluorochrome, Mca and Dpa quencher. The active enzyme (not bound by the Nanobody construct) is able to cleave the substrate, resulting in the release of the fluorochrome and resulting in a fluorescent signal (excitation wavelength 320 nm/emission wavelength 405 nm), while the neutralized enzyme (bound by the Nanobody construct) is not able to. Starting within one minute after adding the substrate, fluorescence was measured kinetically every minute for all 120 cycles (2 hours) using a Tecan F200 plate reader.

The resulting process curves (fluorescent signal vs. reaction time) were analyzed using the GraphPad Prism software by superimposing the linear part of each process curve with a straight line (Y=slope * X+Y-cut-off segment on the axis). Obtained slopes or reaction rates (v _i ) were normalized to the mean slope of all controls (uninhibited reaction rate, v ₀ ), plotted as a function of 949 polypeptide concentration, and fitted using a non-symmetrical 5-parameter logistic curve (5PL) fit to obtaining inhibition curves.

Under these assay conditions, the resulting IC ₅₀ value is equal to the inhibition constant K _I .

The results of quantitative analysis are presented in Table 5.3. Two to six independent experiments were repeated based on the interaction. Based on 6 independent replicate experiments, the inhibition constant of the 949 polypeptide against human cdMMP13 was determined to be 27.86 nM (95% CI: 25.67-30.23 nM). Based on 3 independent replicates, the KI for activated human pro- _MMP13 was determined to be 2.92 nM (95% CI: 0.61-14.02 nM). The KI values for cynomolgus and rat _MMP13 are highly consistent with human KI values for the catalytic domain (cd) as well as for activated pro- _MMP13 (pro), indicating cross-reactivity of polypeptide 949 with cynomolgus and rat MMP13.

Table 5.3 Summary of 949 polypeptide affinity results for MMP13 catalytic domain and activated human, cynomolgus and rat pro-MMP13

Table 5.3 MMP13 form N Mean value K _I (nM) CV (%) 95% CI for K _I (nM) Man cd 6 27.86 7.7 25.67–30.23 pro 3 2.92 52.7 0.61–14.02 Javanese macaque cd 3 20.21 1.6 19.43–21.03 pro 2 3.72 5.5 2.27–6.11 Rat cd 3 26.59 9.2 21.19–33.36 pro 2 13.02 31.9 0.70–241.59

Mean K _I values as well as 95% confidence intervals (CI) were calculated by determining the total means. Cd: catalytic domain; pro: activated pro-MMP13; CV: coefficient of variation.

Functional inhibition of human, cynomolgus and rat cdMMP13 as well as activated pro- _MMP13 by polypeptide 949 was evaluated simultaneously in experiments that were performed to measure KI (see above). Fig. 2 is a representative graphical illustration of the results. These results demonstrate a dose-dependent and complete functional inhibition of the enzymatic activity of cdMMP13 and pro-MMP13 forms of all tested species by the Nanotel 949 construct.

5.4 Affinity of polypeptide 949 for human, cynomolgus and rat Aggrecan G1-IGD-G2

Determination of the affinity of the 949 polypeptide for recombinant human, cynomolgus, and rat Aggrecan G1-IGD-G2 was performed using surface plasmon resonance (SPR) on a ProteOn XPR36 multi-interaction analyzer (BioRad, serial number 670BR6176). Briefly, recombinant Aggrecan G1-IGD-G2 (“ligand”) was immobilized on the ligand lanes of a GLC sensor chip (Biorad cat # 176-5011) by amine coupling at a concentration of 10 μg/ml in 10 mM acetate pH 4.5 and a flow rate 100 µl/min to immobilize at 400 resonance units (RU). Polypeptide 949 (“C010100949”; 'analyte') was injected along the analyte bands of the sensor chip at a continuous flow of 45 μl/min for 2 minutes at various concentrations (0.5–1–2–4–6–10 nM) in 1x HBS -P+pH 7.4 as working buffer (GE Healthcare cat#R-1006-71). The dissociation time was 10 min. After each injection of the sample, the surfaces were restored using 10 mm glycine, pH 1.5. The interaction between Aggrecan G1-IGD-G2 and the 949 polypeptide was defined as an increase in the refractive index that occurs as a result of mass changes on the chip due to the binding of the 949 polypeptide to Aggrecan. This change in refractive index causes a change in the intensity and angle of the reflected light, which is quantitatively measured in real time and displayed on the sensogram as response units (RU) versus time. Kinetic constants were calculated from acquired sensograms using ProteOn Manager 3.1.0 version 3.1.0.6 and Langmuir's 'kinetic k _a /k _d simultaneous' model.

The results are presented in Table 5.4. Based on 2-4 independent replicate experiments, the mean binding affinities (K _D ) of the 949 polypeptide for captured Aggrecan G1-IGD-G2 were determined to be 14.0 pM, 16.0 pM and 16.6 pM for human Aggrecan, Javanese macaque and rat, respectively.

Since the mode of action (MoA) of the CAP building blocks of the 949 polypeptide is based solely on Aggrecan binding, which is directly reflected in the SPR-based affinity determination, further functional analysis of the CAP building blocks is not relevant. From these SPR data, it can be concluded that the 949 polypeptide shows complete cross-reactivity to cynomolgus and rat Aggrecan with no difference in affinity compared to human.

Table 5.4 Summary of 949 affinity results for human, cynomolgus and rat Aggrecan G1-IGD-G2.

Table 5.4 N Mean value K _D (pM) 95% CI for K _D (pM) Man 4 14.0 5.9–33.7 Javanese macaque 2 16.0 NC (*) Rat 3 16.6 7.0–39.5

Mean K _D values as well as 95% confidence intervals (CI) were calculated by determining the total means. N: number of independent experiments; K _D : dissociation constant; NC: didn't count. (*) For the cynomolgus macaque, 2 independent experiments showed the same result of 16.0 pM, hence no CI could be calculated because the ProteOn program did not provide any information on standard errors for K _D .

Example 6 Inhibition of cartilage destruction ex vivo in a bovine explant model

Polypeptide 949 (M-A-C-C: 62C02-2F03-114F08-114F08) was tested in bovine cartilage explant assays for inhibition of cartilage degradation.

Briefly, the polypeptide was incubated with bovine cartilage explants cultured for 5 days with IL-1α to induce an OA-like process of cartilage destruction. Within 5 days, the destruction of mainly Aggrecan occurs, while the destruction of Collagen occurs only after about two weeks, that is, after the termination of the experiments described in this application. At the end of the experiment, GAG (predominantly from Aggrecan) was quantified and released from the degrading cartilage into the culture supernatant. The effectiveness of inhibition of GAG release by the polypeptide was calculated in comparison with a reference small molecule inhibitor, which completely inhibited IL-1α-stimulated GAG release under these conditions.

The results are presented in Fig. 3.

It can be seen that the 949 polypeptide (NB949 in Fig.) completely inhibits the destruction of bovine cartilage in a dose-dependent manner.

Example 7 Inhibition of cartilage destruction ex vivo in the human explant model

Polypeptide 949 (M-A-C-C: 62C02-2F03-114F08-114F08) was tested in human cartilage explant assays for inhibition of cartilage degradation. Briefly, the polypeptide was incubated with human cartilage explants that had been cultured for 7 days with IL-1β+Oncostatin M (OSM). Within 7 days, the destruction of mainly Aggrecan occurs, while the destruction of Collagen occurs only after about two weeks, that is, after the termination of the experiments described in this application. At the end of the experiment, GAG (predominantly from Aggrecan) was quantified and released from the degrading cartilage into the culture supernatant. IC50 was calculated based on the negative control level (= GAG release from explants without additional stimulus) and IL-1 β+OSM level as an indication of maximum GAG loss. GAG is shown as GAG release (in μg) per mg cartilage of each individual explant.

The results are presented in Fig. 4.

The data show that the Nanobody construct inhibits GAG release from human cartilage OA in a dose-dependent manner, with a calculated IC ₅₀ value of 0.03724 μM.

