Aggrecan-binding immunoglobulin

Abstract

To provide: immunoglobulins that specifically bind to aggrecan, and particularly polypeptides and nucleic acids encoding such polypeptides; methods for preparing such polypeptides; and compositions, particularly pharmaceutical compositions, that comprise such polypeptides for prophylactic, therapeutic or diagnostic purposes.SOLUTION: In particular, the immunoglobulins of the present invention inhibit the activity of aggrecan.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Background Osteoarthritis is one of the most common causes of physical disability worldwide. 30 million Americans suffer from it, making it the most common joint disorder. By 2025, it is predicted that over 20% of the US population will suffer from it. The disease can occur in all joints, most frequently in the knees, hips, fingers, and spine. Osteoarthritis (OA) can be defined as a group of diverse conditions characterized by a combination of joint symptoms, signs resulting from cartilage loss, and changes in adjacent tissues including bone, tendons, and muscles. OA is characterized by progressive erosion of articular cartilage (the cartilage that covers the bone). Eventually, the disease results in total destruction of the articular cartilage, sclerosis of the underlying bone, bone spur formation, etc., all of which lead to loss of movement and pain. Pain is the most prominent symptom of OA and is, very frequently, the reason patients seek medical assistance.

[0003] Aggrecan is the major proteoglycan in articular cartilage (Kiani et al. 2002 Cell Research 12:19-32). This molecule is important in the proper function of articular cartilage as it provides a hydrated gel structure that gives cartilage its load-bearing properties. Aggrecan is a large multi-module molecule (2317 amino acids) expressed by chondrocytes. Its core protein consists of three globular domains (G1, G2 and G3), as well as a large extended region between G2 and G3 for the addition of glycosaminoglycan chains. This extended region contains two domains, one of which is substituted with keratan sulfate chains (KS domain) and one with chondroitin sulfate chains (CS domain). The CS domain has 100-150 glycosaminoglycan (GAG) chains attached to it. Aggrecan forms a large complex with hyaluronan, where 50-100 aggrecan molecules interact with one hyaluronan molecule via the G1 domain and a link protein. Due to water uptake (resulting from 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 matrix content of aggrecan and the amount of chondroitin sulfate bound to the intact core protein.

[0004] OA is characterized by 1) the degradation of aggrecan, which progressively releases domains G3 and G2 (resulting in "shrinkage" of the cartilage) and ultimately domain G1, and 2) the degradation of collagen, which irreversibly destroys the cartilage structure.

[0005] Ageing, obesity and joint injury have been identified as risk factors for osteoarthritis (OA), but the cause of OA is unknown and there are currently no pharmacological treatments that can halt disease progression or cure the joint. For large joints, drugs can be injected into the joint to help limit potential side effects such as pain. Treatment strategies primarily aim to reduce pain and improve joint function. Fasnumab, a non-opioid anti-NGF pain treatment, has been shown to bring about improvements in key pain scores during phase II / III trials. Duloxetine is approved for the treatment of chronic knee pain due to OA and is conditionally recommended by the American College of Rheumatology. Strontium ranelate has been found in large multi-centre studies not only to significantly reduce the rate of joint space narrowing in patients with symptomatic knee OA, but also to improve pain scores compared with placebo. However, at this point, the biological agents interleukin-1 receptor antagonist and anti-tumour necrosis factor antibody have not been shown to be effective or to change the course of OA (Smelter Hochberg 2013 Current Opin. Rheumatol. 25:310). Thus, many such treatments are ineffective and / or associated with side effects. Ultimately, patients will undergo total knee or hip replacement if they are unable to control their pain.

[0006] Pharmacological treatment starts with oral administration of paracetamol, which may also be combined with NSAIDS or COX-2 inhibitors and weak opioids. The main drawbacks of oral drug administration are limited bioavailability at the site of interest and the risk of side effects such as liver damage, gastrointestinal (GI) ulceration, GI bleeding and constipation.

[0007] Because OA is a localized disease, intra-articular drug administration offers an excellent opportunity for improved treatment. However, most newly developed disease-modifying osteoarthritis drugs (DMOADs) have a short residence time in the joint, even when administered intra-articularly (Edwards 2011 Vet. J. 190:15-21; Larsen et al. 2008 J Pham Sci 97:4622-4654). Intra-articular (IA) delivery of therapeutic proteins has been limited by their rapid clearance from the joint cavity and lack of retention in cartilage. The residence time of drugs in the synovial fluid of the joint is often less than 24 hours. Due to the rapid clearance of most IA-injected drugs, frequent injections may be required to maintain effective concentrations (Owen et al. 1994 Br. J. Clin Pharmacol. 38:349-355). However, frequent IA injections are undesirable due to the pain and discomfort they can cause, which burdens patient compliance, as well as the risk of leading to joint infections.

[0008] Loffredo et al. tested whether targeted delivery to cartilage by fusion with a heparin-binding domain is sufficient to prolong the in vivo function of insulin-like growth factor 1 (IGF-1). Heparin is present in mast cells. However, the natural role of heparin is unknown, although it is widely used as an antithrombotic agent (Loffredo et al. 2014 Arthritis Rheumatol. 66:1247-1255). There remains a need for additional cartilage anchoring proteins (CAPs). Summary of the Invention

[0009] Summary of the Invention The inventors hypothesized that the efficacy of therapeutic agents could be significantly increased by coupling them to a moiety (also referred to herein as a "cartilage anchor protein" or "CAP") that "anchores" the drug in the joint, resulting in increased drug retention, but which should not impair the efficacy of the therapeutic agent. This anchoring concept increases not only the drug's efficacy but also its operational specificity for the affected joint by reducing toxicity and side effects and thereby expanding the number of potentially useful drugs. The inventors further hypothesized that an aggrecan binder could potentially function as an anchor, but that aggrecan is heavily glycosylated and degraded in the various disorders affecting cartilage in the joint. Furthermore, given the cost and extensive testing in various animal models required before a drug can enter the clinic, such an aggrecan binder should preferentially have broad cross-reactivity, e.g., it should bind to aggrecan from a variety of species.

[0010] Using a variety of sophisticated immunization, screening and characterization methods, the inventors were able to identify a number of aggrecan-binding agents with excellent selectivity, stability and / or specificity characteristics that allow for long-term retention and activity in the joint.

[0011] The present invention therefore relates to immunoglobulin single variable domains (ISVs) that specifically bind to aggrecan, preferably wherein said ISV specifically binds to human aggrecan (SEQ ID NO: 125), and / or wherein said ISV specifically binds to dog aggrecan (SEQ ID NO: 126), bovine aggrecan (SEQ ID NO: 127), rat aggrecan (SEQ ID NO: 128), porcine (core) aggrecan (SEQ ID NO: 129), mouse aggrecan (SEQ ID NO: 130), rabbit aggrecan (SEQ ID NO: 131), cynomolgus monkey aggrecan (SEQ ID NO: 132) and / or rhesus monkey aggrecan (SEQ ID NO: 133), and even more preferably wherein said ISV does not substantially bind to neurocan (SEQ ID NO: 134) and / or brevican (SEQ ID NO: 135).

[0012] In one aspect, the present invention relates to an ISV as described herein, wherein the ISV has greater than 10 fold, greater than 100 fold, preferably greater than 1000 fold selectivity for binding to aggrecan over neurocan and / or brevican, and / or said ISV preferably binds to cartilaginous tissue such as cartilage and / or meniscus, and / or said ISV has a stability of at least 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months at 37°C in synovial fluid (SF), and / or said ISV has a stability of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, have a cartilage retention of at least 2 RU, such as 5 or 6 RU, and / or the ISV penetrates at least 5 μm into cartilage, such as at least 10 μm, 20 μm, 30 μm, 40 μm, 50 μm or even more, and / or the ISV consists essentially of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanised VHH sequence, a camelised VH sequence or a VHH sequence obtained by affinity maturation.

[0013] In one aspect, the invention relates to an ISV as described herein that consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is selected from the group consisting of SEQ ID NOs: 24, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 109. CDR2 is selected from the group consisting of SEQ ID NOs: 42, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 110; and CDR3 is selected from the group consisting of SEQ ID NOs: 60, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 111.

[0014] In one aspect, the present invention relates to an ISV as described herein, wherein the ISV binds to the G1 domain of aglycan, preferably the ISV has a pI greater than 8, and / or the ISV has a * 10 -2 s -1 less than K off and / or the ISV has a * 10 -6 EC less than M 50 and has.

[0015] In one aspect, the present invention relates to an ISV as described herein, consisting essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), where; i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 24, 20, or 21; or b) an amino acid sequence having a difference of 5, 4, 3, 2, or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 24, where at position 2, S is changed to R, F, I, or T; at position 3, T is changed to I; at position 5, I is changed to S; at position 6, I is changed to S, T, or M; at position 7, N is changed to Y or R; at position 8, V is changed to A, Y, T, or G; at position 9, V is changed to M; and / or at position 10, R is changed to G, K, or A; and / or, ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 42, 38, or 39; or d) an amino acid sequence having 5, 4, 3, 2, or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 42, wherein at position 1, T is changed to A or G; S or N is inserted between position 3 and position 4 (position 2a, Table 1.3B); at position 3, S is changed to R, W, N, or T; at position 4, S is changed to T or G; at position 5, G is changed to S; at position 6, G is changed to S or R; at position 7, N is changed to S, T, or R; at position 8, A is changed to T; and / or at position 9, N is changed to D or Y; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 60, 56 or 57; or f) an amino acid sequence having 5, 4, 3, 2 or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 60, wherein at position 1, P is changed to G, R, D or E or is absent; at position 2, T is changed to R, L, P or V or is absent; at position 3, T is changed to M, S or R or is absent; at position 4, H is changed to D, Y, G or T; at position 5, Y is changed to F, V, T or G; and at position 6, G is changed to L, D, S, Y or W. at position 8, V is changed to G, T, H, R, L, or Y; at position 9, Y is changed to R, A, S, D, or G; at position 10, Y is changed to N, E, G, W, or S; W is inserted between positions 10 and 11 (position 10a, Table 1.3C); at position 11, G is changed to S, K, or Y; at position 12, P is changed to E or D or is absent; and / or at position 13, Y is changed to L or is absent.

[0016] In one aspect, the invention relates to an ISV as described herein, wherein said ISV is selected from the group of ISVs wherein CDR1 is selected from the group consisting of SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, 37 and 109; CDR2 is selected from the group consisting of SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, 55 and 110; and CDR3 is selected from the group consisting of SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73, 74 and 111.

[0017] In one aspect, the invention relates to an ISV as described herein, wherein said ISV comprises: - CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; - CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; - CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; - CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; - CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; - CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; - CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; - CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 71; - CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 72; - CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 73; and - CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 74, selected from the group of ISVs.

[0018] In one aspect, the present invention relates to an ISV as described herein, consisting essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), wherein; i) CDR1 is selected from the group consisting of: a) SEQ ID NOs: 24 and 109; or b) an amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 24, wherein at position 7, N is changed to S; and / or at position 9, V is changed to M; and / or ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 42 and 110; or d) an amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 42, wherein at position 1, T is changed to A; at position 3, S is changed to R; at position 4, S is changed to T; at position 8, A is changed to T; and / or at position 9, N is changed to D; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NOs: 60 and 111; or f) an amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 60, wherein at position 4, H is changed to R; and / or at position 8, V is changed to D.

[0019] In one aspect, the present invention relates to an ISV as described herein, where the ISV is such that CDR1 is selected from the group consisting of SEQ ID NO: 24 and 109; CDR2 is selected from the group consisting of SEQ ID NO: 42 and 110; and CDR3 is selected from the group consisting of SEQ ID NO: 60 and 111, and is selected from the group of ISVs.

[0020] In one aspect, the present invention relates to an ISV as described herein, where the ISV belongs to epitope bin 1 or epitope bin 4, and preferably the ISV consists essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), where; i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 36; and b) an amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 36, where at position 3, T is changed to S; at position 6, T is changed to S; at position 8, T is changed to A; and / or at position 9, M is changed to V; and / or ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 54; and d) an amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 54, where at position 1, A is changed to I; at position 4, W is changed to R; at position 7, G is changed to R; and / or at position 8, T is changed to S; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 73; and f) an amino acid sequence having 5, 4, 3, 2, or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 73, wherein at position 1, R is changed to G; at position 2, P is changed to R or L; at position 3, R is changed to L or S; at position 5, Y is changed to R; at position 6, Y is changed to S or A; at position 7, Y is changed to T or is absent; at position 8, S is changed to P; at position 9, L is changed to H or R; at position 10, Y is changed to P or A; at position 11, S is changed to A or Y; at position 12, Y is changed to D; at position 13, D is changed to F; at position 14, Y is changed to G or is absent; and / or S is inserted after position 14.

[0021] In one aspect, the present invention relates to an ISV as described herein, wherein said ISV is selected from the group of ISVs wherein CDR1 is selected from the group consisting of SEQ ID NOs: 20, 29 and 36; CDR2 is selected from the group consisting of SEQ ID NOs: 38, 47 and 54; and CDR3 is selected from the group consisting of SEQ ID NOs: 56, 65 and 73.

[0022] In one aspect, the invention relates to an ISV as described herein, wherein said ISV cross-blocks the binding of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation to the G1 domain of aggrecan.

[0023] In one aspect, the present invention relates to an ISV, a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence or a VHH sequence obtained by affinity maturation which binds to epitope bin 1 of the G1-domain of aggrecan and competes with an ISV as described herein for binding to the G1-domain of aggrecan.

[0024] In one aspect, the invention relates to an ISV as described herein, consisting essentially of four framework regions (FR1-FR4, respectively) and three complementarity determining regions (CDR1-CDR3, respectively), wherein: i) CDR1 is selected from the group consisting of: a) SEQ ID NO:24; and b) an amino acid sequence having two or one amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO:24, wherein at position 2, S is changed to I or F; at position 5, I is changed to S; and at position 6, I is changed to S or M. at position 7, N is changed to R or Y; at position 8, V is changed to A or Y; at position 9, V is changed to M; and / or at position 10, R is changed to K; and / or ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 42; and d) an amino acid sequence having 5, 4, 3, 2, or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 42, wherein at position 1, T is changed to A or G; and an N is inserted between position 2 and position 3 (position 2a, Table 2.3B); at position 7, N has been changed to R; at position 8, A has been changed to T; and / or at position 9, N has been changed to D; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 60; and f) an amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 60, wherein at position 1, P is absent; at position 2, T has been changed to R or is absent; at position 3, T has been changed to M or is absent; at position 4, H has been changed to D or Y; at position 5, Y has been changed to F or V; at position 6, G has been changed to L or D; at position 8, V has been changed to G or T; at position 9, Y has been changed to R; at position 10, Y has been changed to N or E; at position 11, G has been changed to S or K; at position 12, P has been changed to E or is absent; and / or at position 13, Y has been changed to L or is absent; preferably CDR1 is selected from the group consisting of SEQ ID NOs: 24, 25 and 27; CDR2 is selected from the group consisting of SEQ ID NOs: 42, 43 and 45; and CDR3 is selected from the group consisting of SEQ ID NOs: 60, 61 and 63; even more preferably, wherein said ISV cross-blocks the binding of an aglycan's G1 domain of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation.

[0025] In one aspect, the invention relates to an ISV, a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single-domain antibody, an immunoglobulin suitable for use as a single-domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, as described herein, which binds to epitope bin 4 of the G1-domain of aggrecan and competes with an ISV as described herein for binding to the G1 domain of aggrecan.

[0026] In one aspect, the invention relates to an ISV as described herein, wherein said ISV is an ISV having SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19, and any one of SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19, and having a sequence identity higher than 80%, such as 90% or 95%, and is selected from the group consisting of ISVs.

[0027] In one aspect, the invention relates to an ISV as described herein, wherein said ISV binds to the G1-IGD-G2 domain of aggrecan, preferably where said ISV has a pI greater than 8 and / or has a K of less than 2 * 10 -2 s -1 and an EC of less than 1 off 10 * 10 -6 M. 50 It has.

[0028] In one aspect, the invention relates to an ISV as described herein, where; i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 32, 30 and 23; and b) an amino acid sequence having a difference of 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 32, where at position 2, R is changed to L; at position 6, S is changed to T; and / or at position 8, T is changed to A; and / or ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 50, 41, 48 and 51; and d) an amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 50, where at position 7, G is changed to S or R; and / or at position 8, R is changed to T; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 68, 59, 66 and 69; and f) an amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 68, where at position 4, R is changed to V or P; at position 6, A is changed to Y; at position 7, S is changed to T; at position 8, S is absent; at position 9, N is changed to P; at position 10, R is changed to T or L; at position 11, G is changed to E; and / or at position 12, L is changed to T or V.Preferably, here, the ISV is selected from the group of ISVs where CDR1 is selected from the group consisting of SEQ ID NO: 32, 30, and 23; CDR2 is selected from the group consisting of SEQ ID NO: 50, 41, 48, and 51; and CDR3 is selected from the group consisting of SEQ ID NO: 68, 59, 66, and 69. Even more preferably, here, the ISV is such that CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69; CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66; and CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59, selected from the group of ISVs.

[0029] In one aspect, the present invention relates to an ISV as described herein, where the ISV is selected from the group consisting of ISVs having SEQ ID NO: 13, 4, 11, and 14, and ISVs having a sequence identity higher than 80%, such as 90% or 95%, with any one of SEQ ID NO: 13, 4, 11, and 14.

[0030] In one aspect, the invention relates to an ISV as described herein, wherein said ISV cross-blocks the binding of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation to the G1-IGD-G2 domain of aggrecan. In one aspect, the present invention relates to an ISV, a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, which binds to the G1-IGD-G2 domain of aggrecan and competes with an ISV as described herein for binding to the G1-IGD-G2 domain of aggrecan.

[0031] In one aspect, the present invention relates to an ISV as described herein, wherein said ISV binds to the G2 domain of aggrecan, preferably wherein said ISV has a pI greater than 8 and / or a pI greater than 2. * 10 -2 s -1 Less than K off and / or one * 10 -6 EC less than M 50 It has.

[0032] In one aspect, the invention relates to an ISV as described herein, wherein: i) CDR1 is selected from the group consisting of: a) SEQ ID NO:28; and b) an amino acid sequence having 5, 4, 3, 2, or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO:28, wherein at position 1, G is changed to R; at position 2, P is changed to S or R; at position 3, T is changed to I; at position 5, S is changed to N; and at position 6, R is changed to N, M, or S. at position 7, Y is changed to R or is absent; at position 8, A is changed to F or is absent; and / or at position 10, G is changed to Y; and / or ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 46; and d) an amino acid sequence having 5, 4, 3, 2, or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 46, wherein at position 1, A is changed to S or Y; and at position 4, W is changed to L. at position 7, G is absent; at position 8, G is changed to A; at position 9, R is changed to S, D, or T; and / or at position 11, Y is changed to N or R; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 64; and f) an amino acid sequence having 5, 4, 3, 2, or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 64, wherein in position 1, A is changed to R or F; in position 2, R is changed to I or L; in position 3, I is changed to H or Q; in position 4, P is changed to G or N; in position 5, V is changed to S; in position 6, R is changed to G, N, or F; in position 7, T is changed to R, W, or Y; in position 8, Y is changed to R or S or is absent; and in position 9, T is changed to S or is absent;At position 10, S is changed to E, K, or is absent; at position 11, E is changed to N, A, or is absent; at position 12, W is changed to D, or is absent; at position 13, N is changed to D, or is absent; at position 14, Y is absent; and / or D and / or N is added after position 14 of SEQ ID NO: 64; preferably here, the ISV is selected from the group of ISVs where CDR1 is selected from the group consisting of SEQ ID NO: 28, 22, 26, and 33; CDR2 is selected from the group consisting of SEQ ID NO: 46, 40, 44, and 52; and CDR3 is selected from the group consisting of SEQ ID NO: 64, 58, 62, and 70, and even more preferably here, the ISV is such that CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64; CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62; and CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70, selected from the group of ISVs.

[0033] In one aspect, the present invention relates to an ISV as described herein, where the ISV is selected from the group consisting of ISVs having SEQ ID NO: 9, 3, 7, and 15, and ISVs having a sequence identity higher than 80%, such as 90% or 95%, with any one of SEQ ID NO: 9, 3, 7, and 15.

[0034] In one aspect, the invention relates to an ISV as described herein, wherein the ISV cross-blocks the binding of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation to the G2 domain of aggrecan. In one aspect, the invention relates to an ISV, a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, which binds to the G2 domain of aggrecan and competes with an ISV as described herein for binding to the G2 domain of aggrecan.

[0035] In one aspect, the present invention relates to an ISV as described herein, wherein the ISV is selected from the group consisting of SEQ ID NOs: 1-19 and 114-118, and an ISV having greater than 80% sequence identity, such as 90% or 95%, to any one of SEQ ID NOs: 1-19 and 114-118.

[0036] In one aspect, the invention relates to a polypeptide comprising at least one ISV as described herein, preferably comprising at least two ISVs as described herein, wherein the at least two ISVs may be the same or different. Preferably, the at least two ISVs are independently selected from the group consisting of SEQ ID NOs: 1-19 and 114-118, more preferably, wherein the at least two ISVs are selected from the group consisting of SEQ ID NOs: 5, 6, 8 and 114-117, or wherein the at least two ISVs are selected from the group consisting of SEQ ID NOs: 13 and 118.

[0037] Preferably, in one aspect, the polypeptide of the invention comprises at least one further ISV, e.g., a therapeutic ISV, which preferably binds to a member of the serine protease family, a cathepsin, a matrix metalloproteinase (MMP) / matrixin, or a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1), and / or ADAMTS11; wherein the at least one further ISV, e.g., a therapeutic ISV, preferably retains activity. Even more preferably, the at least one further ISV, e.g. a therapeutic ISV, inhibits the activity of a member of the serine protease family, a cathepsin, a matrix metalloproteinase (MMP) / matrixin or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and / or ADAMTS11.

[0038] In one aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide has a stability of at least 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months at 37°C in synovial fluid (SF), and / or has cartilage retention of at least 2 RU, such as at least 3, 4, 5 or 6 RU in a cartilage retention assay, and / or penetrates at least 5 μm into cartilage, such as at least 10 μm, 20 μm, 30 μm, 40 μm, 50 μm or even more.

[0039] In one aspect, the present invention relates to a polypeptide as described herein further comprising a serum protein binding moiety or a serum protein, preferably the serum protein binding moiety binds to serum albumin; even more preferably the serum protein binding moiety is an ISV that binds to serum albumin; even more preferably, the ISV that binds to serum albumin consists essentially of four framework regions (FR1 - FR4 respectively) and three complementarity determining regions (CDR1 - CDR3 respectively), where CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR; even more preferably the ISV that binds to serum albumin includes Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (see Table C). In one aspect, the present invention relates to a polypeptide as described herein further comprising a serum protein binding moiety or a serum protein, where the serum protein binding moiety is a non-antibody-based polypeptide. In one aspect, the present invention relates to a polypeptide as described herein further comprising PEG.

[0040] In one aspect, the present invention relates to a polypeptide as described herein, where the ISVs are directly linked to each other or are linked via a linker. In one aspect, the present invention relates to a polypeptide as described herein, where the first ISV and / or the second ISV and / or optionally the third ISV and / or optionally the fourth ISV and / or optionally the ISV that binds to the serum albumin are linked via a linker; preferably the linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS linkers (see Table D).

[0041] In one aspect, the invention relates to a polypeptide as described herein, wherein the polypeptide is selected from the group of polypeptides and / or constructs comprising an ISV that binds to a target as shown respectively in Table E-1 and Table E-2, and an ISV that binds to one or two aggrecan as shown.

[0042] In one aspect, the invention relates to a construct comprising or consisting essentially of an ISV as described herein, or a polypeptide as described herein, which optionally further comprises one or more other groups, residues, moieties or linking units, which are optionally linked via one or more peptidic linkers; preferably the one or more other groups, residues, moieties or linking units are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, linking units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins.

[0043] In one aspect, the invention relates to a nucleic acid encoding an ISV as described herein, a polypeptide as described herein, or a construct as described herein. In one aspect, the invention relates to an expression vector comprising a nucleic acid as described herein. In one aspect, the invention relates to a host or host cell comprising a nucleic acid as described herein, or an expression vector as described herein.

[0044] In one aspect, the present invention relates to a method for generating an ISV as described herein or a polypeptide as described herein, said method comprising at least the following steps: a) expressing a nucleic acid as described herein in a suitable host cell or host organism, or in another suitable expression system; optionally, subsequently: b) isolating and / or purifying an ISV as described herein, or a polypeptide as described herein.

[0045] In one aspect, the present invention relates to a composition comprising at least one ISV as described herein, a polypeptide as described herein, a construct as described herein, or a nucleic acid as described herein; preferably said composition is a pharmaceutical composition, which preferably further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and / or compounds.

[0046] In one aspect, the present invention relates to a composition as described herein, an ISV as described herein, a polypeptide as described herein, or a construct as described herein for use as a medicament. Preferably, the composition, ISV, polypeptide or construct as described herein is for use in preventing or treating arthrosis and chondrodystrophy, arthritis diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondrogenesis, costochondritis, spondyloepimetaphyseal dysplasia, intervertebral disc hernia, lumbar intervertebral disc degeneration disease, degenerative joint disease, and relapsing polychondritis and the like.

[0047] In one aspect, the invention relates to a method for preventing or treating arthropathies and chondrodystrophies, arthritic diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or detachments, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis, wherein said method comprises administering to a subject in need thereof at least a pharmaceutically active amount, for a person in need thereof, of a composition, ISV, polypeptide or construct as described herein.

[0048] In one aspect, the present invention relates to a method for reducing and / or inhibiting the efflux of a compound, polypeptide or construct from cartilaginous tissue, said method comprising administering to a person in need thereof a pharmaceutically active amount of at least one polypeptide as described herein, a compound or construct as described herein, or a composition as described herein.

[0049] In one aspect, the present invention relates to a method for inhibiting and / or blocking ADAMTS5 activity and / or MMP13 activity, said method comprising administering to a person in need thereof a pharmaceutically active amount of at least one polypeptide as described herein, a construct as described herein, or a composition as described herein.

[0050] In one aspect, the present invention relates to the use of an ISV as described herein, a polypeptide as described herein, a construct as described herein, or a composition as described herein, in the preparation of a pharmaceutical composition for treating or preventing arthrosis and chondrodystrophy, arthritic diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondrogenesis, costochondritis, spondyloepiphyseal dysplasia, intervertebral disc hernia, lumbar intervertebral disc degenerative disease, degenerative joint disease, and relapsing polychondritis.

[0051] Other aspects, advantages, applications, and uses of the polypeptides and compositions will become apparent from further disclosure herein. Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety herein, both above and below. Nothing herein should be construed as an admission that the present invention has no right to antedate such disclosure by virtue of prior invention.

Brief Description of the Drawings

[0052] [Figure 1] Examples of autoradiography images of sections of rat joints 2 or 4 weeks after injection of the 125I-labeled ALB26-CAP construct. For both the results 2 weeks after injection and the results 4 weeks after injection: left panel: histological section; right panel: autoradiography.

[0053] [Figure 2] Representative MARG image. Specific MARG staining appears as black particles on the image and is indicated by the arrow.

[0054] [Figure 3]Inhibition of cartilage degradation by nanobodies in a rat MMT model using anti-MMP13-CAP nanobody (C010100754) or anti-ADAMTS5-CAP nanobody (C010100954). Treatment was initiated by IA injection 3 days after surgery. Histopathology was performed 42 days after surgery. The medical and overall substantial cartilage degeneration width and the percentage reduction of cartilage degeneration were determined. 20 animals per group were used.

[0055] [Figure 4] Serum concentration (mean concentration in ng / ml) versus time after the first dose (h) of the polypeptide in osteoarthritis rats and healthy rats that received a single intra-articular injection of 400 μg of nanobody per joint (right knee). Dots represent individual concentrations in healthy animals; triangles represent individual concentrations in OA animals; and lines represent mean concentrations.

Mode for Carrying Out the Invention

[0056] Detailed Description Unless otherwise specified or defined, all terms used have their ordinary meaning in the art, which will be apparent to those skilled in the art. By reference, for example, standard handbooks such as Sambrook et al. (Molecular Cloning: A Laboratory Manual (2 nd . Ed.) Vols. 1-3, Cold Spring Harbor Laboratory Press, 1989), F. Ausubel et al. (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 1987), Lewin (Genes II, John Wiley & Sons, New York, N.Y., 1985), Old et al. (Principles of Gene Manipulation: An Introduction to Genetic Engineering (2 ndedition) University of California Press, Berkeley, CA, 1981), Roitt et al. th Ed.) Mosby / Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's Essential Immunology (10 th Ed.) Blackwell Publishing, UK, 2001), and Janeway et al. (Immunobiology (6 th (Ed.) Garland Science Publishing / Churchill Livingstone, New York, 2005), as well as the general background art cited therein.

[0057] Unless otherwise stated, all methods, steps, techniques and operations not specifically described in detail can be and have been performed in a manner known per se, as would be apparent to one skilled in the art. References include, for example, standard handbooks and the general background art described herein and the references cited therein, as well as the following reviews, for example: Presta (Adv. Drug Deliv. Rev. 58 (5-6): 640-56, 2006), Levin and Weiss (Mol. Biosyst. 2(1): 49-57, 2006), Irving et al. (J. Immunol. Methods 248(1-2): 31-45, 2001), Schmitz et al. (Placenta 21 Suppl. A: S106-12, 2000), Gonzales et al. (Tumor Biol. 26(1): 31-43, 2005), which describe protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0058] As used herein, the term "sequence" (e.g., "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "V HH(such as in the terms "array" or "protein sequence") should generally be understood to encompass both the appropriate amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless the context requires a more limited interpretation.

[0059] Depending on the context, an amino acid sequence is interpreted to mean a single amino acid or an unbranched sequence of two or more amino acids. A nucleotide sequence is interpreted to mean an unbranched sequence of three or more nucleotides.

[0060] An amino acid is an L - amino acid commonly found in naturally occurring proteins. Amino acid residues will be indicated according to the standard three - letter or one - letter amino acid code. See, for example, Table A - 2 on page 48 of WO 08 / 020079. Amino acid sequences containing D - amino acids are not intended to be encompassed by this definition. Any amino acid sequence containing amino acids that are modified after translation can initially be described as an amino acid sequence translated using the symbols shown in this Table A - 2, together with the position of the modification, for example hydroxylation or glycosylation, although these modifications may not be explicitly shown in the amino acid sequence. Any peptide or protein that can be represented as a sequence modified by ligation, cross - linking, and end - caps, non - peptidyl bonds, etc. is encompassed by this definition.

[0061] The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure, and each has the same meaning for purposes of this disclosure. Each term refers to an organic compound consisting of a linear chain of two or more amino acids. The compound may have 10 or more amino acids; 25 or more amino acids; 50 or more amino acids; 100 or more amino acids; 200 or more amino acids; and even 300 or more amino acids. Those of skill in the art will understand that while there is no art-recognized cutoff point for the number of amino acids that distinguishes a polypeptide from a protein, polypeptides generally contain fewer amino acids than proteins; polypeptides can be made by chemical synthesis or recombinant methods; and proteins are generally made by recombinant methods in vitro or in vivo, all as known in the art.

[0062] A nucleic acid or amino acid sequence is considered to be "(essentially) isolated" when, for example, it has been separated from at least one other component, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity, or trace component, with which it is normally associated in the source or medium, as compared to the reaction medium or culture medium from which it was obtained. A nucleic acid or amino acid sequence is considered to be "(essentially) isolated" when it has been purified, in particular by at least two-fold, in particular by at least 10-fold, more particularly by at least 100-fold, and up to 1000-fold or more. A nucleic acid or amino acid that is "(essentially) isolated" is preferably essentially homogeneous, as determined by using a suitable technique, such as a suitable chromatographic technique, e.g., polyacrylamide gel electrophoresis.

[0063] When a nucleotide sequence or an amino acid sequence is said to "contain" another nucleotide sequence or amino acid sequence, or to "consist essentially of" another nucleotide sequence or amino acid sequence, respectively, it may mean that the latter nucleotide sequence or amino acid sequence is incorporated into the former nucleotide sequence or amino acid sequence, but more commonly this means that the former nucleotide sequence or amino acid sequence generally contains, within that sequence, a stretch of nucleotide or amino acid residues having the same nucleotide sequence or amino acid sequence as the latter sequence, regardless of the method by which the former sequence was actually produced or obtained (e.g., it may be by any suitable method described herein). As a non-limiting example, when a polypeptide of the present invention is said to contain an immunoglobulin single variable domain ("ISV"), it may mean that the immunoglobulin single variable domain sequence is incorporated into the sequence of the polypeptide of the present invention, but more commonly this generally means that the polypeptide of the present invention contains the sequence of the ISV within its sequence, regardless of the method by which the polypeptide of the present invention was produced or obtained. Also, when a nucleic acid or nucleotide sequence is said to contain another nucleotide sequence, the former nucleic acid or nucleotide sequence recited preferably has, when expressed as an expression product (e.g., a polypeptide), an amino acid sequence encoded by the latter nucleotide sequence as part of the expression product (in other words, the latter nucleotide sequence is in the same reading frame as the larger nucleic acid or nucleotide sequence recited as the former).Also, when a construct of the invention is said to comprise a polypeptide or ISV, this can mean that the construct includes at least the polypeptide or ISV, respectively, but more usually this means that the construct includes groups, residues (e.g., amino acid residues), moieties and / or binding units in addition to the polypeptide or ISV, regardless of how the polypeptide or ISV is linked to the groups, residues (e.g., amino acid residues), moieties and / or binding units and regardless of how the construct is produced or obtained.

[0064] "Consisting essentially of" corresponds to an ISV for use in the methods of the invention being either exactly the same as an ISV of the invention, or having a limited number of amino acid residues, such as 1 to 20 amino acid residues, for example 1 to 10 amino acid residues, preferably 1 to 6 amino acid residues, for example 1, 2, 3, 4, 5 or 6 amino acid residues, at the amino terminus, carboxy terminus, or both the amino terminus and carboxy terminus of the ISV.

[0065] When comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a 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 of the second nucleotide sequence] by [the total number of nucleotides of the first nucleotide sequence] and multiplying by [100%]. When comparing the first nucleotide sequence, deletions, insertions, substitutions or additions of nucleotides in the second nucleotide sequence are considered differences at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings with a known computer algorithm for sequence alignment, such as Blast v2.0 of NCBI. Several other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768. Usually, for the purpose of determining the percentage of "sequence identity" between two nucleotide sequences according to the calculation method outlined above, the nucleotide sequence with the largest number of nucleotides is taken as the "first" nucleotide sequence and the other nucleotide sequence as the "second" nucleotide sequence.

[0066] For the purpose of comparing two or more amino acid sequences, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be calculated 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 [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%]. When comparing the first amino acid sequence, deletions, insertions, substitutions or additions of amino acid residues in the second amino acid sequence are considered as differences at a single amino acid residue (position), i.e., "amino acid differences" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as those described above for determining the degree of sequence identity of nucleotide sequences, with standard settings as well. Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is taken as the "first" amino acid sequence and the other amino acid sequence as the "second" amino acid sequence.

[0067] Also, when determining the degree of sequence identity between two amino acid sequences, one of ordinary skill in the art may consider so-called "conservative" amino acid substitutions, i.e., substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure and that generally, or essentially, does not affect the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art from, for example, WO 04 / 037999, GB 335768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300, and the (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings of WO 04 / 037999, or, for example, WO 98 / 49185, as well as further cited references therein.

[0068] Such conservative substitutions are preferably those in which one amino acid in the following groups (a) to (e) is replaced with another amino acid residue in the same group: (a) small aliphatic, non-polar or low-polarity residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val, and cysteine; and (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gin, or Glu; Met to Leu, Tyr, or Ile; Phe to Me, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0069] Any amino acid substitutions applied to the polypeptides described herein may also be determined by, for example, a frequency analysis of amino acid changes between homologous proteins of different species developed by Schulz et al. ("Principles of Protein Structure", Springer-Verlag, 1978), a structure-forming potential analysis developed by, for example, Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45-149, 1978), Eisenberg et al. (Proc. Natl. Acad Sci. USA 81: 140-144, 1984), Kyte and Doolittle (J. Molec. Biol. 157: 105-132, 1981), or Goldman et al. (Ann. Rev. Biophys. Chem. 15: 321-353, 1982). 1986), the entire contents of which are incorporated herein by reference. Information regarding the primary, secondary and tertiary structure of nanobodies is provided in the detailed description herein and in the general background art cited above. In this regard, the V from llamas may also be used. HH The crystal structure of the domain is disclosed, for example, by Desmyter et al. (Nature Structural Biology, 3: 803, 1996), Spinelli et al. (Natural Structural Biology, 3: 752-757, 1996) or Decaniere et al. (Structure, 7 (4): 361, 1999). H In the domain, V H / V L Detailed information on some of the amino acid residues that form the interface of and possible camelizing substitutions at those positions is available in the prior art documents cited above.

[0070] Amino acid and nucleic acid sequences are considered to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length.

[0071] When comparing two amino acid sequences, the term "difference(s) in amino acid(s)" refers to the insertion, deletion, or substitution of a single amino acid residue at a position within the first sequence as compared to the second sequence, i.e., it is understood that the two amino acid sequences may contain one, two, or more such amino acid differences. Further still, in the amino acid sequences and / or polypeptides of the present invention, the term "difference(s) in amino acid(s)" refers to the insertion, deletion, or substitution of a single amino acid residue at the positions of the CDR sequences specified in b), d), or f), as compared to the CDR sequences of a), c), or e), respectively; it is understood that the CDR sequences of b), d), and f) may each contain 1, 2, 3, 4, or up to 5 such amino acid differences as compared to the CDR sequences of a), c), or e).

[0072] "Difference(s) in amino acid(s)" may be 1, 2, 3, 4, or up to 5 substitutions, deletions, insertions, or any combination thereof, provided that it improves the properties of the proteoglycan binder of the present invention, e.g., the polypeptide of the present invention, or at least does not impair the desired properties, or the balance or combination of desired properties, of the proteoglycan binder of the present invention, e.g., the polypeptide of the present invention. In this regard, the resulting proteoglycan binder of the present invention, e.g., the polypeptide of the present invention, should bind to proteoglycan with the same, substantially the same, or higher affinity, as compared to a polypeptide containing one or more CDR sequences that does not have at least 1, 2, 3, 4, or up to 5 substitutions, deletions, or insertions, when the affinity is measured by surface plasmon resonance (SPR).

[0073] In this regard, the amino acid sequences of the CDRs according to b), d), and / or f) may be amino acid sequences derived from the amino acid sequences according to a), c), and / or e), respectively, by affinity maturation using one or more techniques of affinity maturation known per se.

[0074] For example, and depending on the host organism used to express the polypeptide of the invention, such deletions and / or substitutions can be designed to remove one or more sites for post-translational modification (e.g., one or more glycosylation sites), and are within the skill of the art.

[0075] As used herein, "nanobody family," "V HH A "family" or "family" refers to a group of Nanobodies and / or Vs that have the same length (i.e., have the same number of amino acids in their sequence). HH It refers to sequences in which the amino acid sequence between position 8 and position 106 (according to Kabat numbering) has an amino acid sequence identity of 89% or more.

[0076] The terms "epitope" and "antigenic determinant", used interchangeably, refer to a portion of a large molecule, such as a polypeptide or protein, that is recognized by an antigen-binding molecule, such as an immunoglobulin, a conventional antibody, an ISV and / or a polypeptide of the invention, more specifically by the antigen-binding site of said molecule. An epitope defines the minimal binding site for an immunoglobulin and thereby represents the specific target of the immunoglobulin. The portion of an antigen-binding molecule (eg, an immunoglobulin, conventional antibody, ISV and / or polypeptide of the invention) that recognizes an epitope is called the "paratope."

[0077] An amino acid sequence (e.g., an ISV, an antibody, a polypeptide of the invention, or a protein or polypeptide or fragment thereof that generally binds to an antigen) that is capable of "binding to," "specifically binding to," "has affinity for," and / or "has specificity for" a particular epitope, antigen, or protein (or at least a portion, fragment, or epitope thereof) is said to be "against" or "targeted by" said epitope, antigen, or protein, or to be a "binding" molecule for such epitope, antigen, or protein, or to be an "anti" epitope, "anti" antigen, or "anti" protein (e.g., "anti" aggrecan).

[0078] Affinity describes the strength or stability of a molecular interaction. Affinity is generally expressed as K D or dissociation constant, which has units of moles / liter (or M). Affinity is also expressed as the association constant, K A can also be expressed as 1 / K D Equals (moles / liter) -1 (or M -1 ) as used herein, the stability of an interaction between two molecules is primarily determined by the K D will be represented by the value of the relation K A =1 / K D Considering the strength of molecular interactions, we calculate their K D Specifying the value of the corresponding K A It will be apparent to those skilled in the art that the method can also be used to calculate the value of K D The value characterizes the strength of molecular interactions in a thermodynamic sense as well, since it is related to the well-known relationship DG = RT.ln(K D )(Equivalently, DG=-RT.ln(K A )) where R equals the gas constant, T equals the absolute temperature, and ln represents the natural logarithm. K for biological interactions that are considered meaningful (e.g., specific) D is typically 10-12 M(0.001nM)~10 -5 The stronger the interaction, the higher its K D is weak.

[0079] K D Also, k off The dissociation rate constant of the complex, expressed as k on It can also be expressed as a ratio to its rate of association, expressed as (hence, K D =k off / k on and K. A =k on / k off ). Off speed k off is in units of s -1 (where s is the SI unit of second). on is in units of M -1 s -1 The on speed is 10 2 M -1 s -1 ~about 10 7 M -1 s -1 and approaches the diffusion-limited association rate constant for the bimolecular interaction. The off-rate is determined by the relationship t 1 / 2 =ln(2) / k off The off-rate is related to the half-life of a given molecular interaction by -6 s -1 (multiple days t 1 / 2 (close to an irreversible complex with -1 (t 1 / 2 =0.69s).

[0080] Specific binding of an antigen-binding protein, such as an ISVD, to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, saturation binding assays and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the various variants thereof known per se in the art; and other techniques described herein.