Example 8 CAP extends ex vivo the effectiveness of polypeptides that inhibit the destruction of cartilage

In order to evaluate the effect of the CAP fragment, which functions to anchor the polypeptide in cartilage, the above described bovine explant assay was modified to include washing steps after a step of incubation (1h) of the cartilage explant with polypeptides (see Fig. 5, upper panel). After the washing steps, cartilage destruction was initiated by the addition of IL-1α. After 7 days of additional incubation, the release of GAG into the culture supernatant was quantified. As a control, the ADAMTS5 inhibitory polypeptide 581 was included, which does not contain a CAP-binding fragment (SEQ ID NO: 65).

The results are shown in FIG. five.

The experiment shows that the control 581 polypeptide, ie the polypeptide lacking the CAP-binding fragment, is almost ineffective in inhibiting ADAMTS5. This is very different from the assays where washing steps were omitted. On the other hand, the 949 polypeptide remained effective even after intensive washing. Considering that GAG release is mediated by ADAMTS5 and not MMP13 under the present assay conditions, this result is strong evidence that the CAP-binding fragment does indeed function to anchor polypeptides in cartilage, and that the CAP-binding fragment does not affect the performance of ADAMTS5 ISVD.

Example 9 Effect of polypeptide 949 in the system of co-cultivation of the synovial layer of bovine cartilage

Knowing that OA does not only affect cartilage, the 949 polypeptide was also tested in a bovine model consisting of synovial membrane (synovial layer) and articular cartilage explants. Briefly, cartilage explants and synovium in a ratio of 1/1 were co-cultured for 28 days with regular medium changes. On a weekly basis, C2M (Col II degradation) and C3M (Col III degradation) were determined in the co-culture supernatant.

The results (as AUC=area under the curve) are shown in FIG. 6.

The data show that co-incubation of cartilage explants with the synovial layer induces the release of C2M (left panel) and C3M (right panel). The 949 polypeptide was incubated at 3 different concentrations (1 nM, 10 nM, 100 nM). The effect of the 949 polypeptide was evaluated in comparison to the Wyeth reference molecule (MSC2310852A-1). The data show that the exemplary 949α polypeptide as well as the reference compound strongly inhibit the release of C2M and C3M.

Example 10 Cartilage Retention Studies

In order to determine the in vivo retention of the Nanobody constructs, a retention study in rat cartilage was conducted using a representative 949β polypeptide. Since the 949 polypeptide has 4 building blocks (MACC), it is expected that the 754 and 954 polypeptides, each with two Aggrecan-binding and one target-specific ISVD, behave essentially similarly.

An ELISA-based ligand binding assay was designed to quantify local concentrations of the Nanobody construct in rat cartilage, while a ligand binding assay (MSD platform) was developed to quantify the Nanobody construct in the bloodstream.

The results are shown in FIG. 7.

In conclusion, exemplary 949 polypeptide is retained in cartilage in vivo up to 112 days after i/a administration, while systemic concentrations (low pg/ml range) were detected only at the first sampling time point (2 hours after i/a administration). ) in healthy rats, with no polypeptide being detectable at later time points (14 days or more).

Therefore, constructs containing anti-Aggrecan ISVD 114F08 are stable and are retained in in vivo models .

Example 11 Rat In Vivo Study 1 MMT DMOAD

To demonstrate the in vivo efficacy of polypeptide 754 (M-C-C; "Nanobody 754"), polypeptide 954 (A-C-C; "Nanobody 954") and polypeptide 949 (M-A-C-C, "Nanobody 949" ), used a model of surgically induced rupture of the medial meniscus (MMT) in rats. Briefly, rats were operated on on one knee to induce OA-like symptoms. Treatment was started 3 days after surgery with one i/a injection. 3 days after surgery, the following doses were administered to the animals: vehicle (buffer), polypeptide 754 (300 μg/rat), polypeptide 949 (4, 40 or 400 μg/rat) and polypeptide 954 (300 μg/rat).

Histopathology was performed 42 days after surgery. The width of the medial tibial cartilage degeneration and the overall degeneration of the medial tibial cartilage, as well as the percentage reduction in cartilage degeneration, were determined. Used 20 animals per group.

The results of determining the width of the medial tibial cartilage degeneration are shown in FIG. eight.

The results demonstrate that the width of the medial tibial cartilage was significantly reduced with all Nanobody constructs after 42 days compared to the vehicle. These results further suggest that

(a) the CAP fragment does not adversely affect the activity of constructs;

(b) CAP-fragment provides the ability to hold constructs;

(c) all constructs have a positive effect on cartilage width; as well as

(d) the combination of an ADAMTS5 inhibitor and an MMP13 inhibitor shows the greatest overall effect, as demonstrated by the 949.beta . polypeptide.

Example 12 Rat In Vivo Confirmation Study 2 MMT DMOAD

The in vivo efficacy studies described in Example 11 (DMOAD Study 1) were essentially repeated with a different administration schedule. In addition, only mild OA was present in DMOAD Study 1 3 days postoperatively. Therefore, polypeptides were now evaluated at a later stage of OA in the MMT model, with treatment starting 7 days postoperatively. Briefly, the width of the degeneration of the medial tibial cartilage in µm was measured on day 42 postoperatively. Again, the 949 polypeptide was used to illustrate the combined effect of ADAMTS5 and MMP13 inhibitors.

In the first series of experiments, consisting of 20 animals per group, each group received one i/a injection (400 μg, 800 μg, vehicle) 7 days after surgery. At the end of treatment, the 400 μg group of 949β polypeptide showed a 21% reduction in the width of medial tibial cartilage degeneration, while the 800 μg group showed a 16% reduction (see Fig. 9). These results confirm the efficiency of the designs.

In the second series of experiments, again consisting of 20 animals per group, each group received two i/a injections on day 7 and day 10 after surgery (200 μg, 400 μg, 800 μg, vehicle). At the end of treatment, the 200 μg 949β polypeptide group showed a 14% reduction in tibia width, the 800 μg group showed a 31% reduction, and the 400 μg group showed a 35% reduction (FIG. 9).

Example 13 Symptomatic benefit in vivo

To evaluate the ability of polypeptides to provide symptomatic benefit, a rat model of surgical OA was used. Briefly, rats underwent ACLt and tMx surgery to induce OA on day 0. In the ACLt and tMx surgical model (anterior cruciate ligament transection with removal of the medial meniscus), constructs were administered i/a at 1 and 8 weeks at various concentrations (100 mcg, 400 mcg and 1000 mcg). One group of rats was administered by i/a injection of Tanezumab (Pfizer), an anti-nerve growth factor (NGF) mAb that was included as a positive control for symptomatic relief in the study. At weeks 2, 5, 9, and 13, symptomatic benefit was determined by gait analysis (on CatWalk) as well as reduction in joint diameter.

The results are shown in FIG. 10.

Intra-articular treatment with polypeptides resulted in a symptomatic effect of up to 43%.

Example 14 Retention of CAP binders in healthy and osteoarthritis rats is the same in vivo

In Example 10, in a cartilage retention study in healthy rats, it was demonstrated that the polypeptides of the invention can be measured in cartilage up to 112 days after intra-articular (i/a) injection. Because cartilage composition can influence cartilage binding and systemic absorption, the pharmacokinetics of the polypeptides of the invention were compared in vivo in osteoarthritis patients and healthy rats by monitoring serum levels of the polypeptides over time.

In particular, a surgically induced medial meniscus tear (MMT) model in rats was used as described in Example 10, but with some modifications. Briefly, the polypeptides of the invention have been linked to anti-MMP13ISV and anti-ADAMTS5 ISVs (designated as "949", "0949" or "C010100949" Nanobodies). Rats were operated on on one knee to induce OA-like symptoms (OA group). Each treatment group (healthy and OA) consisted of 15 animals and received one i/a injection of 400 μg/30 μl Nanotel on day 7 (healthy) or 7 days after surgery (MMT). Serum samples were collected from anesthetized rats on day 0, day 7 (at 0 hours=pre-dose sample), day 8 (various times post-treatment up to 24 hours), day 9 (48 hours post-treatment), day 10 (3 days post-treatment), day 14 (7 days post-treatment), day 21 (14 days post-treatment), and day 42 (35 days post-treatment). The collected serum samples were used to determine the concentrations of the polypeptide in electrochemiluminescent PK-analysis (ECL), followed by non-compartmental analysis.

Serum retention of polypeptides in healthy and OA rats is shown in FIG. eleven.

The results demonstrate that there are no obvious differences in serum concentrations of polypeptides between healthy and OA rats. Therefore, these results confirm that cartilage degradation has no effect on the pharmacokinetics of the polypeptides of the invention.

Table A–1
Name and short description ("ID"), SEQ ID NO: ("SEQ") and amino acid sequences of the polypeptides of the invention ID SEQ Subsequence ALX-1011 949 one EVQLVESGGGVVQPGGSLRLSCAASGFAFSAAYMSWVRQAPGKGLEWVSSISDDGSKTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNTGYGATTTRPGRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 62C02
MMP13 2 EVQLVESGGGVVQPGGSLRLSCAASGFAFSAAYMSWVRQAPGKGLEWVSSISDDGSKTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNTGYGATTTRPGRYWGQGTLVTVSS 2F03
ATS5 3 EVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSS 114F08
cap 4 EVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSS MCC 754 five EVQLVESGGGVVQPGGSLRLSCAASGFAFSAAYMSWVRQAPGKGLEWVSSISDDGSKTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNTGYGATTTRPGRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA ACC
954 6 EVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 114F08–Alb "054" 41 EVQLVESGGGLVQAGGSLRLSCAASGSTFIINVVRWYRRTPGKQRELVATISSGGNANYVDSVRGRFSISRDGAKNAVDLQMNGLKPEDTAVYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSAVMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb–114F08 –114F08 "118" 42 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSAVMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSTFIINVVRWYRRTPGKQRELVATISSGGNANYVDSVRGRFSISRDGAKNAVDLQMNGLKPEDTAVYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSTFIINVVRWYRRTPGKQRELVATISSGGNANYVDSVRGRFSISRDGAKNAVDLQMNGLKPEDTAVYYCNVPTTHYGGVYYGPYWGQGTLVTVSS Alb–Alb "030" 43 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSAVMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSAVMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 949–9GS
“973” 62 EVQLVESGGGVVQPGGSLRLSCAASGFAFSAAYMSWVRQAPGKGLEWVSSISDDGSKTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNTGYGATTTRPGRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 754–9GS 63 EVQLVESGGGVVQPGGSLRLSCAASGFAFSAAYMSWVRQAPGKGLEWVSSISDDGSKTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNTGYGATTTRPGRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 954–9GS 64 EVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA pp 581 65 DVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA

Table B
Other amino acid sequences: Name and short description ("ID"), SEQ ID NO: ("SEQ") and amino acid sequences ("sequences") ID SEQ Subsequence MMP13
human 66 MHPGVLAAFLFLSWTHCRALPLPSGGDEDDLSEEDLQFAERYLRSYYHPTNLAGILKENAASSMTERLREMQSFFGLEVTGKLDDNTLDVMKKPRCGVPDVGEYNVFPRTLKWSKMNLTYRIVNYTPDMTHSEVEKAFKKAFKVWSDVTPLNFTRLHDGIADIMISFGIKEHGDFYPFDGPSGLLAHAFPPGPNYGGDAHFDDDETWTSSSKGYNLFLVAAHEFGHSLGLDHSKDPGALMFPIYTYTGKSHFMLPDDDVQGIQSLYGPGDEDPNPKHPKTPDKCDPSLSLDAITSLRGETMIFKDRFFWRLHPQQVDAELFLTKSFWPELPNRIDAAYEHPSHDLIFIFRGRKFWALNGYDILEGYPKKISELGLPKEVKKISAAVHFEDTGKTLLFSGNQVWRYDDTNHIMDKDYPRLIEEDFPGIGDKVDAVYEKNGYIYFFNGPIQFEYSIWSNRIVRVMPANSILWC ADAMTS5
human 67 MLLGWASLLLCAFRLPLAAVGPAATPAQDKAGQPPTAAAAAQPRRRQGEEVQERAEPPGHPHPLAQRRRSKGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDGSVGIAGFVPAGGGTSAPWRHRSHCFYRGTVDGSPRSLAVFDLCGGLDGFFAVKHARYTLKPLLRGPWAEEEKGRVYGDGSARILHVYTREGFSFEALPPRASCETPASTPEAHEHAPAHSNPSGRAALASQLLDQSALSPAGGSGPQTWWRRRRRSISRARQVELLLVADASMARLYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEETFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPRNNGRYCTGKRAIYRSCSLMPCPPNGKSFRHEQCEAKNGYQSDAKGVKTFVEWVPKYAGVLPADVCKLTCRAKGTGYYVVFSPKVTDGTECRLYSNSVCVRGKCVRTGCDGIIGSKLQYDKCGVCGGDNSSCTKIVGTFNKKSKGYTDVVRIPEGATHIKVRQFKAKDQTRFTAYLALKKKNGEYLINGKYMISTSETIIDINGTVMNYSGWSHRDDFLHGMGYSATKEILIVQILATDPTKPLDVRYSFFVPKKSTPKVNSVTSHGSNKVGSHTSQPQWVTGPWLACSRTCDTGWHTRTVQCQDGNRKLAKGCPLSQRPSAFKQCLLKKC Aggrekan
human 68 MTTLLWVFVTLRVITAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEVQSLRSNDSGVYRCEVMHGIEDSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGHVYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYVHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAPEIGATAFAEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGPTRYSLTFEEAQQACPGTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSTETYDVYCFVDRLEGEVFFATRLEQFTFQEALEFCESHNATATTGQLYAAWSRGLDKCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGISAVPSPGEEEGGTPTSPSGVEEWIVTQVVPGVAAVPVEEETTAVPSGETTAILEFTTEPENQTEWEPAYTPVGTSPLPGILPTWPPTGAETEESTEGPSATEVPSASEEPSPSEVPFPSEEPSPSEEPFPSVRPFPSVELFPSEEPFPSKEPSPSEEPSASEEPYTPSPPEPSWTELPSSGEESGAPDVSGDFTGSGDVSGHLDFSGQLSGDRASGLPSGDLDSSGLTSTVGSGLTVESGLPSGDEERIEWPSTPTVGELPSGAEILEGSASGVGDLSGLPSGEVLETSASGVGDLSGLPSGEVLETTAPGVEDISGLPSGEVL ETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETTAPGVEEISGLPSGEVLETTAPGVDEISGLPSGEVLETTAPGVEEISGLPSGEVLETSTSAVGDLSGLPSGGEVLEISVSGVEDISGLPSGEVVETSASGIEDVSELPSGEGLETSASGVEDLSRLPSGEEVLEISASGFGDLSGVPSGGEGLETSASEVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDLVGSASGDLDLGKLPSGTLGSGQAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTIGISGAGEISGLPSSELDISGRASGLPSGTELSGQASGSPDVSGEIPGLFGVSGQPSGFPDTSGETSGVTELSGLSSGQPGVSGEASGVLYGTSQPFGITDLSGETSGVPDLSGQPSGLPGFSGATSGVPDLVSGTTSGSGESSGITFVDTSLVEVAPTTFKEEEGLGSVELSGLPSGEADLSGKSGMVDVSGQFSGTVDSSGFTSQTPEFSGLPSGIAEVSGESSRAEIGSSLPSGAYYGSGTPSSFPTVSLVDRTLVESVTQAPTAQEAGEGPSGILELSGAHSGAPDMSGEHSGFLDLSGLQSGLIEPSGEPPGTPYFSGDFASTTNVSGESSVAMGTSGEASGLPEVTLITSEFVEGVTEPTISQELGQRPPVTHTPQLFESSGKVSTAGDISGATPVLPGSGVEVSSVPESSSETSAYPEAGFGASA APEASREDSGSPDLSETTSAFHEANLERSSGLGVSGSTLTFQEGEASAAPEVSGESTTTSDVGTEAPGLPSATPTASGDRTEISGDLSGHTSQLGVVISTSIPESEWTQQTQRPAETHLEIESSSLLYSGEETHTVETATSPTDASIPASPEWKRESESTAAAPARSCAEEPCGAGTCKETEGHVICLCPPGYTGEHCNIDQEVCEEGWNKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGEPPVVEHARTFGQKKDRYEINSLVRYQCTEGFVQRHMPTIRCQPSGHWEEPRITCTDATTYKRRLQKRSSRHPRRSRPSTAH