[0081] The affinity of a molecular interaction between two molecules can be measured via various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559), where one molecule is immobilized on a biosensor chip and the other molecule is detected by a kinetic energy of k on , k off , and ultimately K D (or K A A flow of a sample over the immobilized molecule under flow conditions that result in a measurement of the affinity (A) value. This can be done, for example, using the well-known BIACORE® instrument (Pharmacia Biosensor AB, Uppsala, Sweden). The Kinetic Exclusion Assay (KINEXA®) (Drake et al. 2004, Analytical Biochemistry 328: 35-43) measures binding events in solution without labeling the binding partner and is based on the kinetic exclusion of complex dissociation. Affinity analysis in solution can also be performed using the GYROLAB® Immunoassay System or ELISA, which provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

[0082] If the measurement process is in some way affected by artifacts related to, for example, the coating of one molecule on a biosensor, which influence the intrinsic binding affinity of the molecule being suggested, the measured K D may correspond to the apparent K D , which will be apparent to those skilled in the art. Also, when one molecule contains more than one recognition site for the other molecule, the apparent K D can be measured. In such situations, the measured affinity can be affected by the avidity of the interaction between the two molecules. In particular, the accurate measurement of K D can be very labor-intensive and thus, often, the apparent K D value is determined to evaluate the binding strength of the two molecules. As long as all measurements are made in a consistent manner (e.g., maintaining universal assay conditions), it should be noted that the apparent K D measurement can be used as an approximation of the true K D . Therefore, in this document, K D and the apparent K D should be treated with equal importance or relevance.

[0083] The term "specificity" refers to the number of various types of antigens or antigenic determinants to which a specific antigen-binding molecule or antigen-binding protein (e.g., the ISVD and / or polypeptide of the present invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity, as described, for example, on pages 53 - 56 of WO 08 / 020079 (incorporated herein by reference), and this document also describes some preferred techniques for measuring the binding between an antigen-binding molecule (such as the polypeptide or ISVD of the present invention) and the corresponding antigen. Typically, an antigen-binding protein (e.g., the ISVD and / or polypeptide of the present invention) is 10 -5 ~10 -12 mol / L or less, preferably 10 -7 ~10 -12 mol / L or less, more preferably 10 -8 ~10-12 Dissociation constant (K in mol / L D ) (i.e., 10 5 ~10 12 L / mol or more, preferably 10 7 ~10 12 L / mol or more, preferably 10 8 ~10 12 L / mol association constant (K A ) bind to their antigens. -4 Any K above mol / L D value (or 10 4 M -1 Any K lower than liters / mol A A value of n) is generally considered to be indicative of non-specific binding. Preferably, a monovalent ISVD of the invention binds to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM (e.g., between 10 and 5 nM or less), such as less than 500 pM, such as 10 to 5 pM or less than 5 pM. Reference is also made to paragraph n) of pages 53-56 of WO 08 / 020079.

[0084] The ISV and / or polypeptide binds to a second target or antigen with an affinity that is at least 10-fold, e.g., at least 100-fold, and preferably at least 1000-fold, or better (as described above, K D value, K A value, K off Velocity and / or K on The ISVD and / or polypeptide is said to be "specific for" a (first) target or antigen when it binds to the first antigen with a K (preferably expressed as a rate) relative to another (second) target or antigen. For example, an ISVD and / or polypeptide may be said to be "specific for" a (first) target or antigen when it binds to the second target or antigen with a K D at least 10 times lower, for example at least 100 times lower, and preferably at least 1000 times lower or even lower than DIt may bind to the first target or antigen at a value. Preferably, when the ISV and / or polypeptide is "specific for" the first target or antigen as compared to the second target or antigen, it is directed (as defined herein) to the first target or antigen but not to the second target or antigen.

[0085] Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known per se, which includes, for example, saturation binding assays and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and various variants thereof known in the art; as well as other techniques described herein.

[0086] A preferred approach that can be used to evaluate affinity is the two-step ELISA (enzyme-linked immunosorbent assay) technique of Friguet et al. 1985 (J. Immunol. Methods 77: 305-19). This method establishes the measurement of binding equilibrium in solution phase and avoids artifacts that can occur in relation to the adsorption of one of the molecules on a support such as plastic. As will be apparent to those skilled in the art, the dissociation constant may be the actual dissociation constant or the apparent dissociation constant. Methods for determining the dissociation constant will be apparent to those skilled in the art and include, for example, the techniques described on pages 53-56 of WO 08 / 020079.

[0087] Finally, it should be noted that in many situations, an experienced scientist may find it convenient to determine the binding affinity by comparing it to several reference molecules. For example, to evaluate the binding strength between molecules A and B, a reference molecule C, which is preferably labeled with another chemical moiety such as a fluorophore for fluorescence detection, a chromophore for light absorption detection, or biotin for streptavidin-mediated ELISA detection, and is known to bind to B, is used for easy detection in ELISA or FACS (fluorescence-activated cell sorting) or other formats. Typically, the reference molecule C is kept at a fixed concentration, and the concentration of A is varied relative to a given concentration or amount of B. As a result, the IC 50 value is obtained corresponding to the concentration of A at which the signal measured for C in the absence of A is halved. Assuming that the K D which is the K Dref of the reference molecule and the total concentration c ref of the reference molecule are known, the apparent K D for the interaction A - B can be obtained from the following equation: K D = IC 50 / (1 + c ref / K Dref ). Note that when c ref << K Dref , K D ≈ IC 50 . Assuming that the measurement of IC 50 is made in a consistent manner for the binding agents being compared (e.g., keeping a fixed c ref ), differences in the strength or stability of molecular interactions can be evaluated by comparing the IC 50 , and this measurement is judged to be equal to K D or the apparent K D throughout this document.

[0088] The maximum half-inhibitory concentration (IC 50) It may also be a measurement of the effectiveness of a compound in inhibiting a biological or biochemical function, for example, a pharmacological effect. This quantitative measurement indicates the amount of polypeptide or ISV (e.g., nanobody) required to inhibit a given biological process (or a component of the process, i.e., enzyme, cell, cell receptor, chemotaxis, de-differentiation, metastasis, invasiveness, etc.) by up to half. In other words, it is the maximum half amount (50%) inhibitory concentration (IC) (50% IC or IC 50 ) of a substance. The IC 50 value for a given antagonist such as the polypeptide or ISV (e.g., nanobody) of the present invention can be calculated by determining the concentration required to inhibit half of the maximum biological response of the agonist. The K D of a drug can be calculated by constructing a dose-response curve and testing the effects (e.g., in reversing agonist activity) of various concentrations of the antagonist (such as the polypeptide or ISV (e.g., nanobody) of the present invention).

[0089] The term maximum half-effective concentration (EC 50 ) refers to the concentration of a compound that induces a response that is half-way between the baseline and the maximum value after a specified exposure time. For the present invention, it is used as a measure of the potency of polypeptides, ISVs (e.g., nanobodies). The EC 50 of a quantitative dose-response curve represents the concentration of the compound at which 50% of its maximum effect is observed. The concentration is preferably expressed in molar concentration units.

[0090] In a biological system, a small change in ligand concentration typically results in a rapid change in response followed by a sigmoid function. The inflection point where the increase in response with increasing ligand concentration slows down is the EC 50 . This can be determined mathematically by the deviation of the best-fit line. Relying on a graph for estimation is most often convenient. When EC 50 is provided in the example section, the experiment should be as accurate as possible for K DIn other words, EC 50 The value is therefore K D The term "average K" can be considered as a D " is the average K obtained in at least one experiment, but preferably more than one, e.g., at least two experiments. D The term "average" refers to the mathematical term "mean" (the sum of the data divided by the number of items in the data).

[0091] It also gives the IC, which is a measure of a compound's inhibition of 50 (50% inhibition). For competitive binding assays and functional antagonist assays, IC 50 is the most common summary measure of a dose-response curve. For agonist / stimulator assays, the most common summary measure is the EC 50 is.

[0092] Inhibition constant (K i ) is an indicator of how potent an inhibitor is; it is the concentration required to produce half-maximal inhibition. The IC, which can vary depending on the experimental conditions, is used to measure the potency of an inhibitor. 50 Unlike K i is often referred to as the inhibition constant of the drug. i is the Cheng-Prusoff equation:

number

[0093] An ISV and / or polypeptide is "specific" for a first target or antigen over another (second) target or antigen when it binds to the first antigen with at least 10-fold, e.g., at least 100-fold, and preferably at least 1000-fold or more affinity (suitably expressed as KD values, KA values, Koff rates, and / or Kon rates as described above) compared to the binding of the ISV and / or polypeptide to the second target or antigen. For example, the ISV and / or polypeptide may bind to the first target or antigen with a KD value that is at least 10-fold lower, e.g., at least 100-fold lower, preferably at least 1000-fold lower, or even lower, compared to the binding of the ISV and / or polypeptide to the second target or antigen. Preferably, when an ISV and / or polypeptide is referred to as being "specific" for a first target or antigen compared to a second target or antigen, it means that it is directed against said first target or antigen (as defined herein) but not against said second target or antigen.

[0094] The terms "(cross)blocking," "(cross)blocked," "(cross)blocking," "competitive binding," "(cross)competing," and "(cross)competing" and "(cross)competition" are used interchangeably herein to refer to the ability of an immunoglobulin, antibody, ISV, polypeptide, or other binding agent to prevent another immunoglobulin, antibody, ISV, polypeptide, or binding agent from binding to a given target. The extent to which an immunoglobulin, antibody, ISV, polypeptide, or other binding agent is able to prevent the binding of another to a target, i.e., whether it is capable of cross-blocking in the present invention, can be determined using competitive binding assays, such as screening purified ISVDs against ISVDs displayed on phage in a competitive ELISA, which are common in the art. Particularly suitable quantitative cross-blocking assays include ELISA and fluorescence-activated cell sorting (FACS) binding assays using aggrecan expressed on cells. In a FACS setup, the degree of cross-blocking can be measured by (reduced) channel fluorescence.

[0095] Methods for determining whether an immunoglobulin, antibody, ISV, polypeptide or other binding agent directed against a target is (cross-)blocking, capable of (cross-)blocking, competitively binding or (cross-)competitive, as defined herein, 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), and / or are methods described herein (see, e.g., Example 2.3).

[0096] An amino acid sequence is said to be "cross-reactive" if it is specific for two different antigens or epitopes (such as, for example, aggrecans from different species of mammals, such as human aggrecan, dog aggrecan, bovine aggrecan, rat aggrecan, porcine aggrecan, mouse aggrecan, rabbit aggrecan, cynomolgus monkey aggrecan, and / or rhesus monkey aggrecan, etc.) with respect to these different antigens or epitopes (as defined herein).

[0097] With respect to the present invention, "modulating" or "to modulate" generally means that, when measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those described herein), the ISV, polypeptide or construct of the present invention reduces or inhibits the activity of a member of the serine protease family, cathepsin, matrix metalloproteinase (MMP) / matrilysin or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1), ADAMTS11 and / or a pro-inflammatory cytokine (such as interleukin-1α and -β, interleukin-6 and TNF-α, etc.). In particular, "modulating" or "to modulate" means that, when measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those described herein), the activity of the aforementioned members is at least 1%, preferably at least 5%, such as at least 10%, or at least 25%, such as at least 50%, at least 60%, at least 70%, at least 80%, or 90%, or more reduced or inhibited compared to the activity of the aforementioned members under the same conditions except for the presence of the immunoglobulin or polypeptide of the present invention in the same assay.

[0098] With respect to the present invention, "enhancing" or "to enhance" generally means increasing, augmenting, or stimulating the activity of a polypeptide or construct of the present invention when measured using a suitable in vitro, cellular, ex vivo, or in vivo assay (such as those described herein). In particular, when measured using a suitable in vitro, cellular, ex vivo, or in vivo assay (such as those described herein), the activity of a polypeptide or construct of the present invention is compared to the activity of a construct or polypeptide in the same assay under the same conditions except for the presence of an aggrecan binder (e.g., an ISV that binds to aggrecan of the present invention), and is increased or enhanced by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, such as 100%, etc.

[0099] The "synergistic effect" of two compounds means that the effect of the combination of the two agents is greater than the sum of their individual effects, and preferably is statistically different from the control drug and the single drug.

[0100] The term "potency" of an ISV or polypeptide of the present invention, as used herein, is a function of the amount of the ISV or polypeptide of the present invention required for its specific effects, such as penetration into cartilage, specific binding to aggrecan, and / or cartilage retention, etc., to occur. It can be readily measured, for example, by methods known to those skilled in the art when used in the examples section.

[0101] In contrast, the "efficacy" of an ISV or polypeptide of the present invention is measured at the point when the concentration of the ISV or polypeptide is saturated, and is the maximum intensity of its own effect. Efficacy indicates the maximum response achievable from an ISV or polypeptide of the present invention. It refers to the ability of the ISV or polypeptide to provide a desired (therapeutic) effect, such as binding to or retention on aggrecan, and / or inhibiting the activity of an ADAMTS family member or an MMP family member, etc.

[0102] The "half-life" of a polypeptide or construct of the present invention refers to the time required for the serum concentration of the construct or polypeptide to decrease by 50% in vivo, e.g., due to degradation of the construct or polypeptide by natural mechanisms and / or clearance or sequestration of the construct or polypeptide. See, for example, paragraph o) on page 57 of WO 08 / 020079. The in vivo half-life of a construct or polypeptide of the present invention may be determined by any method known per se, such as pharmacokinetic analysis. Suitable methods will be apparent to those skilled in the art and may, for example, generally be those described in paragraph o) on page 57 of WO 08 / 020079. As described in paragraph o) on page 57 of WO 08 / 020079, half-life can be expressed using parameters such as t½-α, t½-β, and area under the curve (AUC). References include, for example, standard handbooks such as Kenneth et al. (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, John Wiley & Sons Inc., 1986), and M Gibaldi and D Perron ("Pharmacokinetics", Marcel Dekker, 2nd Rev. Edition, 1982). The term "increased half-life" or "increased half-life", as also described, for example, in WO 08 / 020079, page 57, paragraph o), refers to an increase in t1 / 2-β, which may or may not be accompanied by an increase in t1 / 2-α and / or AUC, or both.

[0103] Unless otherwise specified, the terms "immunoglobulin" and "immunoglobulin sequence," when used herein to refer to either a heavy chain antibody or a traditional four-chain antibody, include a full-sized antibody, its individual chains, and all portions, domains, or fragments thereof, including but not limited to, antigen-binding domains or fragments (e.g., V and V sequences, respectively). HH Domain or V H / V LIt is used as a general term that includes (and encompasses) a domain).

[0104] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that has the ability to maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for an individual functional characteristic of a protein and can often be added to, moved to, or removed from another protein without loss of function of the rest of the protein and / or the domain.

[0105] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (such as that of a conventional 4-chain antibody or a chain of a heavy-chain antibody) or, essentially, a polypeptide consisting of such a globular region. Immunoglobulin domains are characterized by having an immunoglobulin fold structure consisting of two layers of sandwiches in which approximately seven antiparallel β-strands are arranged as two β-sheets, which is stabilized by optionally conserved disulfide bonds, and which is characteristic of antibody molecules.

[0106] As used herein, the term "immunoglobulin variable domain" means, in the art and as described below in this specification, an immunoglobulin domain that consists essentially of four "framework regions" respectively referred to as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", wherein said framework regions are interrupted by three "complementary determining regions" or "CDRs", which are respectively referred to as "complementary determining region 1" or "CDR1", "complementary determining region 2" or "CDR2", and "complementary determining region 3" or "CDR3" in the art and as described below in this specification. Thus, the general structure or sequence of an immunoglobulin variable domain can be shown as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain(s) that confer(s) antigen specificity to an antibody by having an antigen-binding site, particularly CDR1, CDR2, and / or CDR3.

[0107] The term "immunoglobulin single variable domain" ("ISV" or "ISVD"), which is used interchangeably with "single variable domain", is defined as a molecule in which the antigen-binding site is present in a single immunoglobulin domain and is thereby formed. This makes the ISV distinct from "conventional" immunoglobulins or fragments thereof in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in a conventional immunoglobulin, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementary determining regions (CDRs) of both VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in the formation of the antigen-binding site.

[0108] Considering the above definitions, the antigen-binding domains of conventional four-chain antibodies (such as immunoglobulin G, immunoglobulin M, immunoglobulin A, immunoglobulin D or immunoglobulin E molecules known in the art), or the antigen-binding domains of Fv fragments such as Fab fragments, F(ab’)2 fragments, disulfide-linked Fv or scFv fragments, or the antigen-binding domains of diabodies (all known) derived from such conventional four-chain antibodies, usually, in these cases, the binding to each epitope of the antigen usually does not occur by only one (single) immunoglobulin domain, but rather by a pair of (associated) immunoglobulin domains, such as the light and heavy chain variable domains, i.e., by VH-VL of the immunoglobulin domains, and only then binds jointly with the epitope of each antigen. Therefore, it is not considered an ISV.

[0109] In contrast, an ISV can specifically bind to an epitope of an antigen without combination with a further immunoglobulin variable domain. The binding site of an ISV is formed by a single VH / VHH or VL domain. Therefore, the antigen-binding site of an ISV is formed by only three CDRs.

[0110] Thus, a single variable domain may be a light chain variable domain sequence (such as a VL sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (such as a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it can form a single antigen-binding unit (i.e., an essentially single variable domain consisting of a single antigen-binding domain that does not need to interact with other variable domains to form a functional antigen-binding unit).

[0111] In one aspect of the present invention, the ISV is a heavy chain variable domain sequence (such as a VH sequence), and more specifically, the ISV may be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0112] For example, the ISV may be a (single) domain antibody (or amino acids suitable for use as a (single) domain antibody), an immunoglobulin suitable for use as a (single) domain antibody, a "dAb" or sdAb, or amino acids suitable for use as a dAb, or a nanobody (as defined herein and including but not limited to VHH); a humanized VHH sequence, a camelized VH sequence, a VHH sequence obtained by affinity maturation, another single variable domain, an immunoglobulin single heavy chain variable domain, or any suitable fragment thereof.

[0113] In particular, the ISV may be a Nanobody® (as defined herein) or a suitable fragment thereof. Nanobody® and Nanobodies® are registered trademarks of Ablynx NV. For a general description of nanobodies, reference may be made further below and also to the prior art, e.g., WO 08 / 020079 (page 16), cited herein.

[0114] "VHH domain" (also known as VHH, V H H domains, VHH antibody fragments and VHH antibodies) were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "light chain-deficient antibodies", Hamers-Casterman et al. Nature 363: 446-448, 1993). The term "VHH domain" refers to these variable domains, which are the heavy chain variable domains (herein referred to as "VHH domains") present in conventional four-chain antibodies. H the light chain variable domain (referred to herein as "VH domain") present in conventional four-chain antibodies; LIt is selected for the purpose of distinction from (also referred to as "domain" or "VL domain"). For references regarding further descriptions of VHH and nanobodies, for example, see the review by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as the following patent applications also mentioned in the background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of 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 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 of Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 of Algonomics N.V and Ablynx N.V.; WO 01 / 90190 by the National Research Council of Canada; WO 03 / 025020 (= EP 1433793) by Institute of Antibodies, and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, and further published patent applications by Ablynx N.V. may be mentioned. You may also refer to the further prior art described in these applications, especially the reference list described on pages 41-43 of International Publication No. 06 / 040153, and the said list and references are incorporated herein by reference.As described in these references, ISVs, nanobodies (in particular VHH sequences and partially humanized nanobodies) can be characterized by the presence of one or more "hallmark residues", which are present in particular in one or more framework sequences. Further explanations regarding ISVs, nanobodies, specifically humanization and / or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions", multivalent constructs (including non-limiting examples of some linker sequences), and different modifications for increasing the half-life of ISVs, nanobodies and their preparation, are described, for example, in WO 08 / 101985 and WO 08 / 142164. As a reference for further general explanations of nanobodies, for example, the prior art described in WO 08 / 020079 (page 16), which is cited herein, can be mentioned.

[0115] "Domain antibodies" (also known as "Dab" (singular or plural), "domain antibodies" and "dAb", the terms "domain antibodies" and "dAb" being used as trademarks of the GlaxoSmithKline group) 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 04 / 068820, as well as in WO 06 / 030220, WO 06 / 003388 and WO 03 / 002609 of Domantis and other published patent applications. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammals, in particular human 4-chain antibodies. In order to bind to an epitope as a single antigen-binding domain (i.e., not by pairs with each of the VL or VH domains), specific selection regarding such antigen-binding needs to be performed, for example, using a library of human single VH or VL domain sequences. Domain antibodies, like VHHs, have a molecular weight of about 13 to about 16 kDa and do not require humanization for, for example, human therapeutic use when they are fully derived from human sequences.

[0116] It should be noted that, since it is not of mammalian origin, it is less preferred in the context of the present invention, but it should be noted that a single variable domain may also be from a specific shark species (see, for example, the so-called "IgNAR domain", WO05 / 18629).

[0117] Thus, in the context of the present invention, the terms "immunoglobulin single variable domain" or "single variable domain" include polypeptides from non-human sources, preferably from animals of the camelidae family, preferably derived from the heavy chain antibodies of camelidae animals. As described above, they may be humanized. Furthermore, the terms may include polypeptides derived from non-camelidae animals (such as mice or humans) that have been "camelized" as described, for example, in 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).

[0118] The amino acid residues of the VHH domain are numbered according to the general numbering for the V domain shown by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), similar to the application to the VHH domain from camelidae animals as shown in Figure 2 of Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999). H domains. HAlternative methods for numbering the amino acid residues of a domain, which can also be applied to VHH domains in the same way, are known in the art. However, in the specification, claims, and drawings of the present application, unless otherwise specified, the Kabat numbering applied to the VHH domain as described above is followed.

[0119] As a point of note, V H Although well-known in the art with respect to the V domain and VHH domain, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be present in the actual sequence, or the actual sequence may contain more amino acid residues than the number considered from Kabat numbering). This generally means that Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in the VH domain and VHH domain is usually in the range of 110 - 120, and often in the range between 112 - 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0120] The determination of the CDR region can be carried out according to various methods. In the determination of CDRs according to Kabat, the FR1 of VHH contains amino acid residues at positions 1 - 30, the CDR1 of VHH contains amino acid residues at positions 31 - 35, the FR2 of VHH contains amino acids at positions 36 - 49, the CDR2 of VHH contains amino acid residues at positions 50 - 65, the FR3 of VHH contains amino acid residues at positions 66 - 94, the CDR3 of VHH contains amino acid residues at positions 95 - 102, and the FR4 of VHH contains amino acid residues at positions 103 - 113.

[0121] However, in the present application, the CDR sequences are determined according to Kontermann and Dubel (Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). According to this method, FR1 contains amino acid residues at positions 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acids at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113 (according to Kabat numbering).

[0122] ISVs such as domain antibodies and nanobodies (including the VHH domain) can be humanized. Specifically, a humanized immunoglobulin single variable domain such as a nanobody (including the VHH domain) may be an immunoglobulin single variable domain generally defined in the previous paragraph, but is a humanized substitution (as defined herein), and / or has at least one corresponding amino acid residue (specifically, at least one framework residue). Potentially useful humanized substitutions are the sequences of the framework regions of the native V HH sequence compared to the corresponding framework sequences of one or more closely related human V H sequences, and then one or more potentially useful humanized substitutions (or combinations thereof) thus determined are introduced into the said V HH sequence according to any of the methods known per se described herein, and the resulting humanized V HHAn array can be confirmed by testing it with respect to affinity for a target, stability, ease and level of expression, and / or other desired characteristics. In this way, while reducing trial and error, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art based on the disclosure herein. Also, based on the foregoing, an immunoglobulin single variable domain, such as a nanobody (including the VHH domain) (the framework region thereof) can be partially humanized or fully humanized.

[0123] ISVs, such as domain antibodies and nanobodies (including VHH domains and humanized VHH domains), can also be affinity matured by introducing one or more mutations within the amino acid sequence of one or more CDRs, and such mutations improve the affinity of the resulting immunoglobulin single variable domain for each antigen compared to the respective parental molecule. The affinity matured immunoglobulin single variable domain molecules of the present invention can be prepared by methods of the prior art, for example, based on the descriptions of 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).

[0124] The process of designing / selecting and / or preparing a polypeptide using an ISV (e.g., a domain antibody or nanobody) as a starting material is also referred to herein as "formatting" the ISV, and the ISV that constitutes part of the polypeptide is said to be a polypeptide in "formatted" or "formatted form". Examples of methods by which an ISV can be formatted, and examples of such formatting, will be apparent to those skilled in the art based on the disclosure herein, and such formatted ISVs constitute a further aspect of the invention.

[0125] For example, but not limited to, one or more ISVs can be used as "binding units", "binding domains" or "building blocks" (these terms are used interchangeably) for the preparation of a polypeptide, which may optionally include one or more further ISVs useful as binding units (i.e., for the same or other epitopes on an aggrecan and / or for one or more other antigens, proteins or targets other than aggrecan).

[0126] The present invention provides an aggrecan binder, e.g., an ISV (also referred to herein as "the ISV of the present invention") and / or a polypeptide (also referred to herein as "the polypeptide of the present invention") that has specificity for and / or binds to an aggrecan. 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 is encoded in humans by the ACAN gene located on chromosome Chr15:q26.1.

[0127] Aggrecan is a large multi-module molecule (2317 amino acids). Its core protein consists of three globular domains (G1, G2, and G3), as well as a large extended region between G2 and G3 to which numerous N-linked oligosaccharides and chondroitin sulfate and keratan sulfate chains are attached. Aggrecan is the major proteoglycan in articular cartilage. It plays an important role in the proper function of articular cartilage by providing a hydrated gel structure through its interaction with hyaluronan and link proteins that confer the overloading properties to cartilage. The G1 domain interacts with hyaluronic acid and link protein to form a stable ternary complex in the extracellular matrix (ECM). The G2 domain is homologous to the tandem repeats of G1 and link protein and is involved in the processing of the product. G3 constitutes the carboxyl terminus of the core protein and enhances glycosaminoglycan modification and secretion of the product. Also, the G3 domain links proteoglycan aggregates to ECM proteins (fibrillin and tenascin). The degradation of aggrecan is thought to be initiated at the C-terminus. The population of aggrecan molecules lacking the G3 domain increases with aging. Aggrecan interacts with laminin, fibronectin, tenascin, and collagen, but is also an enzymatic substrate for various A Disintegrin And Metalloprotease with Thrombo-spondin Motifs (ADAMTS), such as ADAMTS4, ADAMTS5, and ADAMTS11, and matrix metalloproteinases (MMP) such as MMP8, MMP13, MMP19, and MMP20.

[0128] In one aspect, the present invention relates to aggrecan binders such as ISV and polypeptides that specifically bind to aggrecan. The aggrecan binders of the present invention are ultimately intended for use as a medicament in humans. Thus, in one aspect, the present invention relates to aggrecan binders, such as ISV and polypeptides, that specifically bind to human aggrecan (SEQ ID NO: 125). The inventors have identified an aggrecan binder with highly improved interspecies cross-reactivity and exquisite selectivity properties.

[0129] Accordingly, in one aspect, the present invention relates to an aggrecan binder such as an ISV or a polypeptide, wherein said aggrecan binder specifically binds to human aggrecan (P16112; SEQ ID NO: 125), canine aggrecan (Q28343; SEQ ID NO: 126), bovine aggrecan (P13608; SEQ ID NO: 127), rat aggrecan (P07897; SEQ ID NO: 128); porcine aggrecan (core; Q29011, SEQ ID NO: 129); mouse aggrecan (Q61282; SEQ ID NO: 130), rabbit aggrecan (G1U677-1; SEQ ID NO: 131); cynomolgus monkey aggrecan (XP_005560513.1; SEQ ID NO: 132) and / or rhesus monkey aggrecan (XP_002804990.1; SEQ ID NO: 133) (see Table B).

[0130] The inventors have surprisingly observed that the aggrecan binders of the present invention, such as the ISV and / or polypeptide of the present invention, have advantageous characteristics over prior art molecules; they are stable in joints, retained in cartilage over a long period of time, and specific for cartilaginous tissues and, for example, do not substantially bind to neurocan (O14594, SEQ ID NO: 134) and / or brevican (Q96GW7, SEQ ID NO: 135) (see Table B).

[0131] Accordingly, in one aspect, the present invention relates to an aggrecan binder such as an ISV or a polypeptide, wherein said aggrecan binder does not substantially bind to neurocan (O14594, SEQ ID NO: 134) and / or brevican (Q96GW7, SEQ ID NO: 135), preferably where said aggrecan is 10 -5 higher than 10 -4 moles / liter, for example 10 D moles / liter of K

[0132] On one side, the present invention relates to an aggrecan binder having a selectivity for binding to aggrecan that is higher by more than 10-fold, higher by more than 100-fold, preferably higher by more than 1000-fold than neurocan and / or brevican, such as an ISV aggrecan binder.

[0133] Preferred aggrecan binders of the present invention include immunoglobulins (e.g., heavy chain antibodies, conventional four-chain antibodies (such as IgG, IgM, IgA, IgD or IgE molecules), Fab fragments, F(ab’)2 fragments, Fv fragments, e.g., disulfide-linked Fv or scFv fragments, or diabodies derived from such conventional four-chain antibodies, or individual chains thereof, and all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, e.g., immunoglobulin single variable domains), monovalent polypeptides of the present invention, or other binders).

[0134] The aggrecan binders of the present invention were observed to have a pI greater than 8, with the sole exception (see Table 2.2). Without being bound by theory, the inventors hypothesized that the high positive charge of aggrecan can affect retention and cartilage penetration even when coupled to another component, such as in a multispecific polypeptide. Accordingly, the present invention relates to an aggrecan binder having a pI greater than 8, such as 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0 or greater than this, such as 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 or 9.8, such as an ISV, polypeptide or construct of the present invention, preferably an ISV of the present invention.

[0135] The binding of the aggrecan binding agent of the present invention, such as the ISV and / or polypeptide of the present invention, to aggrecan can be measured in a variety of binding assays generally known in the art. Typical assays include (but are not limited to) fluorescent ligand binding assays, fluorescence-activated cell sorting (FACS), radioactive ligand binding assays, surface plasmon resonance (SPR), plasmon waveguide resonance (PWR), SPR imaging for affinity-based biosensors, Whispering gallery type microresonators (WGM), resonant waveguide gratings (RWG), Biolayer Interferometry Biosensor (BIB) assays, nuclear magnetic resonance (NMR), X-ray crystallography, thermal denaturation assays (TDA), isothermal titration calorimetry (ITC), ELISA, and whole cell ligand binding assays such as surface acoustic wave (SAW) biosensor and RWG biosensor assays. A preferred assay for measuring the binding of the aggrecan binding agent of the present invention, such as the ISV and / or polypeptide of the present invention, to aggrecan is SPR, for example, SPR as described in the examples, where the binding of the aggrecan binding agent of the present invention, such as the ISV and / or polypeptide of the present invention, to aggrecan was determined. Some preferred K D values will be apparent from further description and examples herein. Another particularly preferred assay is ELISA as detailed in the examples (see Examples 1.2 and 2.4).

[0136] The binding of the aggrecan binding agent of the present invention to aggrecan can also preferably be measured in a binding assay that preserves the three-dimensional structure of the aggrecan target. Typical assays include (but are not limited to) assays in which aggrecan is exposed on the surface of cells (e.g., CHO cells).

[0137] In an embodiment of the present invention, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, for binding to the aggrecan, preferably when measured by surface plasmon resonance, is at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 , 10 7 M -1 s -1 , at least about 10 8 M -1 s -1 , at least about 10 9 M -1 s -1 , and at least about 10 10 M -1 s -1 selected from the group consisting of, and has an on-rate constant (K on ).

[0138] In an embodiment of the present invention, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, for binding to the aggrecan, preferably when measured by surface plasmon resonance, is about 10 -3 s -1 or less, about 10 -4 s -1 or less, about 10 -5 s -1 or less, about 10 -6 s -1 or less, about 10 -7 s -1 or less, about 10 -8 s -1 or less, about 10 -9 s -1 or less, and about 10 -10 s -1an off-rate constant (K) selected from the group consisting of: off )

[0139] In embodiments of the invention, the aggrecan binding agents of the invention, such as ISVs and / or polypeptides of the invention, have an average K D value, e.g., an average K below 90 nM D values, even more preferably an average K of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM, or even lower, such as less than 4, 3, 2, or 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM, or even lower, such as less than 10 pM. D Preferably, the aggrecan binds to the aggrecan at a value of K D is determined by SPR, for example as determined by Proteon. Some preferred ECs for binding of the immunoglobulins and / or polypeptides of the present invention to aggrecan 50 Values will become apparent from further description and examples herein.

[0140] In an ELISA binding assay, aggrecan binding agents of the invention, e.g., ISVs and / or polypeptides of the invention, which preferably bind to the G1 domain and / or the G1-IGD-G2 domain, exhibit binding to human aggrecan of 10 -8 M or less, preferably 10 -9 M or less, or even 10 -10 EC below M 50 For example, in such an ELISA binding assay, the immunoglobulins and / or polypeptides of the invention may have a binding activity of 10 or more in human aggrecan. -10 M~10 -8 M, e.g. 10 -9 M~10 -8 M, or 10 -10 M~10 -9 M's EC 50 It may have a value.

[0141] In such an ELISA binding assay, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, which preferably binds to the G1 domain and / or the G1-IGD-G2 domain, when binding to cynomolgus (cyno) aggrecan, has an EC -7 value of 10 -8 M or less, preferably 10 -9 M or less, more preferably 10 -10 M or less, or even 10 50 M or less. For example, in such an ELISA binding assay, the polypeptide of the present invention, when binding to cyno aggrecan, has an EC -10 value of 10 -7 M to 10 -10 M, for example 10 -8 M to 10 -10 M, 10 -9 M to 10 50 M. It may have such an EC

[0142] In such an ELISA binding assay, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, which preferably binds to the G1 domain and / or the G1-IGD-G2 domain, when binding to rat aggrecan, has an EC -6 value of 10 -7 M or less, preferably 10 -8 M or less, preferably 10 -9 M or less, more preferably 10 -10 M or less, or even 10 50 M or less. For example, in such an ELISA binding assay, the polypeptide of the present invention, when binding to rat aggrecan, has an EC -10 value of 10 -6 M to 10 -10 M, for example 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 50 M. It may have such an EC

[0143] In such an ELISA binding assay, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, which preferably binds to the G1 domain and / or the G1-IGD-G2 domain, may have an EC value of 10 -6 M or less, preferably 10 -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or less, or even 10 -10 M or less when binding to canine aggrecan. For example, in such an ELISA binding assay, the polypeptide of the present invention may have an EC value of 10 50 M to 10 -10 M, such as 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 50 M when binding to canine aggrecan.

[0144] In such an ELISA binding assay, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, which preferably binds to the G1 domain and / or the G1-IGD-G2 domain, may have an EC value of 10 -6 M or less, preferably 10 -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or less, or even 10 -10 M or less when binding to bovine aggrecan. For example, in such an ELISA binding assay, the polypeptide of the present invention may have an EC value of 10 50 M to 10 -10 M, such as 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 50It may have a value.

[0145] As used herein, the term "cartilaginous tissue" refers to cartilage including elastic cartilage, hyaline cartilage and fibrocartilage, which are defined by the ratio of cells (chondrocytes) to the intercellular space, as well as the relative amounts of collagen and proteoglycan. "Articular cartilage" is the cartilage found on the surface of the joints of bones and is exclusively hyaline cartilage. The meniscus consists entirely of fibrocartilage. Aggrecan is the major proteoglycan in the extracellular matrix (ECM) and accounts for approximately 50% of the total protein content (the other approximately 50% being collagen II and some minor proteins, such as collagen IX, etc.).

[0146] The aggrecan binder of the present invention showed preference for cartilaginous tissues in joints such as cartilage and meniscus over non-cartilaginous tissues such as synovium, tendon, and / or epimysium. Accordingly, the present invention relates to an aggrecan binder such as an ISV or a polypeptide, wherein said aggrecan binder preferably binds to cartilaginous tissues such as cartilage and / or meniscus, preferably at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or even more as compared to non-cartilaginous tissues.

[0147] A joint is understood to be a region where two or more bones meet. Most joints are mobile and allow the bones to move. A joint consists of cartilage, synovium, ligaments, tendons, bursae and synovial fluid. Some joints also have menisci.

[0148] As shown in the examples, the aggrecan binder of the present invention has various cartilage retention characteristics, which enables customization of retention in joints according to specific needs (see Example 2.2). Preferably, the aggrecan binder has the ability to be retained in cartilage for an extended period after a relatively short exposure of the aggrecan binder to cartilage that can be expected by intra-articular injection. Cartilage retention can be measured via an ex vivo cartilage retention assay as described in the example section. The degree of retention can be measured by visual inspection of a Western blot or via quantification by densitometry. The scale used to determine the degree of retention can be defined by those in the art, for example, a scale of 0 - 6 RU (Retention Unit), where 0 in this assay indicates no retention and 6 indicates complete retention. If necessary, the scale can be quantified using the aggrecan binder of the present invention, where a score is assigned to each aggrecan binder, for example, complete retention and no retention are fixed. In an alternative, the scale can be set by various intermediate scores assigned via the aggrecan binder of the present invention, for example, two 114F08 = 6 RU, and a dummy aggrecan binder, for example, ALB26 - ALB26 = 0 RU; or an aggrecan binder containing two 114F08 = 6; an aggrecan binder containing 608A05 = 5; aggrecan binder 604G01 = 4; an aggrecan binder containing two 601D02 = 3; an aggrecan binder containing two 606A07 = 2; aggrecan binder 112A01 = 1; and a dummy aggrecan binder, for example, ALB26 - ALB26 = 0. (See Table 2.2). Accordingly, the present invention relates to an aggrecan binder such as an ISV and / or polypeptide according to the present invention, wherein the aggrecan binder has a cartilage retention of at least 2 RU, such as at least 3, 4, 5, or 6 RU, in a cartilage retention assay.

[0149] The aggrecan binder of the present invention should preferably be stable. As a first requirement, the biophysical properties of the aggrecan binder were tested as detailed in Example 3, where it was shown that these aggrecan binders exhibited advantageous stability characteristics, as indicated by a high melting temperature and the absence of signs of aggregation and multimerization. Next, the aggrecan binder was tested for its long-term activity in joints by incubation at 37 °C in synovial fluid (see Example 6). No degradation of any of the constructs could be detected. This indicates that the constructs were stable under conditions mimicking the in vivo situation.

[0150] In one aspect, the present invention relates to an aggrecan binder, such as an ISV, wherein the aggrecan binder has a stability of at least 3 days, 4 days, 5 days, 6 days, 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months at 37 °C in synovial fluid (SF).

[0151] The present invention provides stretches of amino acid residues (SEQ ID NOs: 20-37 and 109, SEQ ID NOs: 38-55 and 110, and SEQ ID NOs: 56-74 and 111; Table A-2) that are particularly suitable for binding to aggrecan. In particular, the present invention provides stretches of amino acid residues that bind to human aggrecan, wherein the binding of the stretch to the aggrecan is retained in the presence of cartilaginous tissue (as described above). These stretches of amino acid residues may be present in and / or incorporated into the construct or polypeptide of the present invention, particularly such that they form part of the antigen-binding site of the polypeptide of the present invention. These stretches of amino acid residues are the CDR sequences or V of the heavy chain antibodies produced against aggrecan HHThey were prepared as arrays. These stretches of amino acid residues are also referred to herein as "the CDR sequence(s) of the present invention" (each, "the CDR1 sequence(s) of the present invention", "the CDR2 sequence(s) of the present invention" and "the CDR3 sequence(s) of the present invention").

[0152] However, in the broadest sense, the present invention should be noted in that, as long as the polypeptide of the present invention can bind to the aggrecan with the desired affinity and potency by these stretches of amino acid residues, these stretches of amino acid residues are not limited to the specific structural role or function that they have in the polypeptide of the present invention. Thus, generally, the present invention in the broadest sense can bind to the aggrecan with a specific affinity, avidity, effectiveness and / or potency, and a suitable combination of one or more CDR sequences described herein, particularly two or more such CDR sequences, is appropriately linked in a form such that, via one or more additional amino acid sequences, the entire polypeptide forms a binding domain and / or binding unit capable of binding to the aggrecan, and provides a polypeptide (also referred to herein as "the polypeptide(s) of the present invention"). However, it should also be noted that even when only one such CDR sequence is present in the polypeptide of the present invention, the ability of the polypeptide of the present invention to bind to the aggrecan is sufficiently provided, and for example, by way of reference again, the so-called "Expedite fragments" described in WO 03 / 050531 can be mentioned.

[0153] In a specific but non-limiting aspect, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, may consist essentially of, or may include, at least one stretch of amino acid residues selected from the group consisting of: i) CDR1 sequence: a) SEQ ID NOs: 24, 32, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37 and 109; and b) an amino acid sequence having a difference of 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 24; and / or ii) CDR2 sequence: c) SEQ ID NOs: 42, 50, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55 and 110; and d) an amino acid sequence having a difference of 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 42; and / or iii) CDR3 sequence: e) SEQ ID NOs: 60, 68, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74 and 111; and f) an amino acid sequence having a difference of 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 60, Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0154] In a further aspect, an aggrecan binder of the invention, such as a polypeptide and / or an ISV of the invention, may comprise a stretch of at least 1 amino acid residue selected from the group consisting of SEQ ID NOs: 20 to 74 and 109 to 111.

[0155] In particular, the aggrecan binder of the present invention, such as the polypeptide and / or ISV of the present invention, may be an aggrecan binder comprising one binding site, wherein said antigen-binding site comprises a stretch of at least one amino acid residue selected from the group consisting of CDR1 sequence, CDR2 sequence and CDR3 sequence (or any suitable combination thereof) as described above. In a preferred aspect, however, the aggrecan binder of the present invention, such as the polypeptide and / or ISV of the present invention, comprises more than one, for example two or more, stretches of amino acid residues selected from the group consisting of the CDR1 sequence of the present invention, the CDR2 sequence of the present invention and / or the CDR3 sequence of the present invention. Preferably, the aggrecan binder of the present invention, such as the polypeptide and / or ISV of the present invention, comprises a stretch of three amino acid residues selected from the group consisting of the CDR1 sequence of the present invention, the CDR2 sequence of the present invention and the CDR3 sequence of the present invention, respectively. Combinations of CDRs described as preferred herein for the aggrecan binder of the present invention, such as the polypeptide and / or ISV of the present invention, are listed in Table A-2, i.e., preferably, a combination of CDRs shown in a single row of said table.

[0156] Representative polypeptides of the present invention having the above CDRs are shown in Table A-1 (SEQ ID NOs: 1-19 and 114-118).

[0157] In a preferred embodiment, the present invention relates to an aggrecan binder of the present invention, such as an ISV and / or polypeptide of the present invention, comprising three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 is selected from the group consisting of SEQ ID NOs: 24, 32, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37 and 109; - CDR2 is selected from the group consisting of SEQ ID NOs: 42, 50, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55 and 110; and - The CDR3 is selected from the group consisting of SEQ ID NOs: 60, 68, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74 and 111. Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0158] In a preferred embodiment, the present invention relates to an aggrecan binder of the present invention, such as an ISV and / or a polypeptide of the present invention, comprising three complementarity determining regions (CDR1 to CDR3 respectively), wherein: - CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; - CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; - CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; - CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; - CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59; - CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; - CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62; - CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; - CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64; - CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; - CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66; - CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69; - CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70; - CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 71; - CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 72; - CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 73; - CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 74; or - CDR1 is SEQ ID NO: 109, CDR2 is SEQ ID NO: 110, and CDR3 is SEQ ID NO: 111; Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0159] In a preferred embodiment, the invention relates to an aggrecan binder such as an ISV, wherein it is selected from the group consisting of SEQ ID NO: 117, 5, 118, 13, 114-116, 1-4, 6-12 and 14-19.