Table C
Various linker sequences (“ID” refers to SEQ ID NO as used in the table) Name ID Amino acid sequence A3 24 AAA 5GS linker 25 GGGGS 7GS linker 26 sggsggs 8GS linker 27 Gggggggs 9GS linker 28 GGGGSGGGS 10GS linker 29 GGGGSGGGGS 15GS linker thirty GGGGSGGGGSGGGGS 18GS linker 31 GGGGSGGGGSGGGGGGGS 20GS linker 32 GGGGSGGGGSGGGGSGGGGS 25GS linker 33 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS linker 34 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS linker 35 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS linker 36 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge section 37 EPKSCDKTHTCPPCP 9GS–G1 hinge section 38 GGGGSGGGSEPKSCDKTHTCPPCP Lama's upper long hinged area 39 epktpkpqpaaa G3 hinge section 40 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

Table A–2
Sequences for CDRs and frameworks, plus preferred combinations as shown in Formula I, namely FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (following terms: “ID” refers to the SEQ ID NO shown) ID nanobody ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 2 62C02 MMP13 7 EVQLVE SGGGVV QPGGSL RLSCAAS eight GFAFSAAYMS nine WVRQAPG KGLEWVS 10 SISDDG SKTY eleven YADSVKGRF TISRDNSKN TVYLQMNSL RPEDTALYY CNT 12 GYGATTTR PGRY 13 WGQGTLV TVSS 3 02F03 ATS5 7 EVQLVE SGGGVV QPGGSL RLSCAAS fourteen GRTVSS YAMG fifteen WFRQAPG KEREFVA 16 GISRSA ERTY 17 YVDSLKGRFT ISRDNSKNTV YLQMNSLRPE DTALYYCAA eighteen DLDPNRIFS REEYAY 13 WGQGTLV TVSS 4 114F08 Aggrekan–CAP 7 EVQLVE SGGGVV QPGGSL RLSCAAS nineteen GSTFII NVVR twenty WYRRAPG KQRELVA 21 TISSGG NAN 22 YVDSVRGRFT ISRDNSKNTV YLQMNSLRPE DTALYYCNV 23 PTTHYGGVY YGPY 13 WGQGTLV TVSS

Table D
Serum albumin-binding ISVD sequences (“ID” refers to SEQ ID NO used in the table), including CDR sequences Name ID Amino acid sequence Alb8 44 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 45 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 46 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 47 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 48 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11 (S112K)–A 49 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 fifty EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82–A 51 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82–AA 52 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82–AAA 53 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82–G 54 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSSG Alb82–GG 55 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82–GGG 56 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG Alb92 57 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 58 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA ALB CDR1 59 SFGMS ALB CDR2 60 SISGSGSDTLYADSVKG ALB CDR3 61 GGSLSSR

--->

SEQUENCE LIST

<110> Merck Patent GmbH

Ablynx N.V.

<120> ADAMTS5, MMP13 and Aggrecan binding polypeptides

<130> MEPAT.0105

<150>EP17174404.8

<151> 2017-06-02

<160> 68

<170> BiSSAP 1.3.6

<210> 1

<211> 593

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ala Ala

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Asp Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Asn Thr Gly Tyr Gly Ala Thr Thr Thr Arg Pro Gly Arg Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

145 150 155 160

Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu

165 170 175

Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Ser Tyr Ala Met Gly Trp

180 185 190

Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly Ile Ser

195 200 205

Arg Ser Ala Glu Arg Thr Tyr Tyr Val Asp Ser Leu Lys Gly Arg Phe

210 215 220

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn

225 230 235 240

Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala Asp Leu

245 250 255

Asp Pro Asn Arg Ile Phe Ser Arg Glu Glu Tyr Ala Tyr Trp Gly Gln

260 265 270

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

305 310 315 320

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

325 330 335

Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr

340 345 350

Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser

355 360 365

Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile

370 375 380

Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

385 390 395 400

Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr His

405 410 415

Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val

420 425 430

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

465 470 475 480

Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

485 490 495

Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg Ala Pro

500 505 510

Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala

515 520 525

Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

530 535 540

Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

545 550 555 560

Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val

565 570 575

Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

580 585 590

Ala

<210> 2

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ala Ala

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Asp Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Asn Thr Gly Tyr Gly Ala Thr Thr Thr Arg Pro Gly Arg Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 124

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 3

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Ala Glu Arg Thr Tyr Tyr Val Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Asp Pro Asn Arg Ile Phe Ser Arg Glu Glu Tyr Ala

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 4

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn

20 25 30

Val Val Arg Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn

85 90 95

Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 5

<211> 434

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ala Ala

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Asp Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Asn Thr Gly Tyr Gly Ala Thr Thr Thr Arg Pro Gly Arg Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

145 150 155 160

Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu

165 170 175

Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp

180 185 190

Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser

195 200 205

Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr

210 215 220

Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser

225 230 235 240

Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr

245 250 255

His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg Ala

340 345 350

Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly Asn

355 360 365

Ala Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp

370 375 380

Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu

385 390 395 400

Asp Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly Gly

405 410 415

Val Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

420 425 430

Ser Ala

<210> 6

<211> 437

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Ala Glu Arg Thr Tyr Tyr Val Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Asp Pro Asn Arg Ile Phe Ser Arg Glu Glu Tyr Ala

100 105 110

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser

165 170 175

Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn Val

180 185 190

Val Arg Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

195 200 205

Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg Gly

210 215 220

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

225 230 235 240

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn Val

245 250 255

Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly Gln

260 265 270

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

305 310 315 320

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

325 330 335

Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr

340 345 350

Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser

355 360 365

Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile

370 375 380

Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

385 390 395 400

Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr His

405 410 415

Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val

420 425 430

Thr Val Ser Ser Ala

435

<210> 7

<211> 25

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 8

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 8

Gly Phe Ala Phe Ser Ala Ala Tyr Met Ser

1 5 10

<210> 9

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 9

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 10

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 10

Ser Ile Ser Asp Asp Gly Ser Lys Thr Tyr

1 5 10

<210> 11

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 11

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

20 25 30

Ala Leu Tyr Tyr Cys Asn Thr

35

<210> 12

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 12

Gly Tyr Gly Ala Thr Thr Thr Arg Pro Gly Arg Tyr

1 5 10

<210> 13

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 13

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 14

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 14

Gly Arg Thr Val Ser Ser Tyr Ala Met Gly

1 5 10

<210> 15

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 15

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala

1 5 10

<210> 16

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 16

Gly Ile Ser Arg Ser Ala Glu Arg Thr Tyr

1 5 10

<210> 17

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 17

Tyr Val Asp Ser Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

20 25 30

Ala Leu Tyr Tyr Cys Ala Ala

35

<210> 18

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 18

Asp Leu Asp Pro Asn Arg Ile Phe Ser Arg Glu Glu Tyr Ala Tyr

1 5 10 15

<210> 19

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 19

Gly Ser Thr Phe Ile Ile Asn Val Val Arg

1 5 10

<210> 20

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 20

Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

1 5 10

<210> 21

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 21

Thr Ile Ser Ser Gly Gly Asn Ala Asn

fifteen

<210> 22

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 22

Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

20 25 30

Ala Leu Tyr Tyr Cys Asn Val

35

<210> 23

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 23

Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr

1 5 10

<210> 24

<400> 24

000

<210> 25

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 25

Gly Gly Gly Gly Ser

fifteen

<210> 26

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 26

Ser Gly Gly Ser Gly Gly Ser

fifteen

<210> 27

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 27

Gly Gly Gly Gly Gly Gly Gly Ser

fifteen

<210> 28

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 28

Gly Gly Gly Gly Ser Gly Gly Gly Ser

fifteen

<210> 29

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 29

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 30

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 31

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 31

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly

1 5 10 15

Gly Ser

<210> 32

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 32

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 33

<211> 25

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 33

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 34

<211> 30

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 34

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 35

<211> 35

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 35

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser

35

<210> 36

<211> 40

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 36

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser

35 40

<210> 37

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 37

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 38

<211> 24

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 38

Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys

1 5 10 15

Thr His Thr Cys Pro Pro Cys Pro

twenty

<210> 39

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 39

Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala

1 5 10

<210> 40

<211> 62

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 40

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

35 40 45

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

50 55 60

<210> 41

<211> 271

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn

20 25 30

Val Val Arg Trp Tyr Arg Arg Thr Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg

50 55 60

Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala Lys Asn Ala Val Asp Leu

65 70 75 80

Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

145 150 155 160

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu

165 170 175

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala Val Met Ser Trp

180 185 190

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser

195 200 205

Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe

210 215 220

Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn

225 230 235 240

Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly

245 250 255

Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser

260 265 270

<210> 42

<211> 427

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala

20 25 30

Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

165 170 175

Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg Thr Pro Gly

180 185 190

Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn

195 200 205

Tyr Val Asp Ser Val Arg Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala

210 215 220

Lys Asn Ala Val Asp Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr

225 230 235 240

Ala Val Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val Tyr

245 250 255

Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

260 265 270

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

290 295 300

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

305 310 315 320

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile

325 330 335

Ile Asn Val Val Arg Trp Tyr Arg Arg Thr Pro Gly Lys Gln Arg Glu

340 345 350

Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser

355 360 365

Val Arg Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala Lys Asn Ala Val

370 375 380

Asp Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

385 390 395 400

Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr

405 410 415

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

420 425

<210> 43

<211> 265

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala

20 25 30

Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

165 170 175

Phe Thr Phe Ser Ser Ala Val Met Ser Trp Val Arg Gln Ala Pro Gly

180 185 190

Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr

195 200 205

Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

210 215 220

Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

225 230 235 240

Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser

245 250 255

Gln Gly Thr Leu Val Thr Val Ser Ser

260 265

<210> 44

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 45

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 45

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 46

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 47

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 48

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 49

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Lys

100 105 110

Val Ser Ser Ala

115

<210> 50

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 51

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 52

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ala

115

<210> 53

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ala Ala

115

<210> 54

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 54

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly

115

<210> 55

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 55

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly

115

<210> 56

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 56

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Gly Gly Gly

115

<210> 57

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 58

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 58

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala

115

<210> 59

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 59

Ser Phe Gly Met Ser

fifteen

<210> 60

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 60

Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 61

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 61

Gly Gly Ser Leu Ser Arg

fifteen

<210> 62

<211> 515

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 62

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ala Ala

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Asp Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Asn Thr Gly Tyr Gly Ala Thr Thr Thr Arg Pro Gly Arg Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

130 135 140

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser

145 150 155 160

Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

165 170 175

Phe Val Ala Gly Ile Ser Arg Ser Ala Glu Arg Thr Tyr Tyr Val Asp

180 185 190

Ser Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

195 200 205

Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr

210 215 220

Tyr Cys Ala Ala Asp Leu Asp Pro Asn Arg Ile Phe Ser Arg Glu Glu

225 230 235 240

Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

245 250 255

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

260 265 270

Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

275 280 285

Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg Ala Pro

290 295 300

Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala

305 310 315 320

Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

325 330 335

Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

340 345 350

Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val

355 360 365

Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

370 375 380

Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

385 390 395 400

Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

405 410 415

Ala Ser Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg

420 425 430

Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly

435 440 445

Asn Ala Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg

450 455 460

Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro

465 470 475 480

Glu Asp Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly

485 490 495

Gly Val Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

500 505 510

Ser Ser Ala

515

<210> 63

<211> 382

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 63

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ala Ala

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Asp Asp Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Asn Thr Gly Tyr Gly Ala Thr Thr Thr Arg Pro Gly Arg Tyr Trp Gly

100 105 110

Gly Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

130 135 140

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile

145 150 155 160

Ile Asn Val Val Arg Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu

165 170 175

Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser

180 185 190

Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val

195 200 205

Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr

210 215 220

Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr

225 230 235 240

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

260 265 270

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr

275 280 285

Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg Ala Pro Gly Lys Gln

290 295 300

Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val

305 310 315 320

Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

325 330 335

Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu

340 345 350

Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly

355 360 365

Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

370 375 380

<210> 64

<211> 385

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 64

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Ala Glu Arg Thr Tyr Tyr Val Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Asp Pro Asn Arg Ile Phe Ser Arg Glu Glu Tyr Ala

100 105 110

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val

130 135 140

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser

145 150 155 160

Thr Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg Ala Pro Gly Lys

165 170 175

Gln Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr

180 185 190

Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

195 200 205

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala

210 215 220

Leu Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr

225 230 235 240

Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

245 250 255

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

260 265 270

Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

275 280 285

Gly Ser Thr Phe Ile Ile Asn Val Val Arg Trp Tyr Arg Arg Ala Pro

290 295 300

Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Ser Ser Gly Gly Asn Ala

305 310 315 320

Asn Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

325 330 335

Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

340 345 350

Thr Ala Leu Tyr Tyr Cys Asn Val Pro Thr Thr His Tyr Gly Gly Val

355 360 365

Tyr Tyr Gly Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

370 375 380

Ala

385

<210> 65

<211> 275

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 65

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Ala Glu Arg Thr Tyr Tyr Val Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Asp Pro Asn Arg Ile Phe Ser Arg Glu Glu Tyr Ala

100 105 110

Tyr Trp Gly Glyn Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

145 150 155 160

Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn Ser

165 170 175

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly

180 185 190

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

195 200 205

Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys

210 215 220

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu

225 230 235 240

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr

245 250 255

Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val

260 265 270

Ser Ser Ala

275

<210> 66

<211> 471

<212> Protein

<213> Homo sapiens

<220>

<223> Amino acid sequence

<400> 66

Met His Pro Gly Val Leu Ala Ala Phe Leu Phe Leu Ser Trp Thr His

1 5 10 15

Cys Arg Ala Leu Pro Leu Pro Ser Gly Gly Asp Glu Asp Asp Leu Ser

20 25 30

Glu Glu Asp Leu Gln Phe Ala Glu Arg Tyr Leu Arg Ser Tyr Tyr His

35 40 45

Pro Thr Asn Leu Ala Gly Ile Leu Lys Glu Asn Ala Ala Ser Ser Met

50 55 60

Thr Glu Arg Leu Arg Glu Met Gln Ser Phe Phe Gly Leu Glu Val Thr

65 70 75 80

Gly Lys Leu Asp Asp Asn Thr Leu Asp Val Met Lys Lys Pro Arg Cys

85 90 95

Gly Val Pro Asp Val Gly Glu Tyr Asn Val Phe Pro Arg Thr Leu Lys

100 105 110

Trp Ser Lys Met Asn Leu Thr Tyr Arg Ile Val Asn Tyr Thr Pro Asp

115 120 125

Met Thr His Ser Glu Val Glu Lys Ala Phe Lys Lys Ala Phe Lys Val

130 135 140

Trp Ser Asp Val Thr Pro Leu Asn Phe Thr Arg Leu His Asp Gly Ile

145 150 155 160

Ala Asp Ile Met Ile Ser Phe Gly Ile Lys Glu His Gly Asp Phe Tyr

165 170 175

Pro Phe Asp Gly Pro Ser Gly Leu Leu Ala His Ala Phe Pro Pro Gly

180 185 190

Pro Asn Tyr Gly Gly Asp Ala His Phe Asp Asp Asp Glu Thr Trp Thr

195 200 205

Ser Ser Ser Lys Gly Tyr Asn Leu Phe Leu Val Ala Ala His Glu Phe

210 215 220

Gly His Ser Leu Gly Leu Asp His Ser Lys Asp Pro Gly Ala Leu Met

225 230 235 240

Phe Pro Ile Tyr Thr Tyr Thr Gly Lys Ser His Phe Met Leu Pro Asp

245 250 255

Asp Asp Val Gln Gly Ile Gln Ser Leu Tyr Gly Pro Gly Asp Glu Asp

260 265 270

Pro Asn Pro Lys His Pro Lys Thr Pro Asp Lys Cys Asp Pro Ser Leu

275 280 285

Ser Leu Asp Ala Ile Thr Ser Leu Arg Gly Glu Thr Met Ile Phe Lys

290 295 300

Asp Arg Phe Phe Trp Arg Leu His Pro Gln Gln Val Asp Ala Glu Leu

305 310 315 320

Phe Leu Thr Lys Ser Phe Trp Pro Glu Leu Pro Asn Arg Ile Asp Ala

325 330 335

Ala Tyr Glu His Pro Ser His Asp Leu Ile Phe Ile Phe Arg Gly Arg

340 345 350

Lys Phe Trp Ala Leu Asn Gly Tyr Asp Ile Leu Glu Gly Tyr Pro Lys

355 360 365

Lys Ile Ser Glu Leu Gly Leu Pro Lys Glu Val Lys Lys Ile Ser Ala

370 375 380

Ala Val His Phe Glu Asp Thr Gly Lys Thr Leu Leu Phe Ser Gly Asn

385 390 395 400

Gln Val Trp Arg Tyr Asp Asp Thr Asn His Ile Met Asp Lys Asp Tyr

405 410 415

Pro Arg Leu Ile Glu Glu Asp Phe Pro Gly Ile Gly Asp Lys Val Asp

420 425 430

Ala Val Tyr Glu Lys Asn Gly Tyr Ile Tyr Phe Phe Asn Gly Pro Ile

435 440 445

Gln Phe Glu Tyr Ser Ile Trp Ser Asn Arg Ile Val Arg Val Met Pro

450 455 460

Ala Asn Ser Ile Leu Trp Cys

465 470

<210> 67

<211> 930

<212> Protein

<213> Homo sapiens

<220>

<223> Amino acid sequence

<400> 67

Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys Ala Phe Arg Leu Pro

1 5 10 15

Leu Ala Ala Val Gly Pro Ala Ala Thr Pro Ala Gln Asp Lys Ala Gly

20 25 30

Gln Pro Pro Thr Ala Ala Ala Ala Ala Gln Pro Arg Arg Arg Gln Gly

35 40 45

Glu Glu Val Gln Glu Arg Ala Glu Pro Pro Gly His Pro His Pro Leu

50 55 60

Ala Gln Arg Arg Arg Ser Lys Gly Leu Val Gln Asn Ile Asp Gln Leu

65 70 75 80

Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly Arg

85 90 95

Arg Phe Leu Leu Asp Leu Glu Arg Asp Gly Ser Val Gly Ile Ala Gly

100 105 110

Phe Val Pro Ala Gly Gly Gly Thr Ser Ala Pro Trp Arg His Arg Ser

115 120 125

His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser Leu Ala

130 135 140

Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val Lys His

145 150 155 160

Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Pro Trp Ala Glu Glu

165 170 175

Glu Lys Gly Arg Val Tyr Gly Asp Gly Ser Ala Arg Ile Leu His Val

180 185 190

Tyr Thr Arg Glu Gly Phe Ser Phe Glu Ala Leu Pro Pro Arg Ala Ser

195 200 205

Cys Glu Thr Pro Ala Ser Thr Pro Glu Ala His Glu His Ala Pro Ala

210 215 220

His Ser Asn Pro Ser Gly Arg Ala Ala Leu Ala Ser Gln Leu Leu Asp

225 230 235 240

Gln Ser Ala Leu Ser Pro Ala Gly Gly Ser Gly Pro Gln Thr Trp Trp

245 250 255

Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu

260 265 270

Leu Val Ala Asp Ala Ser Met Ala Arg Leu Tyr Gly Arg Gly Leu Gln

275 280 285

His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His

290 295 300

Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val

305 310 315 320

Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr

325 330 335

Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly

340 345 350

Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu

355 360 365

Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val

370 375 380

Gly Thr Ile Cys Ser Pro Glu Arg Ser Cys Ala Val Ile Glu Asp Asp

385 390 395 400

Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu

405 410 415

Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu Thr Phe Gly Ser

420 425 430

Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala

435 440 445

Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Ile Thr Glu Phe Leu

450 455 460

Asp Asp Gly His Gly Asn Cys Leu Leu Asp Leu Pro Arg Lys Gln Ile

465 470 475 480

Leu Gly Pro Glu Glu Leu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln

485 490 495

Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp

500 505 510

Val Cys Ala Arg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val

515 520 525

Cys Leu Thr Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys

530 535 540

Gly Arg Ile Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys

545 550 555 560

Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Ser Trp

565 570 575

Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg

580 585 590

His Cys Asn Asn Pro Ala Pro Arg Asn Asn Gly Arg Tyr Cys Thr Gly

595 600 605

Lys Arg Ala Ile Tyr Arg Ser Cys Ser Leu Met Pro Cys Pro Pro Asn

610 615 620

Gly Lys Ser Phe Arg His Glu Gln Cys Glu Ala Lys Asn Gly Tyr Gln

625 630 635 640

Ser Asp Ala Lys Gly Val Lys Thr Phe Val Glu Trp Val Pro Lys Tyr

645 650 655

Ala Gly Val Leu Pro Ala Asp Val Cys Lys Leu Thr Cys Arg Ala Lys

660 665 670

Gly Thr Gly Tyr Tyr Val Val Phe Ser Pro Lys Val Thr Asp Gly Thr

675 680 685

Glu Cys Arg Leu Tyr Ser Asn Ser Val Cys Val Arg Gly Lys Cys Val

690 695 700

Arg Thr Gly Cys Asp Gly Ile Ile Gly Ser Lys Leu Gln Tyr Asp Lys

705 710 715 720

Cys Gly Val Cys Gly Gly Asp Asn Ser Ser Cys Thr Lys Ile Val Gly

725 730 735

Thr Phe Asn Lys Lys Ser Lys Gly Tyr Thr Asp Val Val Arg Ile Pro

740 745 750

Glu Gly Ala Thr His Ile Lys Val Arg Gln Phe Lys Ala Lys Asp Gln

755 760 765

Thr Arg Phe Thr Ala Tyr Leu Ala Leu Lys Lys Lys Asn Gly Glu Tyr

770 775 780

Leu Ile Asn Gly Lys Tyr Met Ile Ser Thr Ser Glu Thr Ile Ile Asp

785 790 795 800

Ile Asn Gly Thr Val Met Asn Tyr Ser Gly Trp Ser His Arg Asp Asp

805 810 815

Phe Leu His Gly Met Gly Tyr Ser Ala Thr Lys Glu Ile Leu Ile Val

820 825 830

Gln Ile Leu Ala Thr Asp Pro Thr Lys Pro Leu Asp Val Arg Tyr Ser

835 840 845

Phe Phe Val Pro Lys Lys Ser Thr Pro Lys Val Asn Ser Val Thr Ser

850 855 860

His Gly Ser Asn Lys Val Gly Ser His Thr Ser Gln Pro Gln Trp Val

865 870 875 880

Thr Gly Pro Trp Leu Ala Cys Ser Arg Thr Cys Asp Thr Gly Trp His

885 890 895

Thr Arg Thr Val Gln Cys Gln Asp Gly Asn Arg Lys Leu Ala Lys Gly

900 905 910

Cys Pro Leu Ser Gln Arg Pro Ser Ala Phe Lys Gln Cys Leu Leu Lys

915 920 925

Lys Cys

930

<210> 68

<211> 2415

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 68

Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Thr Ala

1 5 10 15

Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

Ile Pro Gln Pro Ser Pro Leu Arg Val Leu Leu Gly Thr Ser Leu Thr

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Val Gln Ser Leu Arg Ser Asn Asp Ser