[0160] The present invention is also not limited to the origin of the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, (or the nucleic acid of the present invention used to express it), nor is it limited to whether the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, or the nucleic acid of the present invention is produced (or has been produced) or obtained (or has been obtained). Thus, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, may be a naturally occurring ISV (from any suitable species), or a synthetic or semi-synthetic ISV and / or polypeptide.

[0161] Furthermore, it will be apparent to those skilled in the art that the one or more CDRs can be "grafted" onto other "frameworks" such as, but not limited to, a human framework or a non-immunoglobulin framework. Suitable frameworks and techniques for such CDR grafting are apparent to those skilled in the art and are known in the art, for example, by U.S. Patent No. 7,180,370, International Publication No. 01 / 27160, European Patent Application Publication No. 0605522, European Patent Application Publication No. 0460167, U.S. Patent No. 7,054,297, Nicaise et al. (Protein Science 13: 1882-1891, 2004), Ewert et al. (Methods 34: 184-199, 2004), Kettleborough et al. (Protein Eng. 4: 773-783, 1991), O'Brien and Jones (Methods Mol. Biol. 207: 81-100, 2003), Skerra (J. Mol. Recognit. 13: 167-187, 2000) and Saerens et al. (J. Mol. Biol. 352: 597-607, 2005), and further citations therein. For example, the technique of grafting mouse or rat CDRs onto a human framework and backbone itself is also known and can be used similarly to provide a chimeric protein comprising one or more of the CDR sequences as defined herein for the monovalent polypeptides of the present invention and one or more human framework regions or sequences.Suitable scaffolds for presenting amino acid sequences will be apparent to those skilled in the art and include, for example, binding scaffolds based on or derived from immunoglobulins (i.e., other than the immunoglobulin sequences described hereinabove), protein scaffolds derived from protein A domains (such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T cell receptors, artificial ankyrin repeats, avimers, and PDZ domains (Binz et al. Nat Biotech 23: 1257, 2005), as well as DNA- or RNA-based binding moieties (such as, but not limited to, DNA or RNA aptamers) (Ulrich et al. Com Chem. High Throughput Screen 9: 619-32, 2006).

[0162] In the aggrecan binders of the invention, such as the ISV and / or polypeptides of the invention, the CDRs may be linked to further amino acid sequences and / or may be linked to each other via amino acid sequences, where the amino acid sequences are preferably framework sequences or amino acid sequences that act as framework sequences or that form a scaffold for presenting the CDRs together.

[0163] In a preferred embodiment, the aggrecan binder of the present invention, such as an ISV and / or a polypeptide, comprises at least three CDR sequences linked to at least two framework sequences, wherein preferably at least one of the three CDR sequences is a CDR3 sequence and the other two CDR sequences are CDR1 or CDR2 sequences, preferably one is a CDR1 sequence and one is a CDR2 sequence. In a particularly preferred and non-limiting embodiment, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein CDR1, CDR2 and CDR3 are as defined herein with respect to the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, and FR1, FR2, FR3 and FR4 are framework sequences. In the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, the framework sequence can be any suitable framework sequence, and examples of suitable framework sequences will be apparent to those skilled in the art based on, for example, standard handbooks and further disclosure herein and the prior art.

[0164] Accordingly, the aggrecan binder of the present invention, such as the ISV and / or polypeptide of the present invention, comprises three complementarity determining regions (CDR1 to CDR3 respectively), where: (i) CDR1 is (a) SEQ ID NO: 24, 32, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37 and 109; and (b) an amino acid sequence having 4, 3, 2 or 1 amino acid(s) difference from the amino acid sequence of SEQ ID NO: 24 or any of SEQ ID NOs: 20-23, 25-37 and 109; is selected from the group consisting of, and / or (ii) CDR2 is (c) SEQ ID NO: 42, 50, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55 and 110; and (d) The amino acid sequence of SEQ ID NO: 42, and an amino acid sequence having a difference of 4, 3, 2, or 1 amino acid(s) from any one of SEQ ID NOs: 38-41, 43-55, and 110; selected from the group consisting of, and / or (iii) CDR3 is (e) SEQ ID NOs: 60, 68, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, and 111; and (f) the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence having a difference of 4, 3, 2, or 1 amino acid(s) from any one of SEQ ID NOs: 56-59, 61-74, and 111; selected from the group consisting of Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3, and FR4 are framework sequences.

[0165] The aggrecan binder of the present invention can map to the G1 region, G1-IGD-G2 region, or G2 region of aggrecan. Accordingly, the present invention relates to an aggrecan binder of the present invention that binds to the G2 domain of aggrecan, such as an ISV and / or a polypeptide. As described in the examples, these aggrecan binders of the present invention, such as an ISV and / or a polypeptide, have various preferred characteristics. Preferably, the aggrecan binder of the present invention, such as an ISV and / or a polypeptide, has a pI greater than 8 and / or a K of less than 2 * 10 -2 s -1 and / or an EC of less than 1 off 10 * 10 -6 M. 50 It has.

[0166] By comparing the aggrecan binders of the present invention, such as the CDRs of the ISV and / or polypeptides of the present invention, a number of acceptable amino acid changes in the CDRs have been revealed while retaining the binding to the G2 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDR of 601D02 used as a reference is represented in Tables 1.5A, 1.5B, and 1.5C.

[0167] In one aspect, the present invention relates to an aggrecan binder of the present invention, such as an ISV and / or polypeptide, wherein; i) CDR1 is a) SEQ ID NO: 28, 22, 26, and 33; and b) an amino acid sequence having a difference of 5, 4, 3, 2, or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 28, wherein the amino acid difference(s) is / are defined as follows: - At position 1, G is changed to R; - At position 2, P is changed to S or R; - At position 3, T is changed to I; - At position 5, S is changed to N; - At position 6, R is changed to N, M, or S; - At position 7, Y is changed to R or is absent; - At position 8, A is changed to F or is absent; and / or - At position 10, G is changed to Y; selected from the group consisting of, and / or ii) CDR2 is c) SEQ ID NO: 46, 40, 44, and 52; and d) an amino acid sequence having a difference of 5, 4, 3, 2, or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 46, wherein the amino acid difference(s) is / are defined as follows: - At position 1, A is changed to S or Y; - At position 4, W has been changed to L; - At position 5, S has been changed to N; - At position 6, S is absent; - At position 7, G is absent; - At position 8, G has been changed to A; - At position 9, R has been changed to S, D or T; and / or - At position 11, Y has been changed to N or R; selected from the group consisting of; and / or iii) CDR3 is e) SEQ ID NO: 64, 58, 62 and 70; and f) an amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 64, wherein the amino acid difference(s) is / are defined as follows: - At position 1, A has been changed to R or F; - At position 2, R has been changed to I or L; - At position 3, I has been changed to H or Q; - At position 4, P has been changed to G or N; - At position 5, V has been changed to S; - At position 6, R has been changed to G, N or F; - At position 7, T has been changed to R, W or Y; - At position 8, Y has been changed to R or S or is absent; - At position 9, T has been changed to S or is absent; - At position 10, S has been changed to E, K or is absent; - At position 11, E has been changed to N, A or is absent; - At position 12, W has been changed to D or is absent; - At position 13, N is changed to D or is absent; - At position 14, Y is absent; and / or - D and N are added after position 14 of SEQ ID NO: 64; selected from the group consisting of Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0168] In one aspect, the present invention - CDR1 is selected from the group consisting of SEQ ID NOs: 28, 22, 26 and 33; - CDR2 is selected from the group consisting of SEQ ID NOs: 46, 40, 44 and 52; and - CDR3 is selected from the group consisting of SEQ ID NOs: 64, 58, 62 and 70, relates to an aggrecan binder of the present invention selected from the group of aggrecan binders, such as an ISV and / or a polypeptide, wherein Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0169] In one aspect, the present invention - CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64; - CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; - CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62; and - CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70; Regarding the aggrecan binder of the present invention selected from the group of aggrecan binders, such as ISV and / or polypeptide Preferably, an aggrecan binder such as ISV and / or polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0170] In one aspect, the present invention relates to an aggrecan binder of the present invention selected from the group of aggrecan binders, such as ISV and / or polypeptide, consisting of an aggrecan binder having a sequence identity higher than 80%, such as 90% or 95%, with any one of SEQ ID NOs: 9, 3, 7 and 15, and SEQ ID NOs: 9, 3, 7 and 15.

[0171] In one aspect, the present invention relates to an aggrecan binder of the present invention, such as ISV and / or polypeptide, which cross-blocks the binding of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single-domain antibody, an immunoglobulin suitable for use as a single-domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, to the G2 domain of aggrecan.

[0172] In one aspect, the present invention relates to a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single-domain antibody, an immunoglobulin suitable for use as a single-domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, which binds to the G2-domain of aggrecan and competes with the ISV and / or polypeptide of the present invention, preferably represented by any one of SEQ ID NOs: 9, 3, 7 and 15, for binding to the G2 domain of aggrecan.

[0173] The present invention also relates to the aggrecan binding agents of the present invention that bind to the G1-IGD-G2 domain of aggrecan, such as ISV and / or polypeptides. As described in the examples, these aggrecan binding agents of the present invention, such as ISV and / or polypeptides, have various favorable characteristics. Preferably, the aggrecan binding agents of the present invention, such as ISV and / or polypeptides, have a pI greater than 8 and / or * 10 -2 s -1 less than K off and / or have an EC of less than 1 * 10 -6 M. 50 have.

[0174] By comparing the CDRs of the aggrecan binding agents of the present invention, such as the ISV and / or polypeptides of the present invention, amino acid changes that are acceptable in the CDRs have been revealed while retaining the binding to the G1-IGD-G2 domain of aggrecan. The variability of the sequences in the CDRs of all clones relative to the CDR of 604F02 used as a reference is represented in Tables 1.4A, 1.4B, and 1.4C.

[0175] In one aspect, the present invention also relates to the aggrecan binding agents of the present invention, such as ISV and / or polypeptides, wherein; i) CDR1 is a) SEQ ID NO: 32, 30, and 23; and b) an amino acid sequence having a difference of 3, 2, or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 32, wherein the amino acid difference(s) is / are defined as follows: - at position 2, R is changed to L; - at position 6, S is changed to T; and / or - at position 8, T is changed to A; selected from the group consisting of, and / or ii) CDR2 is c) SEQ ID NOs: 50, 41, 48 and 51; and d) An amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 50, wherein the amino acid difference(s) is / are defined as follows: - At position 7, G is changed to S or R; and / or - At position 8, R is changed to T; selected from the group consisting of; and / or iii) CDR3 is e) SEQ ID NOs: 68, 59, 66 and 69; and f) An amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 68, wherein the amino acid difference(s) is / are defined as follows: - At position 4, R is changed to V or P; - At position 6, A is changed to Y; - At position 7, S is changed to T; - At position 8, S is absent; - At position 9, N is changed to P; - At position 10, R is changed to T or L; - At position 11, G is changed to E; and / or - At position 12, L is changed to T or V; selected from the group consisting of, Preferably, an aglycan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0176] In one aspect, the invention provides - CDR1 is selected from the group consisting of SEQ ID NOs: 32, 30 and 23; - CDR2 is selected from the group consisting of SEQ ID NO: 50, 41, 48 and 51; and - CDR3 is selected from the group consisting of SEQ ID NO: 68, 59, 66 and 69, Regarding the aggrecan binder of the present invention, such as ISV and / or polypeptide, Preferably, an aggrecan binder such as ISV and / or polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0177] In one aspect, the present invention - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69; - CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66; and - CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59; Regarding the aggrecan binder of the present invention, such as ISV and / or polypeptide, which is selected from the group of aggrecan binders, Preferably, an aggrecan binder such as ISV and / or polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0178] In one aspect, the present invention relates to an aggrecan binder having SEQ ID NO: 118, 13, 4, 11 and 14, and an aggrecan binder selected from the group consisting of an aggrecan binder having any one of SEQ ID NO: 118, 13, 4, 11 and 14 and having a sequence identity higher than 80%, such as 90% or 95%.

[0179] In one aspect, the present invention relates to an aggrecan binder of the present invention, such as an ISV and / or polypeptide, which cross-blocks the binding of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, to the G1-IGD-G2 domain of aggrecan.

[0180] In one aspect, the present invention relates to a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, which binds to the G1-IGD-G2 domain of aggrecan and competes for the binding to the G1-IGD-G2 domain of aggrecan, preferably represented by any one of SEQ ID NOs: 118, 13, 4, 11, and 14, of an aggrecan binder of the present invention, such as an ISV and / or polypeptide of the present invention.

[0181] In a particularly preferred embodiment, the present invention relates to an aggrecan binder of the present invention, such as an ISV and / or polypeptide of the present invention, which binds to the G1 domain of aggrecan. As described in the examples, these aggrecan binders of the present invention, such as an ISV and / or polypeptide of the present invention, have various preferred features. Preferably, the aggrecan binder of the present invention, such as an ISV and / or polypeptide, has a pI greater than 8 and / or has a K less than 2 * 10 -2 s -1 and / or has an EC less than 1 off 10 * 10 -6 M. 50 It has.

[0182] By comparing the aggrecan binders of the present invention, such as the CDRs of the ISV and / or polypeptides of the present invention, a number of acceptable amino acid changes in the CDRs were revealed while retaining the binding to the G1 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDR of 114F08 used as a reference is represented in Tables 1.3A, 1.3B, and 1.3C.

[0183] In a preferred aspect, the present invention relates to an aggrecan binder of the present invention, such as an ISV and / or polypeptide of the present invention, comprising three complementarity-determining regions (CDR1 to CDR3 respectively), wherein: i) CDR1 is a) SEQ ID NO: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, and 37; and b) an amino acid sequence having a difference of 5, 4, 3, 2, or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 24, wherein the amino acid difference(s) is / are defined as follows: - At position 2, S is changed to R, F, I, or T; - At position 3, T is changed to I; - At position 5, I is changed to S; - At position 6, I is changed to S, R, or M; - At position 7, N is changed to Y or R; - At position 8, V is changed to A, Y, T, or G; - At position 9, V is changed to M; and / or - At position 10, R is changed to G, K, or A; selected from the group consisting of, and / or ii) CDR2 is c) SEQ ID NO: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, and 55; and d) An amino acid sequence having a difference of 5, 4, 3, 2, or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 42, wherein the amino acid difference(s) is / are defined as follows: - At position 1, T is changed to A or G; - S or N is inserted between positions 3 and 4 (position 2a, Table 1.3B); - At position 3, S is changed to R, W, N, or T; - At position 4, S is changed to T or G; - At position 5, G is changed to S; - At position 6, G is changed to S or R; - At position 7, N is changed to S, T, or R; - At position 8, A is changed to T; and / or - At position 9, N is changed to D or Y; selected from the group consisting of; and / or iii) CDR3 is e) SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73, and 74; and f) An amino acid sequence having a difference of 5, 4, 3, 2, or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) is / are defined as follows: - At position 1, P is changed to G, R, D, or E or is absent; - At position 2, T is changed to R, L, P, or V or is absent; - At position 3, T is changed to M, S, or R or is absent; - At position 4, H is changed to D, Y, G, or T; - At position 5, Y is changed to F, V, T, or G; - At position 6, G is changed to L, D, S, Y, or W; - R, T, Y, or V is inserted between positions 6 and 7 (position 6a, Table 1.3C); - At position 7, G is changed to P or S; - At position 8, V is changed to G, T, H, R, L, or Y; - At position 9, Y is changed to R, A, S, D, or G; - At position 10, Y is changed to N, E, G, W, or S; - W is inserted between positions 10 and 11 (position 10a, Table 1.3C); - At position 11, G is changed to S, K, or Y; - At position 12, P is changed to E or D or is absent; and / or - At position 13, Y is changed to L or is absent; selected from the group consisting of Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3, and FR4 are framework sequences.

[0184] In a preferred aspect, the invention relates to an aggrecan binder of the invention, such as an ISV and / or a polypeptide, wherein CDR1 is selected from the group consisting of SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, 37, and 109; CDR2 is selected from the group consisting of SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, 55, and 110; and CDR3 is selected from the group consisting of SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73, 74, and 111; preferably, the aggrecan binder, such as an ISV and / or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3, and FR4 are framework sequences.

[0185] In a preferred aspect, the present invention - CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; - CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; - CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; - CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; - CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; - CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; - CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; - CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 71; - CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 72; - CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 73; - CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 74; and - CDR1 is SEQ ID NO: 109, CDR2 is SEQ ID NO: 110, and CDR3 is SEQ ID NO: 111; Regarding the aggrecan binder of the present invention, selected from the group of aggrecan binders, such as ISV and / or polypeptide, Preferably, the aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0186] In the Examples section, exemplary clone 114F08 is shown to have particularly preferred characteristics. Clone 114F08 is grouped (see Table A-2 and Tables 3.3A, 3.3B and 3.3C) based on similarity in the CDRs (which reflects similarity in functional characteristics) and further includes clones 114A09 (SEQ ID NO: 114) and 114B04 (SEQ ID NO: 115), representing a family or set of clones. Thus, in another particularly preferred aspect, the invention relates to an aggrecan binder of the invention, such as an ISV and / or a polypeptide, comprising three complementarity determining regions (CDR1 to CDR3 respectively), where: i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 24 and 109; and b) an amino acid sequence having 2 or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 24, where the amino acid difference(s) is / are defined as follows: - at position 7, N is changed to S; and / or - at position 9, V is changed to M; and / or ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 42 and 110; and d) an amino acid sequence having 5, 4, 3, 2 or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 42, where the amino acid difference(s) is / are defined as follows: - at position 1, T is changed to A; - at position 3, S is changed to R; - at position 4, S is changed to T; - At position 8, A has been changed to T; and / or - At position 9, N has been changed to D; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 60 and 111; and f) an amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) is / are defined as follows: - At position 4, H has been changed to R; and / or - At position 8, V has been changed to D; Preferably, an aggrecan binder such as ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0187] In one aspect, the present invention relates to - CDR1 is selected from the group consisting of SEQ ID NO: 24 and 109; - CDR2 is selected from the group consisting of SEQ ID NO: 42 and 110; and - CDR3 is selected from the group consisting of SEQ ID NO: 60 and 111, an aggrecan binder of the present invention selected from the group of aggrecan binders, such as ISV and / or a polypeptide, Preferably, an aggrecan binder such as ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0188] Furthermore, in the examples section, it has been shown that an aggrecan binder that binds to the G1 region of aggrecan and belongs to epitope bin 1 or epitope bin 4 is particularly effective in a cartilage retention assay. In one aspect, the present invention relates to an aggrecan binder of the present invention belonging to epitope bin 1 or epitope bin 4, such as ISV and / or a polypeptide.

[0189] By comparing the CDRs of the aggrecan binder of the present invention belonging to epitope bin 1, such as the ISV and / or polypeptide of the present invention, a number of amino acid changes that can be tolerated in the CDRs were revealed while retaining the binding to the G1 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDR of 608A05 used as a reference is represented in Tables 2.3D, 2.3E, and 2.3F.

[0190] In a preferred aspect, the present invention relates to an aggrecan binder of the present invention, such as ISV and / or a polypeptide, comprising three complementarity determining regions (CDR1 to CDR3 respectively), where; i) CDR1 is a) SEQ ID NO: 36, 20, and 29; and b) an amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 36, where the amino acid difference(s) is / are defined as follows: - At position 3, T is changed to S; - At position 6, T is changed to S; - At position 8, T is changed to A; and / or - At position 9, M is changed to V; selected from the group consisting of, and / or ii) CDR2 is c) SEQ ID NO: 54, 38, and 37; and d) An amino acid sequence having differences of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 54, wherein the difference(s) of the amino acid(s) is / are defined as follows: - At position 1, A is changed to I; - At position 4, W is changed to R; - At position 7, G is changed to R; and / or - At position 8, T is changed to S; selected from the group consisting of; and / or iii) CDR3 is e) SEQ ID NOs: 73, 56 and 65; and f) An amino acid sequence having differences of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 73, wherein the difference(s) of the amino acid(s) is / are defined as follows: - At position 1, R is changed to G; - At position 2, P is changed to R or L; - At position 3, R is changed to L or S; - At position 5, Y is changed to R; - At position 6, Y is changed to S or A; - At position 7, Y is changed to T or absent; - At position 8, S is changed to P; - At position 9, L is changed to H or R; - At position 10, Y is changed to P or A; - At position 11, S is changed to A or Y; - At position 12, Y is changed to D; - At position 13, D is changed to F; - At position 14, Y is changed to G or absent; and / or - After position 14, S is inserted; selected from the group consisting of Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0191] In one aspect, the present invention relates to - CDR1 is selected from the group consisting of SEQ ID NOs: 20, 29 and 36; - CDR2 is selected from the group consisting of SEQ ID NOs: 38, 47 and 54; and - CDR3 is selected from the group consisting of SEQ ID NOs: 56, 65 and 73; Regarding the aggrecan binder of the present invention, for example an ISV and / or a polypeptide, selected from the group of aggrecan binders Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0192] In one aspect, the present invention relates to an aggrecan binder of the present invention belonging to epitope bin 1, for example an ISV and / or a polypeptide, which cross-blocks the binding of a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, to the G1 domain of aggrecan.

[0193] In one aspect, the present invention relates to a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, which binds to epitope bin 1 of the G1-domain of aggrecan and competes for binding to the G1 domain of aggrecan with an aggrecan binder of the present invention, such as belonging to epitope bin 1, preferably an ISV and / or polypeptide represented by any one of SEQ ID NOs: 1, 10, and 18.

[0194] By comparison of the CDRs of the aggrecan binders of the present invention belonging to epitope bin 4, such as the ISV and / or polypeptides of the present invention, a number of amino acid changes have been revealed that are acceptable in the CDRs while retaining binding to the G1 domain of aggrecan. The variability of the sequences in the CDRs of all clones relative to the CDR of 114F08 used as a reference is shown in Tables 2.3A, 2.3B, and 2.3C.

[0195] In one aspect, the present invention relates to an aggrecan binder of the present invention, such as an ISV and / or polypeptide, comprising three complementarity determining regions (CDR1 to CDR3 respectively), wherein: i) CDR1 is a) SEQ ID NOs: 24, 25, and 27; and b) an amino acid sequence having a difference of 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 24, wherein the amino acid difference(s) is / are defined as follows: - At position 2, S is changed to I or F; - At position 5, I is changed to S; - At position 6, I is changed to S or M; - At position 7, N is changed to R or Y; - At position 8, V is changed to A or Y; - At position 9, V is changed to M; and / or - At position 10, R is changed to K; selected from the group consisting of; and / or ii) CDR2 is c) SEQ ID NOs: 42, 43 and 45; and d) an amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 42, wherein the amino acid difference(s) is / are defined as follows: - At position 1, T is changed to A or G; - An N is inserted between positions 2 and 3 (position 2a, Table 2.3B); - At position 7, N is changed to R; - At position 8, A is changed to T; and / or - At position 9, N is changed to D; selected from the group consisting of; and / or iii) CDR3 is e) SEQ ID NOs: 60, 61 and 63; and f) an amino acid sequence having a difference of 5, 4, 3, 2 or 1 amino acid(s) from the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) is / are defined as follows: - At position 1, P is absent; - At position 2, T is changed to R or is absent; - At position 3, T is changed to M or is absent; - At position 4, H is changed to D or Y; - At position 5, Y is changed to F or V; - At position 6, G is changed to L or D; - At position 8, V is changed to G or T; - At position 9, Y has been changed to R; - At position 10, Y has been changed to N or E; - At position 11, G has been changed to S or K; - At position 12, P has been changed to E or is absent; and / or - At position 13, Y has been changed to L or is absent; selected from the group consisting of Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0196] In one aspect, the present invention - CDR1 is selected from the group consisting of SEQ ID NOs: 24, 25 and 27; - CDR2 is selected from the group consisting of SEQ ID NOs: 42, 43 and 45; and - CDR3 is selected from the group consisting of SEQ ID NOs: 60, 61 and 63; Regarding the aggrecan binder of the present invention, which is selected from the group of aggrecan binders, such as an ISV and / or a polypeptide, Preferably, an aggrecan binder such as an ISV and / or a polypeptide comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0197] In one aspect, the present invention relates to an aggrecan binder of the present invention belonging to epitope bin 4, such as an ISV and / or polypeptide, which is a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, and which cross-blocks the binding of aggrecan to the G1 domain.

[0198] In one aspect, the present invention relates to a domain antibody, an immunoglobulin suitable for use as a domain antibody, a single domain antibody, an immunoglobulin suitable for use as a single domain antibody, a dAb, an immunoglobulin suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence obtained by affinity maturation, which binds to the epitope bin 4 of the G1-domain of aggrecan and competes for the binding of aggrecan to the G1 domain with an aggrecan binder of the present invention belonging to epitope bin 4, such as an ISV and / or polypeptide represented by any one of SEQ ID NOs: 117, 114, 115, 116, 5, 6, and 8.

[0199] In one aspect, the present invention relates to an aggrecan binder represented by SEQ ID NOs: 117, 118, 116, 114, 115, 5, 13, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19, and an aggrecan binder of the present invention, such as an ISV and / or polypeptide, selected from the group consisting of ISVs having a sequence identity higher than 80%, such as higher than 90%, or 95%, or even higher, with any one of SEQ ID NOs: 117, 118, 116, 114, 115, 5, 13, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19.

[0200] In one specific and non-limiting aspect, the aggrecan binder of the present invention may be a stretch of amino acid residues containing an immunoglobulin fold, or an aggrecan binder capable of forming an immunoglobulin fold (i.e., by folding) under suitable conditions (such as physiological conditions). By reference, in particular, the review by Halaby et al. (J. Protein Eng. 12: 563-71, 1999) is cited. Preferably, when folded to appropriately form an immunoglobulin fold, the stretch of amino acid residues can appropriately form an antigen-binding site for binding to aggrecan. Thus, in a preferred aspect, the aggrecan binder of the present invention is an immunoglobulin, such as, for example, an immunoglobulin single variable domain.

[0201] Thus, the framework sequence is preferably an immunoglobulin framework sequence (a suitable combination thereof), or a framework sequence derived from an immunoglobulin framework sequence (e.g., by sequence optimization (such as humanization or camelization)). For example, the framework sequence may be an immunoglobulin single variable domain, such as a light chain variable domain (e.g., V L sequence), and / or a framework sequence derived from a heavy chain variable domain (e.g., V H sequence). In one particularly preferred aspect, the framework sequence is a framework sequence derived from the V HH sequence (the framework sequence may optionally be partially or completely), or a camelized conventional V H sequence, as defined herein.

[0202] The framework sequence is preferably such that the monovalent polypeptide of the present invention is an ISV, such as a domain antibody (or an amino acid sequence suitable for use as a domain antibody), a single domain antibody (or an amino acid suitable for use as a single domain antibody), a "dAb" (or an amino acid suitable for use as a dAb), a nanobody®, a V HH sequence, a humanized V HHArray, camelized V H An array, or a V obtained by affinity maturation HH It may be such as an array. Also, suitable framework arrays will be apparent to those skilled in the art based on, for example, standard handbooks and the further disclosure of this specification and the prior art.

[0203] Another particularly preferred class of ISVs of the present invention is a naturally occurring V H Corresponding to the amino acid sequence of the domain, but "camelized", i.e., a naturally occurring V from a conventional four-chain antibody H One or more amino acid residues in the amino acid sequence of the domain are replaced by one or more of the amino acid residues occurring at the corresponding position(s) in the V HH Domain, including an ISV having an amino acid sequence. This can be done in a manner known per se to those skilled in the art, for example based on the description in this specification. Such "camelization" substitutions preferably occur in the V H -V L Form an interface and / or at the positions of the amino acids present therein, and / or are inserted at the so-called camelid hallmark residues, which are well known to those skilled in the art and defined, for example, in WO 94 / 04678 and Davies and Riechmann (1994 and 1996). Preferably, the V H Sequence is preferably a V from a mammal H Sequence, more preferably a human V H Sequence, for example a V H 3 sequence. However, it should be noted that such camelized ISVs of the present invention can be obtained in any suitable manner known per se and are thus not strictly limited to polypeptides obtained using polypeptides containing a naturally occurring V H Domain as a starting material.

[0204] For example, as further described herein, both "humanization" and "camelization" each provide a naturally occurring V HH domain or a nucleotide sequence encoding a V H domain, and then one or more codons in said nucleotide sequence are changed in a manner known per se such that the new nucleotide sequence encodes a "humanized" or "camelized" ISV of the invention respectively. This nucleic acid can then be expressed in a manner known per se to provide the desired ISV of the invention. Alternatively, based on the amino acid sequence of each naturally occurring V HH domain or V H domain, the amino acid sequence of the desired humanized or camelized ISV of the invention can be designed respectively, and then synthesized de novo using techniques known per se for peptide synthesis. Also, based on the amino acid sequence or nucleotide sequence of each naturally occurring V HH domain or V H domain, the nucleotide sequence encoding the desired humanized or camelized ISV of the invention can be designed respectively, and then synthesized de novo using techniques known per se for nucleic acid synthesis, and thereafter the nucleic acid thus obtained can be expressed in a manner known per se to provide the desired ISV of the invention.

[0205] In particular, the framework sequences present in the aggrecan binders of the invention, such as the ISV and / or polypeptide of the invention, may include one or more of the hallmark residues as defined, for example, in WO 08 / 020079 (Tables A-3 to A-8), such that the aggrecan binder of the invention becomes a nanobody. Some preferred but non-limiting examples of such framework sequences (suitable combinations thereof) will become apparent from further disclosure herein (see, for example, Table A-2). Generally, nanobodies (in particular, V HHAn array and a partially humanized nanobody can be characterized, in particular, by the presence of one or more "hallmark residues" in one or more of the framework arrays (for example, as further described in WO 08 / 020079, pages 61 line 24 to page 98 line 3). As used herein, "represented by" with respect to any sequence number is equal to "comprising or consisting of" said sequence number, preferably equal to "consisting of" said sequence number.

[0206] Furthermore, the present invention has the (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 provides an aggrecan binder comprising at least one ISV having the amino acid sequence of wherein FR1 to FR4 each refer to framework regions 1 to 4, wherein CDR1 to CDR3 each refer to complementarity determining regions 1 to 3, and this is: i) having 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 NOs: 117, 116, 118, 116, 115, 114 and 1-19 (see Table A-2), wherein for the purpose of determining the degree of amino acid identity, the amino acid residues forming the CDR sequences are ignored. In this regard, reference is also made to Table A-2 listing the framework 1 sequences (SEQ ID NOs: 119, 120 and 75-84), framework 2 sequences (SEQ ID NOs: 121 and 85-93), framework 3 sequences (SEQ ID NOs: 123, 124, 122, 94-104 and 112-113) and framework 4 sequences (SEQ ID NOs: 105-108) of the immunoglobulin single variable domains of SEQ ID NOs: 117, 118, 116, 115, 114 and 1-19; or ii) a combination of framework sequences as represented in Table A-2; and here; iii) Preferably, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from hallmark residues as described in Tables A-3 to A-8 of WO 08 / 020079, for example.

[0207] Accordingly, the present invention relates to an ISV and / or a polypeptide, wherein said ISV consists essentially of four framework regions (FR1 to FR4 respectively) and said three complementarity determining regions CDR1 to CDR3. For example, an ISV that specifically binds to aggrecan consists of four framework regions (FR1 to FR4 respectively) and said three complementarity determining regions CDR1 to CDR3; a therapeutic ISV, for example, an ISV that binds to a member of the serine protease family, cathepsin, matrix metalloproteinase (MMP) / matrilysin or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and / or ADAMTS11 consists of four framework regions (FR1 to FR4 respectively) and said three complementarity determining regions CDR1 to CDR3; an ISV that binds to serum albumin consists essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively).

[0208] The aglycan binders of the present invention, such as the ISV and / or polypeptides of the present invention, may also contain certain mutations / amino acid residues described in the following co-pending U.S. provisional applications all entitled "Improved immunoglobulin variable domains": US 61 / 994,552 filed on May 16, 2014; US 61 / 014,015 filed on Jun. 18, 2014; US 62 / 040,167 filed on Aug. 21, 2014; and US 62 / 047,560 filed on Sep. 8, 2014 (all assigned to Ablynx N.V.).

[0209] In particular, the aglycan binders of the present invention, such as the ISV and / or polypeptides of the present invention, may preferably contain: (i) K or Q at position 112; or (ii) K or Q at position 110 in combination with V at position 11; or (iii) T at position 89; or (iv) L at position 89 and K or Q at position 110; or (v) V at position 11 and L at position 89; or any suitable combination of (i) to (v).

[0210] As also described in the above co-pending U.S. provisional applications, when the aglycan binders of the present invention, such as the ISV and / or polypeptides of the present invention, contain a mutation (or a suitable combination thereof) according to one of (i) to (v) above: - The amino acid residue at position 11 is preferably selected from L, V or K (and most preferably V); and - The amino acid residue at position 14 is preferably suitably selected from A or P; and - The amino acid residue at position 41 is preferably suitably selected from A or P; and - The amino acid residue at position 89 is preferably suitably selected from T, V or L; and - The amino acid residue at position 108 is preferably suitably selected from Q or L; and - The amino acid residue at position 110 is preferably suitably selected from T, K or Q; and - The amino acid residue at position 112 is preferably suitably selected from S, K or Q.

[0211] As described in the above-mentioned co-pending US provisional application, said mutations are effective in preventing or reducing the binding of so-called "existing antibodies" to the ISVs, polypeptides and constructs of the present invention. For this purpose, the aggrecan binders of the present invention, such as the ISVs and / or polypeptides of the present invention, may also optionally (optionally in combination with said mutations) have a C-terminal extension (X) n (wherein n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)), for which reference is again made to said US provisional application as well as WO 12 / 175741. In particular, the aggrecan binders of the present invention, such as the ISVs and / or polypeptides of the present invention, may contain such a C-terminal extension when it forms the C-terminal end of a protein, polypeptide or other compound or construct containing it (again, as further described in said US provisional application as well as WO 12 / 175741).

[0212] The aggrecan binders of the present invention may be immunoglobulins, such as ISVs, derived from any suitable source in any suitable manner, for example, naturally occurring V HHAn array (i.e., from a suitable species of camelid), which may be a synthetic or semi-synthetic amino acid sequence, which may be, but is not limited to, a "humanized" (as defined herein) nanobody or VHH sequence, a "camelized" (as defined herein) immunoglobulin sequence (and in particular, a camelized heavy chain variable domain sequence), and affinity maturation (starting, for example, from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, techniques such as PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences well known to those skilled in the art, to obtain nanobodies; or any suitable combination of any of the foregoing as further described herein. Also, if the immunoglobulin contains a V HH sequence, the immunoglobulin may be suitably humanized, as further described herein, to provide one or more additional (partially or fully) humanized immunoglobulins of the invention. Similarly, if the immunoglobulin contains a synthetic or semi-synthetic sequence (such as a partially humanized sequence), the immunoglobulin may optionally be further suitably humanized, as described herein again, to provide one or more additional (partially or fully) humanized immunoglobulins of the invention.

[0213] In one aspect, the invention provides an aggrecan binder of the invention, such as ISV, selected from the group consisting of SEQ ID NOs: 117, 118, 116, 115, 114 and 1-19.

[0214] The ISV can be used as a "building block" for the preparation of polypeptides, which may optionally include one or more additional "building blocks", such as ISVs, for example those having a therapeutic mechanism of action, such as therapeutic ISVs, against the same or different epitopes on the aggrecan and / or against one or more other antigens, proteins or targets other than the aggrecan.

[0215] Generally, a protein or polypeptide or construct that contains or consists essentially of a single building block, a single ISV or a single nanobody will be referred to herein as a "monovalent" protein or polypeptide or "monovalent construct", respectively. A polypeptide or construct that contains two or more building blocks or binding units (such as ISVs, etc.) will also be referred to herein as a "multivalent" polypeptide or construct, and the building blocks / ISVs present in such a polypeptide or construct will also be referred to herein as being in "multivalent form". For example, a "bivalent" polypeptide may contain an array of two ISVs optionally linked via a linker, while a "trivalent" polypeptide may contain three ISVs optionally linked via two linker arrays; while a "tetravalent" polypeptide may contain four ISVs optionally linked via three linker arrays, etc.

[0216] In a multivalent polypeptide or construct, two or more ISVs, such as nanobodies, may be the same or different and may be directed against the same antigen or antigenic determinant (e.g., against the same moiety(ies) or epitope(s), or against different moieties or epitopes), or against different antigens or antigenic determinants; or any suitable combination thereof. A polypeptide or construct comprising at least two components (e.g., ISVs etc.) in which at least one component is directed against a first antigen (i.e., an aglycan) and at least one component is directed against a second antigen (i.e., something different from the aglycan, such as a therapeutic target etc.) is also referred to as a “multispecific” polypeptide or multispecific construct respectively, and the components (e.g., ISVs etc.) present in such a polypeptide or construct will also be referred to herein as being in “multispecific form”. Thus, for example, a “bispecific” polypeptide of the invention is a polypeptide comprising at least one ISV directed against a first antigen (i.e., an aglycan) and at least one further ISV directed against a second antigen (i.e., something different from the aglycan, such as a therapeutic target etc.), while a “trispecific” polypeptide of the invention is a polypeptide comprising at least one ISV directed against a first antigen (i.e., an aglycan), at least one further ISV directed against a second antigen (i.e., something different from the aglycan, such as a therapeutic target etc.), and at least one further ISV directed against a third antigen (i.e., something different from both the aglycan and the second antigen); and so on.

[0217] "Multiparatopic" polypeptides and "multiparatopic" constructs, such as "biparatopic" polypeptides or constructs and "triparatopic" polypeptides or constructs, each contain or consist essentially of two or more components having different paratopes.

[0218] Thus, the ISVs of the invention that bind to aggrecan may be in a substantially isolated form (as defined herein), or they may form part of a construct or polypeptide, which may contain or consist essentially of one or more ISVs that bind to aggrecan, and which may optionally further contain one or more additional amino acid sequences (all optionally linked via one or more suitable linkers). The invention relates to a polypeptide or construct that binds to aggrecan and contains or consists essentially of at least one ISV according to the invention, such as one or more ISVs of the invention (or suitable fragments thereof).

[0219] One or more of the ISVs of the present invention can be used as binding units or components in such polypeptides or constructs to provide, respectively, monovalent, multivalent or multi-paratope type polypeptides or constructs of the present invention, all as described herein. The present invention thus also relates to polypeptides which are monovalent constructs, comprising or consisting essentially of one monovalent polypeptide or ISV of the present invention. The present invention thus also relates to polypeptides or constructs which are respectively multivalent polypeptides or multivalent constructs, such as, for example, divalent or trivalent polypeptides or constructs comprising or consisting essentially of two or more ISVs of the present invention (for multivalent and multispecific polypeptides comprising one or more VHH domains and their preparation, reference is also made, for example, to Conrath et al. (J. Biol. Chem. 276: 7346-7350, 2001) and to, for example, WO 96 / 34103, WO 99 / 23221 and WO 2010 / 115998).

[0220] The present invention further relates to multivalent polypeptides (also herein referred to as "the multivalent polypeptide(s) of the present invention"), comprising or consisting (essentially) of at least one ISV, for example one or two ISVs (or suitable fragments thereof), directed against aggrecan, preferably human aggrecan, and one further ISV.

[0221] In one aspect, in its most simplistic form, the multivalent polypeptide or construct of the present invention is a bivalent polypeptide or construct of the present invention comprising a first ISV, such as a nanobody, directed against an aggrecan and an identical second ISV, such as a nanobody, directed against an aggrecan, wherein the first and second ISVs, such as nanobodies, may optionally be linked via a linker sequence (as defined herein). In another form, the multivalent polypeptide or construct of the present invention is a trivalent polypeptide or construct of the present invention comprising a first ISV, such as a nanobody, directed against an aggrecan, an identical second ISV, such as a nanobody, directed against an aggrecan, and a third ISV, such as a nanobody, directed against an antigen different from the aggrecan, such as a therapeutic target, wherein the first, second, and third ISVs, such as nanobodies, may optionally be linked via one or more, particularly two, linker sequences.

[0222] In another aspect, the multivalent polypeptide or construct of the present invention may be a bispecific polypeptide or construct of the present invention comprising a first ISV, such as a nanobody, directed against an aggrecan and a second ISV, such as a nanobody, directed against a second antigen, such as a therapeutic target, wherein the first and second ISVs, such as nanobodies, may optionally be linked via a linker sequence (as defined herein); while the multivalent polypeptide or construct of the present invention may also be a trispecific polypeptide or construct of the present invention comprising a first ISV, such as a nanobody, directed against an aggrecan, a second ISV, such as a nanobody, directed against a second antigen, such as a therapeutic target, and a third ISV, such as a nanobody, directed against a third antigen, such as a therapeutic target, different from the second antigen, wherein the first, second, and third ISVs, such as nanobodies, may optionally be linked via one or more, particularly two, linker sequences.

[0223] In a preferred aspect, the polypeptide or construct of the invention is, respectively, a trivalent bispecific polypeptide or construct. The trivalent bispecific polypeptide or construct of the invention, in its simplest form, comprises two identical ISVs, such as nanobodies, against aggrecan, and a third ISV, such as a nanobody, directed against another antigen, such as a therapeutic target, of the trivalent polypeptide or construct of the invention (as defined herein), where the first, second, and third ISVs, such as nanobodies, may optionally be linked via one or more, in particular two, linker sequences.

[0224] In a preferred aspect, the polypeptide or construct of the invention is, respectively, a trivalent bispecific polypeptide or construct. The trivalent bispecific polypeptide or construct of the invention comprises two ISVs, such as nanobodies, against aggrecan (where the ISVs against aggrecan may be the same or different), and a third ISV, such as a nanobody, directed against another antigen, such as a therapeutic target, of the trivalent polypeptide or construct of the invention (as defined herein), where the first, second, and third ISVs, such as nanobodies, may optionally be linked via one or more, and in particular two, linker sequences. Particularly preferred trivalent bispecific polypeptides or constructs according to the invention are those described in the examples herein and shown in Tables E-1 and E-2.

[0225] In another aspect, the polypeptide of the invention is a bispecific polypeptide or construct. The bispecific polypeptide or construct of the invention, in its simplest form, is a bivalent polypeptide or construct of the invention (as defined herein) that includes an ISV against aggrecan, such as a nanobody, and a second ISV, such as a nanobody, directed against another antigen, such as a therapeutic target, wherein the first and second ISVs, such as nanobodies, may be sequences optionally linked via a linker.