115 120 125

Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly His Val Tyr Leu

275 280 285

Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Val His Ala Asn Gln Thr Gly

325 330 335

Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu

370 375 380

Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr

385 390 395 400

Val Lys Pro Ile Phe Glu Val Ser Pro Ser Pro Leu Glu Pro Glu Glu

405 410 415

Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Ala Glu Val

420 425 430

Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro

435 440 445

Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln

450 455 460

Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe

465 470 475 480

His Tyr Arg Pro Gly Pro Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala

485 490 495

Gln Gln Ala Cys Pro Gly Thr Gly Ala Val Ile Ala Ser Pro Glu Gln

500 505 510

Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp

515 520 525

Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro

530 535 540

Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val

545 550 555 560

Arg Pro Ser Thr Glu Thr Tyr Asp Val Tyr Cys Phe Val Asp Arg Leu

565 570 575

Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln

580 585 590

Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Ala Thr Thr Gly

595 600 605

Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala Gly

610 615 620

Trp Leu Ala Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr Pro Arg Pro

625 630 635 640

Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro

645 650 655

Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe Cys

660 665 670

Phe Arg Gly Ile Ser Ala Val Pro Ser Pro Gly Glu Glu Glu Gly Gly

675 680 685

Thr Pro Thr Ser Pro Ser Gly Val Glu Glu Trp Ile Val Thr Gln Val

690 695 700

Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala Val

705 710 715 720

Pro Ser Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro Glu

725 730 735

Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Val Gly Thr Ser Pro

740 745 750

Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Ala Glu Thr Glu

755 760 765

Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Pro Ser Ala Ser Glu

770 775 780

Glu Pro Ser Pro Ser Glu Val Pro Phe Pro Ser Glu Glu Pro Ser Pro

785 790 795 800

Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu Leu

805 810 815

Phe Pro Ser Glu Glu Pro Phe Pro Ser Lys Glu Pro Ser Pro Ser Glu

820 825 830

Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Glu Pro

835 840 845

Ser Trp Thr Glu Leu Pro Ser Ser Gly Glu Glu Ser Gly Ala Pro Asp

850 855 860

Val Ser Gly Asp Phe Thr Gly Ser Gly Asp Val Ser Gly His Leu Asp

865 870 875 880

Phe Ser Gly Gln Leu Ser Gly Asp Arg Ala Ser Gly Leu Pro Ser Gly

885 890 895

Asp Leu Asp Ser Ser Gly Leu Thr Ser Thr Val Gly Ser Gly Leu Thr

900 905 910

Val Glu Ser Gly Leu Pro Ser Gly Asp Glu Glu Arg Ile Glu Trp Pro

915 920 925

Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu Glu

930 935 940

Gly Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu

945 950 955 960

Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro

965 970 975

Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser

980 985 990

Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu

995 1000 1005

Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro

1010 1015 1020

Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr

1025 1030 1035 1040

Thr Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val

1045 1050 1055

Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser

1060 1065 1070

Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly

1075 1080 1085

Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp

1090 1095 1100

Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly

1105 1110 1115 1120

Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala

1125 1130 1135

Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu

1140 1145 1150

Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly

1155 1160 1165

Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu

1170 1175 1180

Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile

1185 1190 1195 1200

Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val

1205 1210 1215

Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala

1220 1225 1230

Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu

1235 1240 1245

Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu

1250 1255 1260

Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro

1265 1270 1275 1280

Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Glu Ile Ser

1285 1290 1295

Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Asp

1300 1305 1310

Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro

1315 1320 1325

Gly Val Glu Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr

1330 1335 1340

Ser Thr Ser Ala Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu

1345 1350 1355 1360

Val Leu Glu Ile Ser Val Ser Gly Val Glu Asp Ile Ser Gly Leu Pro

1365 1370 1375

Ser Gly Glu Val Val Glu Thr Ser Ala Ser Gly Ile Glu Asp Val Ser

1380 1385 1390

Glu Leu Pro Ser Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Glu

1395 1400 1405

Asp Leu Ser Arg Leu Pro Ser Gly Glu Glu Val Leu Glu Ile Ser Ala

1410 1415 1420

Ser Gly Phe Gly Asp Leu Ser Gly Val Pro Ser Gly Gly Glu Gly Leu

1425 1430 1435 1440

Glu Thr Ser Ala Ser Glu Val Gly Thr Asp Leu Ser Gly Leu Pro Ser

1445 1450 1455

Gly Arg Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser

1460 1465 1470

Gly Leu Pro Ser Gly Lys Glu Asp Leu Val Gly Ser Ala Ser Gly Asp

1475 1480 1485

Leu Asp Leu Gly Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln Ala

1490 1495 1500

Pro Glu Thr Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly

1505 1510 1515 1520

Val Asp Leu Gly Ser Gly Pro Ser Gly Leu Pro Asp Phe Ser Gly

1525 1530 1535

Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser Thr Leu Val

1540 1545 1550

Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu Glu Gly Arg Gly Thr

1555 1560 1565

Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser Ser Glu

1570 1575 1580

Leu Asp Ile Ser Gly Arg Ala Ser Gly Leu Pro Ser Gly Thr Glu Leu

1585 1590 1595 1600

Ser Gly Gln Ala Ser Gly Ser Pro Asp Val Ser Gly Glu Ile Pro Gly

1605 1610 1615

Leu Phe Gly Val Ser Gly Gln Pro Ser Gly Phe Pro Asp Thr Ser Gly

1620 1625 1630

Glu Thr Ser Gly Val Thr Glu Leu Ser Gly Leu Ser Ser Gly Gln Pro

1635 1640 1645

Gly Val Ser Gly Glu Ala Ser Gly Val Leu Tyr Gly Thr Ser Gln Pro

1650 1655 1660

Phe Gly Ile Thr Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp Leu

1665 1670 1675 1680

Ser Gly Gln Pro Ser Gly Leu Pro Gly Phe Ser Gly Ala Thr Ser Gly

1685 1690 1695

Val Pro Asp Leu Val Ser Gly Thr Thr Ser Gly Ser Gly Glu Ser Ser

1700 1705 1710

Gly Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Ala Pro Thr Thr

1715 1720 1725

Phe Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu Pro

1730 1735 1740

Ser Gly Glu Ala Asp Leu Ser Gly Lys Ser Gly Met Val Asp Val Ser

1745 1750 1755 1760

Gly Gln Phe Ser Gly Thr Val Asp Ser Ser Gly Phe Thr Ser Gln Thr

1765 1770 1775

Pro Glu Phe Ser Gly Leu Pro Ser Gly Ile Ala Glu Val Ser Gly Glu

1780 1785 1790

Ser Ser Arg Ala Glu Ile Gly Ser Ser Leu Pro Ser Gly Ala Tyr Tyr

1795 1800 1805

Gly Ser Gly Thr Pro Ser Ser Phe Pro Thr Val Ser Leu Val Asp Arg

1810 1815 1820

Thr Leu Val Glu Ser Val Thr Gln Ala Pro Thr Ala Gln Glu Ala Gly

1825 1830 1835 1840

Glu Gly Pro Ser Gly Ile Leu Glu Leu Ser Gly Ala His Ser Gly Ala

1845 1850 1855

Pro Asp Met Ser Gly Glu His Ser Gly Phe Leu Asp Leu Ser Gly Leu

1860 1865 1870

Gln Ser Gly Leu Ile Glu Pro Ser Gly Glu Pro Pro Gly Thr Pro Tyr

1875 1880 1885

Phe Ser Gly Asp Phe Ala Ser Thr Thr Asn Val Ser Gly Glu Ser Ser

1890 1895 1900

Val Ala Met Gly Thr Ser Gly Glu Ala Ser Gly Leu Pro Glu Val Thr

1905 1910 1915 1920

Leu Ile Thr Ser Glu Phe Val Glu Gly Val Thr Glu Pro Thr Ile Ser

1925 1930 1935

Gln Glu Leu Gly Gln Arg Pro Val Thr His Thr Pro Gln Leu Phe

1940 1945 1950

Glu Ser Ser Gly Lys Val Ser Thr Ala Gly Asp Ile Ser Gly Ala Thr

1955 1960 1965

Pro Val Leu Pro Gly Ser Gly Val Glu Val Ser Ser Val Pro Glu Ser

1970 1975 1980

Ser Ser Glu Thr Ser Ala Tyr Pro Glu Ala Gly Phe Gly Ala Ser Ala

1985 1990 1995 2000

Ala Pro Glu Ala Ser Arg Glu Asp Ser Gly Ser Pro Asp Leu Ser Glu

2005 2010 2015

Thr Thr Ser Ala Phe His Glu Ala Asn Leu Glu Arg Ser Ser Gly Leu

2020 2025 2030

Gly Val Ser Gly Ser Thr Leu Thr Phe Gln Glu Gly Glu Ala Ser Ala