[0226] In a preferred aspect, the multivalent polypeptide or construct of the invention comprises or consists essentially of two or more ISVs directed against aggrecan. In one aspect, the invention relates to a polypeptide or construct that comprises or consists essentially of at least two ISVs (or suitable fragments thereof) that bind to at least two aggrecans according to the invention, such as 2, 3 or 4 ISVs. The two or more ISVs may optionally be linked via one or more peptidic linkers.

[0227] The two or more ISVs present in the multivalent polypeptide or construct of the invention may consist of light chain variable domain sequences (e.g., V L sequences), or of heavy chain variable domain sequences (e.g., V H sequences); they may consist of heavy chain variable domain sequences derived from conventional 4-chain antibodies or from heavy chain antibodies. In a preferred aspect, they may consist of domain antibodies (or amino acids suitable for use as domain antibodies), single domain antibodies (or amino acids suitable for use as single domain antibodies), "dAbs" (or amino acids suitable for use as dAbs), nanobodies® (including, but not limited to, V HH ), humanized V HH sequences, camelized V H sequences; or V obtained by affinity maturationHH It consists of arrays. Two or more ISVs may consist of partially or fully humanized nanobodies or partially or fully humanized VHHs.

[0228] In one aspect of the invention, the first ISV and the second ISV present in the polypeptide or construct of the invention of the multi-paratope type (preferably biparatope type or triparatope type) do not (cross) compete with each other for binding to aggrecan, and thus belong to different families. Accordingly, the present invention relates to a multi-paratope type (preferably biparatope type) polypeptide or construct comprising two or more ISVs, wherein each ISV belongs to a different family. In one aspect, the first ISV of this multi-paratope type (preferably biparatope type) polypeptide or construct of the invention does not cross-block the binding of the second ISV of this multi-paratope type (preferably biparatope type) polypeptide or construct of the invention to aggrecan, and / or the first ISV is not cross-blocked from binding to aggrecan by the second ISV. In another aspect, the first ISV of the multi-paratope type (preferably biparatope type) polypeptide or construct of the invention cross-blocks the binding of the second ISV of this multi-paratope type (preferably biparatope type) polypeptide or construct of the invention to aggrecan, and / or the first ISV is cross-blocked from binding to aggrecan by the second ISV.

[0229] In a preferred aspect, the polypeptide or construct of the present invention comprises or consists essentially of two or more ISVs, and at least one of these ISVs is directed against an agrin. In a particularly preferred aspect, the polypeptide or construct of the present invention comprises or consists essentially of three or more ISVs, and at least two of these ISVs are directed against an agrin. It will be understood that the at least two ISVs directed against the agrin may be the same or different, may be directed against the same epitope or different epitopes of the agrin, may belong to the same epitope bin or different epitope bins, and / or may bind to the same domain or different domains of the agrin.

[0230] In a preferred aspect, the polypeptide or construct of the present invention comprises or consists essentially of at least two ISVs, where the at least two ISVs may be the same or different, and they are independently selected from the group consisting of SEQ ID NO: 117, 118, 116, 115, and 1 to 19, more preferably, the at least two ISVs are selected from the group consisting of SEQ ID NO: 117, 5, 6, 8, 114 to 116, and / or the at least two ISVs are selected from the group consisting of SEQ ID NO: 118 and 13.

[0231] In a further aspect, the present invention relates to a multi-paratope type (preferably biparatope type) polypeptide or construct comprising two or more immunoglobulin single variable domains directed against an agrin and binding to the same epitope(s) as bound by any one of SEQ ID NO: 117, 118, 114, 115, 116, and 1 to 19.

[0232] The final form of the molecule for clinical use is understood to comprise one or two components that bind to aggrecan, such as an ISV, and one or more components that have a mode of therapeutic action, such as an ISV, and possibly additional moieties. In the examples section, it is shown that such forms retain both the properties of aggrecan binding and retention, as well as a therapeutic effect, such as an enzymatic and / or inhibitory function. One or more components that have a mode of therapeutic action, such as an ISV, may be any component that has a therapeutic effect in a disease in which aggrecan is involved, such as an arthritis disease, osteoarthritis, spondyloepiphyseal dysplasia, lumbar intervertebral disc degeneration disease, osteoarthritic disease, rheumatoid arthritis, osteochondritis dissecans, aggrecanopathies, where aggrecan directs, anchors, and / or retains other, e.g., therapeutic, components at a desired site (such as in a joint, etc.) (a "therapeutic component" or "therapeutic ISV"). The invention thus relates to a polypeptide or construct according to the invention in which one or more additional components, such as additional ISVs, retain activity.

[0233] The invention relates to a polypeptide or construct comprising or consisting essentially of at least one ISV according to the invention that binds to aggrecan, such as one or more ISVs of the invention (or suitable fragments thereof), and at least one additional ISV, particularly a therapeutic ISV, wherein said at least one additional ISV preferably binds to a therapeutic target, such as a member of the serine protease family, cathepsin, matrix metalloproteinase (MMP) / matrilysin or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and / or ADAMTS11.

[0234] In one aspect, the invention relates to a polypeptide or construct of the invention that consists essentially of, or comprises, at least one ISV that binds to aggrecan and at least one additional ISV having a therapeutic effect, such as a therapeutic component. The therapeutic effect can be any desired effect that alleviates, treats, or prevents a disease that will be described in more detail below. Preferably, the additional ISV, such as a therapeutic ISV, inhibits or reduces protease activity, e.g., of a therapeutic target, i.e., a member of the serine protease family, cathepsin, matrix metalloproteinase (MMP) / matrilysin, or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1), and / or ADAMTS11, by inhibiting or reducing its activity. Inhibiting or reducing activity can be achieved by binding to the protease or the active site of the protease or by modifying its structure to prevent and / or reduce the hydrolysis of the target protein of the protease or protease.

[0235] In one aspect, the invention relates to a polypeptide or construct of the invention selected from the polypeptides and constructs of Tables E-1 and E-2. In one aspect, the invention relates to an ISV, polypeptide, or construct of the invention having stability of at least 7 days, such as 14 days, 21 days, 1 month, 2 months, or even 3 months, at 37°C in synovial fluid (SF). In one aspect, the invention relates to an ISV, polypeptide, or construct of the invention having cartilage retention of at least 2 RU, such as at least 3, 4, 5, or 6 RU, in a cartilage retention assay. In one aspect, the present invention relates to the ISV, polypeptide or construct of the present invention that penetrates into cartilage, at least 5 μm, such as at least 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, or even more.

[0236] The stability of the polypeptide, construct or ISV of the present invention can be measured by conventional assays known to those skilled in the art. As a typical assay (without limitation), for example, as detailed again in the Examples section (see Example 6), an assay in which the activity of the polypeptide, construct or ISV is determined, then incubated in synovial fluid for a desired period, and then the activity is determined.

[0237] The desired activity of the therapeutic component in the multivalent polypeptide or construct of the present invention can be measured by conventional assays known to those skilled in the art. As a typical assay, an assay in which GAG release is assayed, as detailed in the Examples section (see Example 8), can be mentioned.

[0238] The relative affinity may depend on the position of the ISVD in the polypeptide. The order (orientation) of the ISVD in the polypeptide of the present invention can be selected according to the needs of those skilled in the art. The order of the individual ISVDs, as well as whether the polypeptide contains a linker, is a matter of design choice. Some orientations with or without a linker may provide favorable binding characteristics compared to other orientations. For example, the order of the first ISV (e.g., ISV1) and the second ISV (e.g., ISV2) in the polypeptide of the present invention may be as follows (from the N-terminus to the C-terminus): (i) ISV1 (e.g., nanobody 1)-[linker]-ISV2 (e.g., nanobody 2)-[C-terminal extension]; or (ii) ISV2 (e.g., nanobody 2)-[linker]-ISV1 (e.g., nanobody 1)-[C-terminal extension]; (where the part between the square brackets, i.e., the linker and the C-terminal extension, is optional). All orientations are encompassed by the present invention. Polypeptides containing the orientation of the ISV that provides the desired binding characteristics can be readily identified, for example, by conventional screening as exemplified in the Examples section. Preferred orders are, from the N-terminus to the C-terminus, as follows: therapeutic ISV-[linker]-ISV that binds to aggrecan-[C-terminal extension], where the part between the square brackets is optional. Another preferred order is, from the N-terminus to the C-terminus, as follows: therapeutic ISV-[linker]-ISV that binds to aggrecan-[linker]-ISV that binds to aggrecan-[C-terminal extension], where the part between the square brackets is optional.

[0239] The aggrecan binders of the present invention, such as the polypeptides and / or ISVs of the present invention, may or may not further contain one or more other groups, residues (such as amino acid residues), moieties or linking units (all of these aggrecan binders, such as polypeptides and / or ISVs (with or without further groups, residues, moieties or linking units) are all referred to as "compound(s) of the present invention", "construct(s) of the present invention" and / or "polypeptide(s) of the present invention"). When present, such further groups, residues, moieties or linking units may or may not provide additional functionality to the aggrecan binder such as a polypeptide and / or ISV, and may or may not modify the properties of the aggrecan binder such as a polypeptide and / or ISV.

[0240] For example, such additional groups, residues, moieties or linking units may be one or more additional amino acid sequences, in which case the resulting polypeptide is a (fusion) polypeptide. In a preferred and non-limiting aspect, the one or more other groups, residues, moieties or linking units are immunoglobulins. Even more preferably, the one or more other groups, residues, moieties or linking units are ISVs selected from the group consisting of domain antibodies, amino acids suitable for use as domain antibodies, single domain antibodies, amino acids suitable for use as single domain antibodies, dAbs, amino acids suitable for use as dAbs, nanobodies (such as, for example, VHH, humanized VHH or camelized VH sequences).

[0241] As described above, additional linking units, such as ISVs having different antigen specificities, can be linked to form multi-specific polypeptides. By linking two or more ISVs with specificities, polypeptides or constructs with dual specificity, triple specificity, etc. can be formed. For example, the polypeptide of the present invention may include two or more ISVs against aggrecan and at least one ISV against another target. All such constructs and their modifications are included in the terms used herein, "compounds of the present invention, constructs of the present invention, and / or polypeptides of the present invention" (as can be easily envisioned by those skilled in the art).

[0242] In the above-mentioned compounds, constructs, and / or polypeptides, one, two, three, or more ISVs, and one or more groups, residues, moieties, or linking units may each be linked directly and / or via one or more appropriate linkers or spacers. For example, when one or more groups, residues, moieties, or linking units are amino acid sequences, the linker may also be an amino acid sequence, and as a result, the resulting polypeptide will be a fusion (protein) or fusion (polypeptide).

[0243] One or more additional groups, residues, moieties, or linking units may be any suitable and / or desired amino acid sequence. The additional amino acid sequence may or may not change the (biological) properties of the polypeptide of the present invention, may or may not affect it, and may or may not confer additional functionality to the polypeptide of the present invention. Preferably, the additional amino acid sequence is one that confers one or more desired properties or functionality to the polypeptide of the present invention.

[0244] Examples of such amino acid sequences will be apparent to those skilled in the art and generally may include any amino acid sequence (including, but not limited to, ScFv and single domain antibodies) that is commonly used in peptide fusions based on normal antibodies and fragments thereof. References include, for example, the review by Holliger and Hudson (Nature Biotechnology 23: 1126-1136, 2005).

[0245] For example, such amino acid sequences may increase half-life, solubility or absorption, decrease immunogenicity or toxicity, eliminate or reduce undesirable side effects, and / or confer other advantageous properties, and / or decrease undesirable properties of the compounds, constructs or polypeptides of the present invention, as compared to the polypeptide of the present invention itself. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see, for example, WO 00 / 27435) or hapten molecules (such as haptens recognized by circulating antibodies, see, for example, WO 98 / 22141).

[0246] In certain aspects of the invention, the constructs or polypeptides of the invention may have a portion that confers an extended half-life as compared to the corresponding constructs or polypeptides of the invention that do not contain such portion. 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 further disclosure herein and include, for example: an ISV or polypeptide of the invention that is chemically modified (e.g., by pegylation) to extend its half-life; an aggrecan binder of the invention, such as an ISV and / or polypeptide of the invention, that contains at least one additional binding site for binding to a serum protein (such as serum albumin); or a polypeptide of the invention that contains at least one amino acid sequence of the invention that is linked to at least one portion (and in particular at least one amino acid sequence) that extends the half-life of the amino acid sequence of the invention.Examples of constructs of the invention, such as polypeptides of the invention, comprising such a half-life extension moiety or ISV, will be apparent to those skilled in the art based on further disclosure herein; and include, by way of example and without limitation: polypeptides in which one or more ISVs of the invention are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof), or to a binding unit capable of binding to one or more serum proteins (for example, a serum protein such as serum albumin (such as human serum albumin), a serum immunoglobulin such as IgG, or transferrin, a domain antibody, an ISV suitable for use as a domain antibody, a single domain antibody, an ISV suitable for use as a single domain antibody, a dAb, an ISV suitable for use as a dAb, or a nanobody, etc.; further description and reference are made to the references described herein); polypeptides in which the amino acid sequence of the invention is linked to an Fc moiety (such as human Fc) or a suitable moiety or fragment thereof; or polypeptides in which one or more ISVs of the invention are suitably linked to a small protein or peptide capable of binding to one or more serum proteins, such as the proteins and peptides described in WO 91 / 01743, WO 01 / 45746, WO 02 / 076489, WO2008 / 068280, WO2009 / 127691 and PCT / EP2011 / 051559.

[0247] In one aspect, the invention provides a construct of the invention, such as a polypeptide, wherein the polypeptide further comprises a serum protein binding moiety or a serum protein. Preferably, the serum protein binding moiety binds to serum albumin, such as human serum albumin.

[0248] In general, the constructs or polypeptides of the present invention having an 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 more than 20-fold longer than the half-life of the corresponding construct or polypeptide of the present invention itself, i.e., the one that does not contain the part that brings about the extended half-life. For example, the construct or polypeptide of the present invention with an extended half-life may have a half-life that is, for example, in humans, longer than 1 hour, preferably longer than 2 hours, more preferably longer than 6 hours, such as longer than 12 hours, or even longer than 24, 48 or 72 hours, compared to the corresponding construct or polypeptide of the present invention itself, i.e., the one that does not contain the part that brings about the extended half-life.

[0249] In a preferred but non-limiting aspect of the present invention, the construct of the present invention, such as the polypeptide of the present invention, has a serum half-life that is extended longer than 1 hour, preferably longer than 2 hours, more preferably longer than 6 hours, such as longer than 12 hours, or even longer than 24, 48 or 72 hours, compared to the corresponding construct or polypeptide of the present invention itself, i.e., the one without the part that brings about the extended half-life, for example, in humans.

[0250] In another preferred but non-limiting aspect of the present invention, such a construct of the present invention, such as the polypeptide of the present invention, exhibits a 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 longer in humans. For example, the compound or polypeptide of the present invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), even more preferably at least about 12 days (such as about 12 to 18 days or longer), or longer than 14 days (such as about 14 to 19 days).

[0251] In a particularly preferred but non-limiting aspect of the invention, the invention provides, in addition to one or more components that bind to aglycan and optionally one or more therapeutic components, at least one component that binds to serum albumin, such as an ISV that binds to serum albumin (such as human serum albumin), as described herein, a construct of the invention, such as a polypeptide of the invention, wherein the ISV that binds to the serum albumin comprises or consists essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), wherein CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR. Preferably, the ISV that binds to the human serum albumin is selected from the group consisting of Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG, Alb92 or Alb223 (see Table C).

[0252] In one aspect, the invention relates to a construct of the invention, such as a polypeptide, comprising a serum protein binding moiety, wherein the serum protein binding moiety is a non-antibody-based polypeptide.

[0253] In one aspect, the invention relates to a compound or construct as described herein, comprising one or more other groups, residues, moieties or linking units, which are preferably selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, linking units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins.

[0254] In one aspect, the invention relates to a construct of the invention, such as a polypeptide, comprising a moiety that results in an extended half-life, where said moiety is PEG. Accordingly, the invention relates to a construct or polypeptide of the invention that comprises PEG.

[0255] Additional amino acid residues may or may not change, modify, or otherwise affect other (biological) properties of the polypeptides of the invention, and may or may not add additional functionality to the polypeptides of the invention. For example, such amino acid residues may be: a) Met residues, for example, as a result of expression in a heterologous host cell or host organism, may include an N-terminal Met residue; b) form a signal sequence or leader sequence that directs secretion of the polypeptide from the host cell after synthesis (e.g., to provide a pre-form, pro-form, or prepro-form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention). Suitable secretory leader peptides will be apparent to those skilled in the art and may be further described herein. Usually, such a leader sequence is linked to the N-terminus of the polypeptide, but the invention is not limited thereto in its broadest sense; c) form a "tag", such as an amino acid sequence or residue that enables or facilitates purification of the polypeptide, using, for example, affinity techniques directed against said sequence or residue. Subsequently, said sequence or residue can be removed (e.g., by chemical or enzymatic cleavage) to provide the polypeptide (for this purpose, the tag may optionally be linked to the amino acid sequence or polypeptide sequence via a cleavable linker sequence or may contain a cleavable motif). Some preferred but non-limiting examples of such residues are multiple histidine residues, glutathione residues, and myc tags (such as AAAEQKLISEEDLNGAA); d) may be one or more amino acid residues that are functionalized and / or can serve as sites for the addition of functional groups. Suitable amino acid residues and functional groups will be apparent to those skilled in the art and include, but are not limited to, the amino acid residues and functional groups for derivatives of the polypeptides of the invention described herein.

[0256] In the constructs of the invention, for example the polypeptides of the invention, two or more components, such as ISVs etc., and optionally one or more other groups, drugs, agents, residues, moieties or linking units may be directly linked to each other (as described in WO 99 / 23221, for example), and / or may be linked to each other via one or more suitable spacers or linkers, or may be any combination thereof. Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be apparent to those skilled in the art and may generally be any linker or spacer used in the art for linking amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing constructs, proteins or polypeptides intended for pharmaceutical use.

[0257] For example, the polypeptide of the present invention may be a trivalent, trispecific polypeptide comprising, for example, one component (such as an ISV) that binds to aggrecan, one therapeutic component (such as an ISV), and one component (such as an ISV) that binds to (human) serum albumin, wherein the first, second, and third components (such as ISVs) may optionally be linked via one or more, particularly two, linker sequences. Further, the present invention provides a construct or polypeptide of the present invention comprising a first ISV and / or a second ISV that binds to aggrecan and / or optionally a third ISV and / or optionally an ISV that binds to serum albumin, wherein the first ISV and / or the second ISV and / or optionally the third ISV and / or optionally the ISV that binds to serum albumin are linked via a linker.

[0258] Some particularly preferred spacers include spacers and linkers used in the art to link antibody fragments or antibody domains. These include the linkers described in the general background art cited above, as well as linkers used in the art to construct, for example, diabodies or ScFv fragments (in this regard, however, in diabodies and in ScFv fragments, the linker sequences used should have a length, degree of flexibility, and other properties that allow the associated V H and V L domains to assemble to form a complete antigen-binding site, but it should be noted that there are no particular limitations on the length or flexibility of the linkers used in the polypeptides of the present invention, since each ISV (such as a nanobody) forms a complete antigen-binding site by itself).

[0259] For example, the linker may be a suitable amino acid sequence, and in particular, an amino acid sequence of 1 to 50, preferably 1 to 30, for example 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, such as (gly x ser y ) z type, such as (gly4ser)3 or (gly3ser2)3 as described in, for example, WO 99 / 42077, and GS30, GS15, GS9 and GS7 linkers (see, for example, WO 06 / 040153 and WO 06 / 122825) described in the applications by Ablynx described herein, and hinge-like regions, such as the hinge region of a naturally occurring heavy chain antibody or a similar sequence (such as those described in WO 94 / 04678). Preferred linkers are represented in Table D (SEQ ID NOs: 154 to 170).

[0260] Other suitable linkers generally include organic compounds or polymers, particularly those suitable for use in proteins for pharmaceutical applications. For example, poly(ethylene glycol) moieties have been used to link antibody domains; see, for example, WO 04 / 081026.

[0261] The length, degree of flexibility, and / or other properties (which are not critical but are usually those for linkers used in ScFv fragments) of the linker(s) used may have some effect on the properties of the final construct of the invention, for example, the properties of the polypeptide of the invention (including, but not limited to, affinity, specificity or avidity for chemokines or one or more other antigens), which is encompassed within the scope of the invention. Based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linker(s) for use in a particular construct of the invention, for example, the polypeptide of the invention, optionally after some limited routine experimentation.

[0262] For example, in the multivalent polypeptides of the invention that include, for example, agricans and components, ISVs or nanobodies directed to another target, the length and flexibility of the linker is preferably such that each component (such as an ISV) of the invention is present in the polypeptide and can bind to its cognate target (e.g., an antigenic determinant on each of the targets). Also, based on the disclosure herein, one of ordinary skill 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 some limited routine experimentation.

[0263] It is also within the scope of the invention for the linker(s) used to impart one or more other advantageous properties or functions to a construct of the invention, such as a polypeptide of the invention, and / or to provide one or more sites for the formation of derivatives and / or for the addition of functional groups (e.g., as described herein for derivatives of the ISVs of the invention). For example, a linker containing one or more charged amino acid residues can provide improved hydrophilic properties, while a linker that forms or contains a small epitope or tag can be used for detection, identification and / or purification purposes. Also, based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linker for use in a particular polypeptide of the invention, optionally after some limited routine experimentation.

[0264] Finally, when two or more linkers are used in a construct such as a polypeptide of the invention, these linkers may be the same or different. Also, based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linker for use in a particular construct or polypeptide of the invention, optionally after some limited routine experimentation.