2035 2040 2045

Ala Pro Glu Val Ser Gly Glu Ser Thr Thr Thr Ser Asp Val Gly Thr

2050 2055 2060

Glu Ala Pro Gly Leu Pro Ser Ala Thr Pro Thr Ala Ser Gly Asp Arg

2065 2070 2075 2080

Thr Glu Ile Ser Gly Asp Leu Ser Gly His Thr Ser Gln Leu Gly Val

2085 2090 2095

Val Ile Ser Thr Ser Ile Pro Glu Ser Glu Trp Thr Gln Gln Thr Gln

2100 2105 2110

Arg Pro Ala Glu Thr His Leu Glu Ile Glu Ser Ser Ser Leu Leu Tyr

2115 2120 2125

Ser Gly Glu Glu Thr His Thr Val Glu Thr Ala Thr Ser Pro Thr Asp

2130 2135 2140

Ala Ser Ile Pro Ala Ser Pro Glu Trp Lys Arg Glu Ser Glu Ser Thr

2145 2150 2155 2160

Ala Ala Ala Pro Ala Arg Ser Cys Ala Glu Glu Pro Cys Gly Ala Gly

2165 2170 2175

Thr Cys Lys Glu Thr Glu Gly His Val Ile Cys Leu Cys Pro Pro Gly

2180 2185 2190

Tyr Thr Gly Glu His Cys Asn Ile Asp Gln Glu Val Cys Glu Glu Gly

2195 2200 2205

Trp Asn Lys Tyr Gln Gly His Cys Tyr Arg His Phe Pro Asp Arg Glu

2210 2215 2220

Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln Gln Ser His Leu

2225 2230 2235 2240

Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe Val Asn Asn Asn Ala

2245 2250 2255

Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp

2260 2265 2270

Phe Arg Trp Ser Asp Gly His Pro Met Gln Phe Glu Asn Trp Arg Pro

2275 2280 2285

Asn Gln Pro Asp Asn Phe Phe Ala Ala Gly Glu Asp Cys Val Val Met

2290 2295 2300

Ile Trp His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr His

2305 2310 2315 2320

Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu Pro Pro

2325 2330 2335

Val Val Glu His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg Tyr Glu

2340 2345 2350

Ile Asn Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg

2355 2360 2365

His Met Pro Thr Ile Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro

2370 2375 2380

Arg Ile Thr Cys Thr Asp Ala Thr Thr Tyr Lys Arg Arg Leu Gln Lys

2385 2390 2395 2400

Arg Ser Ser Arg His Pro Arg Arg Ser Arg Pro Ser Thr Ala His

2405 2410 2415

<---

Claims (39)

1. A polypeptide for inhibition of disintegrin A-mediated and thrombospondin metalloproteinase 5 (ADAMTS5) mediated aggrecan cleavage, where the specified polypeptide includes at least 3 ISVD, in which the first ISVD specifically binds to matrix metalloproteinase 13 (MMP13), the second ISVD specifically binds to ADAMTS5, and the third ISVD specifically binds to aggrecan, where the specified ISVD, specifically binding to MMP13, contains 3 complementarity determining regions (CDR1-CDR3, respectively), in which (i) CDR1 is SEQ ID NO: 8; (ii) CDR2 is SEQ ID NO: 10 and (iii) CDR3 is SEQ ID NO: 12; and where the specified ISVD specifically binding to ADAMTS5 contains 3 complementarity determining regions (CDR1-CDR3, respectively), in which (i) CDR1 is SEQ ID NO: 14 [GRTVSSYAMG]; (ii) CDR2 is SEQ ID NO: 16 [GISRSAERTY] and (iii) CDR3 is SEQ ID NO: 18 [DLDPNRIFSREEYAY]; and contains 3 complementarity-determining regions (CDR1-CDR3, respectively), in which (i) CDR1 is SEQ ID NO: 19 or an amino acid sequence that has up to 2 amino acid differences from SEQ ID NO: 19; (ii) CDR2 is SEQ ID NO: 21 or an amino acid sequence that has up to 3 amino acid differences from SEQ ID NO: 21; and (iii) CDR3 is SEQ ID NO: 23 or an amino acid sequence that has up to 2 amino acid differences from SEQ ID NO: 23. 2. The polypeptide of claim 1, wherein said polypeptide contains a fourth ISVD that specifically binds to aggrecan. 3. A polypeptide according to claim 1 or 2, wherein said ISVD specifically binding to MMP13 comprises or consists of SEQ ID NO: 2. 4. The polypeptide according to any one of paragraphs. 1-3, wherein said ISVD specifically binding to ADAMTS5 comprises or consists of SEQ ID NO: 3. 5. The polypeptide according to any one of paragraphs. 1-4, wherein said aggrecan-specific ISVD comprises or consists of SEQ ID NO: 4. 6. A polypeptide according to any one of the preceding claims, wherein the sequences of said third and fourth ISVDs are the same. 7. The polypeptide according to any one of paragraphs. 1-5, where these ISVDs are connected to each other via a linker. 8. The polypeptide of claim 7 wherein said linker is selected from the group consisting of SEQ ID NO: 24-40, preferably SEQ ID NO: 28 [9GS] or SEQ ID NO: 35 [35GS]. 9. The polypeptide of claim 1, wherein said first ISVD specifically binds to MMP13, said second ISVD specifically binds to ADAMTS5, said third ISVD specifically binds to aggrecan, and said fourth ISVD specifically binds to aggrecan, and wherein said polypeptide comprises or consists of SEQ ID NO: 1 or 62, or includes or consists of a polypeptide that shares at least 95% sequence identity with SEQ ID NO: 1 or 62. 10. A polypeptide according to any one of the preceding claims, where (i) said ISVD specifically binding to MMP13 essentially consists of 4 framework regions (FR1-FR4, respectively) and said 3 complementarity-determining regions CDR1-CDR3; and/or (ii) said ISVD specifically binding to ADAMTS5 essentially consists of 4 framework regions (FR1-FR4, respectively) and said 3 complementarity-determining regions CDR1-CDR3; and/or (iii) said ISVD specifically binding to aggrecan essentially consists of 4 framework regions (FR1-FR4, respectively) and said 3 complementarity-determining regions CDR1-CDR3. 11. A construct for inhibiting ADAMTS5-mediated aggrecan cleavage, wherein said construct comprises a polypeptide according to any one of the preceding claims, and wherein said construct further comprises one or more other groups selected from the group consisting of whey protein or a fragment thereof, an Fc portion and a protein, or peptide that can bind to whey proteins. 12. A construct for inhibiting ADAMTS5-mediated aggrecan cleavage, wherein said construct comprises a polypeptide according to any one of the preceding claims, and wherein said construct further comprises one or more other groups that are selected from the group consisting of a polyethylene glycol molecule, a marker, a toxin, a label, and a radiochemical . 13. Nucleic acid encoding a polypeptide according to any one of paragraphs. 1-10 or the design according to item 11. 14. An expression vector comprising a nucleic acid according to claim 13. 15. The host cell for obtaining the polypeptide according to any one of paragraphs. 1-10, including a nucleic acid according to claim 13 or an expression vector according to claim 14. 16. Pharmaceutical composition for the treatment of at least one ADAMTS5-associated disease, comprising an effective amount of a polypeptide according to any one of paragraphs. 1-10, or a construct according to claim 11 or 12, or a nucleic acid according to claim 13. 17. The method of obtaining a polypeptide according to any one of paragraphs. 1-10, at least comprising the steps of: a) expression in a suitable host cell, host organism, or suitable nucleic acid expression system according to claim 13; optionally followed by b) isolation and/or purification of the polypeptide according to any one of paragraphs. 1–10. 18. The pharmaceutical composition of claim 16 which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant and optionally includes one or more additional pharmaceutically active polypeptides and/or compounds. 19. The use of the composition according to p. 16 or 18, the polypeptide according to any one of paragraphs. 1-10 or constructs according to claim 11 or 12 as a medicine. 20. The use of the composition according to p. 16 or 18, the polypeptide according to any one of paragraphs. 1-10 or constructs according to claim 11 or 12 for preventing a symptom or treating a disease selected from the group consisting of arthropathies and chondrodystrophies, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia , costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, aggrecanopathy, NASH, chronic periodontitis, and abdominal aortic aneurysms. 21. A method for preventing a symptom or treating a disease or disorder in an individual, comprising administering a polypeptide according to any one of paragraphs. 1-10, designs according to paragraph 11 or 12 or composition according to any one of paragraphs. 16 or 18 to said individual, wherein said disease or disorder is selected from the group consisting of arthropathy, chondrodystrophy, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, disc herniation, lumbar disc degeneration, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans, aggrecanopathy, NASH, chronic periodontitis, and abdominal aortic aneurysms.

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