[0265] Typically, for ease of expression and production, the constructs of the present invention, such as the polypeptides of the present invention, will be linear polypeptides. However, the present invention is, in the broadest sense, not limited thereto. For example, when the constructs of the present invention, such as the polypeptides of the present invention, contain three or more components, ISVs or nanobodies, they can be linked by using a linker having three or more "arms", and each of the "arms" is linked to a component, ISV or nanobody so as to provide a "star-shaped" construct. Also, although usually less preferred, it is also possible to use cyclic constructs.

[0266] Accordingly, the present invention relates to constructs of the present invention, such as polypeptides of the present invention, wherein the ISVs are directly linked to each other or are linked via a linker. Accordingly, the present invention relates to constructs of the present invention, such as polypeptides of the present invention, wherein the first ISV and / or the second ISV and / or optionally the ISV that binds to serum albumin are linked via a linker.

[0267] Accordingly, the present invention relates to constructs of the present invention, such as polypeptides of the present invention, wherein the linker is selected from the group consisting of linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, 35GS, poly-A, 8GS, 40GS, G1 hinge, 9GS-G1 hinge, llama upper long hinge region and G3 hinge. Accordingly, the present invention relates to constructs of the present invention, such as polypeptides of the present invention, wherein the polypeptide is selected from the group consisting of the polypeptides of Table E-1 and Table E-2.

[0268] Compounds, constructs and / or polypeptides comprising the ISVs or polypeptides of the present invention that further contain tags or other functional moieties (such as toxins, labels, radiochemicals, etc.) are also encompassed by the present invention.

[0269] Other groups, residues, moieties or linking units may be, for example, chemical groups, residues or moieties that are either biologically and / or pharmacologically active or inactive alone. For example, but not limited to, such groups may form "derivatives" of the polypeptides of the present invention when linked to one or more ISVs or polypeptides of the present invention.

[0270] Accordingly, the present invention most broadly also encompasses compounds, constructs and / or polypeptides that are derivatives of the polypeptides of the present invention. Such derivatives can generally be obtained by one or more modifications in the polypeptides of the present invention and / or in one or more of the amino acid residues forming the polypeptides of the present invention, specifically by chemical and / or biological (e.g., enzymatic) modifications.

[0271] Examples of such modifications, as well as examples of amino acid residues in the polypeptide sequences that can be modified by such modifications and the modes, methods and techniques that can be used to introduce such modifications and their potential uses and advantages, will be apparent to those skilled in the art (see Zangi et al., Nat Biotechnol 31(10):898-907, 2013).

[0272] For example, such modifications may involve the introduction of one or more functional groups, residues or moieties, particularly one or more (functional) groups, residues or moieties, into the interior and surface of the polypeptides of the present invention (e.g., by covalent linkage or any other suitable method), thereby conferring one or more desired properties or functionalities on the polypeptides of the present invention. Examples of such functional groups will be apparent to those skilled in the art.

[0273] For example, such modifications may involve the introduction of one or more functional groups (e.g., by covalent linkage or any other suitable method), thereby increasing the half-life, solubility and / or absorbability of the polypeptide of the present invention, decreasing the immunogenicity and / or toxicity of the polypeptide of the present invention, eliminating or reducing any undesirable side effects of the polypeptide of the present invention, and / or conferring other advantageous properties, and / or decreasing the undesirable properties of the polypeptide of the present invention, or resulting in any combination of two or more of the foregoing. Examples of such functional groups and the techniques for introducing them will be apparent to those skilled in the art and typically include any of the functional groups and techniques described in the general prior art cited above, as well as functional groups and known techniques for the modification of pharmaceutical proteins, particularly for the modification of antibodies or antibody fragments (including ScFv and single domain antibodies). By way of reference, for example, Remington (Pharmaceutical Sciences, 16 th ed., Mack Publishing Co., Easton, PA, 1980) may be mentioned. Such functional groups may be directly linked (e.g., covalently) to the polypeptide of the present invention, or optionally linked via a suitable linker or spacer, which will also be apparent to those skilled in the art.

[0274] One specific example is a derivative peptide of the present invention in which the polypeptide of the present invention is chemically modified (e.g., by PEGylation) to increase its half-life. This is one of the most widely used techniques for increasing the half-life and / or reducing the immunogenicity of pharmaceutical proteins, and includes the addition of a suitable pharmacologically acceptable polymer such as poly(ethylene glycol) (PEG) or its derivatives (e.g., methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, for example, the PEGylation used in the fields of antibodies and antibody fragments, such as (single) domain antibodies and ScFv, can be used. References include, 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 International Publication No. 04 / 060965). For example, various reagents for PEGylation of proteins are also commercially available from Nektar Therapeutics (USA).

[0275] Preferably, site-specific PEGylation via cysteine residues in particular is used (see, for example, Yang et al. (Protein Engineering 16: 761-770, 2003)). For example, in this case, it is possible to add PEG to cysteine residues naturally present in the polypeptide of the present invention, and the polypeptide of the present invention may be modified such that one or more cysteine residues for PEG addition are optimally introduced, or an amino acid sequence containing one or more cysteine residues for PEG addition may be fused to the N-terminus and / or C-terminus of the polypeptide of the present invention. In any case, protein engineering techniques known to those skilled in the art can be directly used.

[0276] Preferably, in the case of the polypeptide of the present invention, PEG having a molecular weight of 5000 or more, for example 10,000 or more and less than 200,000, for example less than 100,000, for example in the range of 20,000 to 80,000 is used.

[0277] Another, usually less preferred modification usually involves N-linked or O-linked glycosylation as part of the post-translational and / or post-translational modifications, depending on the host cell used to express the polypeptide of the present invention.

[0278] Depending on the intended use of the marker polypeptide of the present invention, one or more detectable markers or other signal generating groups or moieties may be introduced as yet another modification. Suitable labels and the techniques for attaching, using, and detecting them will be apparent to those skilled in the art and include, for example, but are not limited to, fluorescent markers (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and fluorescent metals such as the lanthanoid series 152Eu or other metals), phosphorescent labels, chemiluminescent labels or bioluminescent labels (luminol, isoluminol, theromatic acridinium esters, imidazole, acridinium salts, oxalate esters, dioxetanes or GFP and its analogs), radioisotopes (e.g., 3 H, 125 I, 32 P, 35 S, 14 C, 51 Cr, 36 Cl, 57 Co, 58 Co, 59 Fe and 75 Se), metals, metal chelates or metal cations (e.g., 99m Tc, 123 I, 111 In, 131 I, 97 Ru, 67 Cu, 67 Ga, 68metal cations such as Ga), or other metals or metal cations (e.g., 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe), as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase). Other suitable labels will be apparent to those skilled in the art and include, for example, moieties detectable using NMR or ESR spectroscopy.

[0279] Such marker polypeptides of the present invention can be used, for example, in in vitro, in vivo, or in situ assays (such as known immunoassays such as ELISA, RIA, EIA, and other "sandwich assays"), and for in vivo diagnostic and imaging purposes (depending on the choice of specific label).

[0280] As will be apparent to those skilled in the art, other modifications may be made, for example, to introduce chelating groups to chelate one of the metals or metal cations mentioned above. Suitable chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0281] As yet another modification, a functional group that is part of a specific binding pair (e.g., biotin-(strept)avidin binding pair) may be introduced. By using such a functional group, the polypeptide of the present invention can be bound to other proteins, polypeptides or chemical substances to which the other half of the binding pair is bound, by so-called formation of the binding pair. For example, the polypeptide of the present invention can be conjugated with biotin and linked to other proteins, polypeptides, compounds or carriers conjugated with avidin or streptavidin. For example, such a conjugated polypeptide of the present invention can be used as a reporter in a diagnostic system in which a detectable signal generator is conjugated to avidin or streptavidin. Such a binding pair can also be used, for example, to bind the polypeptide of the present invention to a carrier such as a carrier suitable for pharmaceutical use. See, for example, the liposome formulation described in Cao and Suresh (Journal of Drug Targeting 8: 257, 2000). Also, a therapeutic active agent may be linked to the polypeptide of the present invention using such a binding pair.

[0282] Other promising chemical and enzymatic modifications will also be apparent to those skilled in the art. Such modifications may be introduced for research purposes (e.g., testing of function-activity relationships). References include, for example, Lundblad and Bradshaw (Biotechnol. Appl. Biochem. 26: 143-151, 1997).

[0283] Preferably, the compound, construct, polypeptide and / or derivative binds to aggrecan by an affinity (optimally, a K D value (actual or apparent), a K A value (actual or apparent), a K on rate and / or a K off rate, or an IC 50 value measured and / or expressed as further described herein). Such compounds, constructs and / or polypeptides of the invention, and derivatives thereof, may be in an essentially isolated form (as defined herein).

[0284] In one aspect, the invention relates to constructs of the invention comprising, or consisting essentially of, an ISV or polypeptide of the invention, and wherein one or more other groups, residues, moieties or linking units are further included, which are optionally linked via one or more peptidic linkers.

[0285] In one aspect, the invention relates to constructs of the invention wherein the one or more other groups, residues, moieties or linking units are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, linking units capable of binding to serum proteins, Fc portions, and small proteins or peptides capable of binding to serum proteins. The invention further relates to methods for preparing the compounds, constructs, polypeptides, nucleic acids, host cells, and compositions described herein.

[0286] The multivalent polypeptides of the invention can generally be prepared by a method comprising at least the step of suitably linking an ISV and / or a monovalent polypeptide of the invention, optionally via one or more suitable linkers, to one or more additional ISVs. The polypeptides of the invention can also be prepared by a method comprising at least the steps of providing a nucleic acid encoding the polypeptide of the invention, expressing the nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be carried out in a manner known per se, which will be apparent to those skilled in the art based, for example, on the methods and techniques further described herein.

[0287] The method for preparing the multivalent polypeptide of the present invention may include at least the step of linking two or more ISVs of the present invention together with, for example, one or more linkers in a suitable manner. The ISVs (and linkers) of the present invention are known in the art and can be coupled by any method further described herein. A preferred technique involves ligating nucleic acid sequences encoding the ISVs (and linkers) of the present invention to prepare a construct of a gene that expresses a multivalent polypeptide. Techniques for ligating amino acids or nucleic acids will be apparent to those skilled in the art, and reference is again made to standard handbooks such as Sambrook et al. and Ausubel et al. mentioned above, as well as the following examples.

[0288] Accordingly, the present invention also relates to the use of the ISVs of the present invention in preparing the multivalent polypeptides of the present invention. The method for preparing a multivalent polypeptide may include linking an ISV of the present invention to at least one additional ISV of the present invention, optionally via one or more linkers. The ISVs of the present invention are then used as binding domains or components in providing and / or preparing a multivalent polypeptide comprising 2 (e.g., in a bivalent polypeptide), 3 (e.g., in a trivalent polypeptide), 4 (e.g., in a tetravalent polypeptide), or more (e.g., in a multivalent polypeptide) components. In this regard, the ISVs of the present invention can be used as binding domains or binding units in providing and / or preparing a multivalent, e.g., bivalent, trivalent or tetravalent polypeptide of the present invention comprising 2, 3, 4 or more components.

[0289] Accordingly, the present invention also relates to the use of the ISV polypeptides of the present invention (as described herein) in preparing multivalent polypeptides. The method for preparing a multivalent polypeptide may include linking an ISV of the present invention to at least one additional ISV of the present invention, optionally via one or more linkers.

[0290] The polypeptides and nucleic acids of the present invention can be prepared using known methods as they are, which will be apparent to those skilled in the art from the further descriptions herein. For example, the polypeptides of the present invention can be prepared using any of the known methods for the preparation of antibodies, specifically, for the preparation of antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments). Some suitable and non-limiting methods for preparing polypeptides and nucleic acids include the methods and techniques described herein.

[0291] A method for producing the polypeptide of the present invention may include the following steps: - expressing in a suitable host cell or host organism (also referred to herein as "the host of the present invention") or in another suitable expression system of the nucleic acid encoding the polypeptide of the present invention (also referred to herein as "the nucleic acid of the present invention"), and then optionally: - isolating and / or generating the polypeptide of the present invention thus obtained. Specifically, such a method may include the following steps: - culturing and / or maintaining the host of the present invention under conditions where the host of the present invention expresses and / or produces at least one polypeptide of the present invention, and then optionally, - isolating and / or purifying the polypeptide of the present invention thus obtained. Accordingly, the present invention also relates to a nucleic acid or nucleotide sequence encoding a polypeptide, ISV or construct of the present invention (also referred to herein as "the nucleic acid of the present invention").

[0292] The nucleic acids of the present invention may be in the form of single-stranded or double-stranded DNA or RNA. According to one aspect of the present invention, the nucleic acids of the present invention are in an essentially isolated form as defined herein. The nucleic acids of the present invention may also be present in and / or be part of a vector, such as an expression vector, such as a plasmid, cosmid or YAC, etc., which may also be in an essentially isolated form. Accordingly, the present invention also relates to an expression vector containing the nucleic acid or nucleotide sequence of the present invention.

[0293] The nucleic acids of the present invention can be prepared or obtained by using known methods as they are, based on the information on the polypeptides of the present invention described herein, and / or can be isolated from suitable natural sources. Also, as will be apparent to those skilled in the art, in order to prepare the nucleic acids of the present invention, several nucleotide sequences, for example, at least two nucleic acids encoding the ISV of the present invention, and for example, nucleic acids encoding one or more linkers can be ligated in a suitable manner. The techniques for generating the nucleic acids of the present invention are apparent to those skilled in the art and include, for example, but not limited to, automated DNA synthesis, site-directed mutagenesis, joining two or more natural and / or synthetic sequences (or two or more portions thereof), introduction of mutations that result in the expression of an incomplete-length expression product, introduction of one or more restriction enzyme sites (for example, creating cassettes and / or regions that can be easily cleaved and / or ligated using appropriate restriction enzymes), and / or introduction of mutations by PCR reactions using one or more "mismatch" primers. These and other techniques are apparent to those skilled in the art and references include the standard handbooks, Sambrook et al. and Ausubel et al. (supra), as well as the following examples.

[0294] In a preferred but non-limiting embodiment, the gene construct of the present invention comprises: a) at least one nucleic acid of the present invention; b) one or more regulatory elements such as a promoter, and optionally a suitable terminator operatively linked; and optionally also, c) one or more further elements of a gene construct known per se; wherein the terms "regulatory element", "promoter", "terminator" and "operatively linked" have their normal meaning in the art.

[0295] The gene construct of the invention can generally be provided by suitably linking the nucleotide sequence(s) of the invention to one or more of the above further elements using techniques described in general handbooks such as, for example, Sambrook et al. and Ausubel et al. mentioned above.

[0296] The nucleic acid of the present invention and / or the gene construct of the present invention can be used for transforming a host cell or a host organism, i.e., for the expression and / or production of the polypeptide of the present invention. Suitable hosts or host cells will be apparent to those skilled in the art and include, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or (non-human) eukaryotic organism, for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be apparent to those skilled in the art, as well as all other host cells or (non-human) hosts known per se. Reference is also made to the general background art cited hereinabove, as well as, for example, WO 94 / 29457; WO 96 / 34103; WO 99 / 42077; Frenken et al. (Res Immunol. 149: 589-99, 1998); Riechmann and Muyldermans (1999), supra; van der Linden (J. Biotechnol. 80: 261-70, 2000); Joosten et al. (Microb. Cell Fact. 2: 1, 2003); Joosten et al. (Appl. Microbiol. Biotechnol. 66: 384-92, 2005); and further references cited herein. Furthermore, the polypeptide of the present invention can also be expressed and / or produced in a cell-free expression system, and suitable examples of such systems will be apparent to those skilled in the art. Suitable techniques for transforming the host or host cell of the present invention will be apparent to those skilled in the art and may depend on the intended host cell / host organism and the gene construct to be used. Again, reference is made to the above-mentioned handbooks and patent applications. The transformed host cell, which may be in the form of a stable cell line, or the host organism, which may be a stable mutant strain or line, forms a further aspect of the present invention. Accordingly, the present invention relates to a host or host cell comprising a nucleic acid according to the present invention, or an expression vector according to the present invention.Preferably, these host cells or host organisms are such that they express, or are capable of expressing (at least), the polypeptide of the invention (and in the case of a host organism, in at least one of its cells, parts, tissues or organs), for example under suitable conditions. The invention also includes further generations, progeny and / or descendants of the host cells or host organisms of the invention, which can be obtained, for example, by cell division or by sexual or asexual reproduction.

[0297] In order to effect / obtain the expression of the polypeptide of the invention, the transformed host cells, or transformed host organisms, are generally maintained, kept and / or cultured under conditions such that the (desired) polypeptide of the invention is expressed / produced. Suitable conditions will be apparent to those skilled in the art and will usually depend on the host cell / host organism used and on the regulatory elements which control the expression of the (appropriate) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications described above in the paragraph on the gene constructs of the invention.

[0298] The polypeptide of the invention can then be isolated from the host cell / host organism and / or from the medium in which the host cell or host organism has been cultured therein, using techniques for the isolation and / or purification of proteins known per se, such as (fractional) chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques (for example using a specific cleavable amino acid sequence fused to the polypeptide of the invention), and / or fractional immunological techniques (i.e. using an antibody against the polypeptide to be isolated).

[0299] In one aspect, the present invention relates to a method for generating a construct, polypeptide or ISV according to the present invention, the method comprising at least the following steps: (a) expressing a nucleic acid sequence according to the present invention in a suitable host cell or host organism or in another suitable expression system; optionally followed by, (b) isolating and / or purifying a construct, polypeptide or ISV according to the present invention. In one aspect, the present invention relates to a composition comprising a construct, polypeptide, ISV or nucleic acid according to the present invention.

[0300] In general, for pharmaceutical use, the constructs, polypeptides and / or ISVDs of the present invention can be formulated as a pharmaceutical preparation or composition comprising at least one construct, polypeptide and / or ISVD of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more pharmaceutically active polypeptides and / or compounds. By way of non-limiting example, such formulations may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (such as by intra-articular administration), for administration by inhalation, by transdermal patch, by graft, by suppository, etc., where intra-articular administration is preferred. Depending on the mode of administration, such suitable dosage forms, which may be solid, semi-solid or liquid, as well as the methods and carriers for use in their preparation, will be apparent to those skilled in the art and are further described herein. Such pharmaceutical preparations or compositions will generally be referred to herein as "pharmaceutical compositions".

[0301] Accordingly, in a further aspect, the present invention relates to a pharmaceutical composition comprising at least at least one construct of the present invention, at least one polypeptide of the present invention, at least one ISV of the present invention, or at least one nucleic acid of the present invention, and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances. In a particular aspect, the present invention relates to a pharmaceutical composition comprising a construct, polypeptide, ISV or nucleic acid according to the present invention, preferably at least one of Tables E-1 or E-2, and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances.

[0302] In general, the constructs, polypeptides, and / or ISVs of the present invention can be formulated and administered in any suitable manner known per se. For example, reference is made to the general background art cited above (and in particular WO 04 / 041862, WO 04 / 041863, WO 04 / 041865, WO 04 / 041867 and WO 08 / 020079), as well as standard handbooks such as Remington’s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins (2005); or Handbook of Therapeutic Antibodies (ed. S. Dubel), Wiley, Weinheim, 2007 (see, for example, pages 252-255).

[0303] In certain aspects, the invention relates to a pharmaceutical composition comprising a construct, polypeptide, ISV or nucleic acid according to the invention, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more further pharmaceutically active polypeptides and / or compounds.

[0304] The constructs, polypeptides, and / or ISVs of the invention can be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing them will be apparent to those skilled in the art and include, for example, formulations suitable for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intracavitary, intraarterial or intrathecal administration) or for topical (e.g., intra-articular, transdermal or intradermal) administration.

[0305] Formulations for parenteral administration can be, for example, sterile solutions, suspensions, dispersions or emulsions suitable for infusion or injection. Suitable carriers or diluents for such formulations include, for example, those described on page 143 of WO 08 / 020079. Usually, an aqueous solution or suspension will be preferred.

[0306] The constructs, polypeptides, and / or ISVs of the invention can also be administered using delivery methods known from gene therapy. See, for example, U.S. Patent No. 5,399,346, which is incorporated by reference for its teachings regarding gene therapy delivery methods. Using gene therapy delivery methods, genes encoding the constructs, polypeptides, and / or ISVs of the invention can be introduced into primary cells that have been transfected with a tissue-specific promoter that targets a particular organ, tissue, graft, tumor, joint or cell, and further, signals and stabilizing sequences for intracellular localization of expression can be introduced.

[0307] The constructs, polypeptides, and / or ISVs of the present invention may also be administered by infusion or injection, intravenously, intra-articularly, or intraperitoneally. Specific examples are as further described on pages 144 and 145 of WO 08 / 020079 or in PCT / EP2010 / 062975 (the entire document). Useful dosages of the constructs, polypeptides, and / or ISVs of the present invention can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art; see, for example, US 4,938,949.

[0308] The amount of the constructs, polypeptides, and / or ISVs of the present invention required for use in treatment will vary not only depending on the particular ISV, polypeptide, compound, and / or construct selected, but also depending on the route of administration, the nature of the condition being treated, the age and condition of the patient, and ultimately will be at the discretion of the attending or treating physician. Also, the dosage of the constructs, polypeptides, and / or ISVs of the present invention will vary depending on the cell, tumor, joint, tissue, graft, or organ that is the target.

[0309] The desired dosage can conveniently be presented as divided doses administered at appropriate intervals in a single dose, for example, as two, three, four, or more sub-doses per day. The sub-doses themselves may be further divided, for example, into a number of individual, spaced administrations. Preferably, the dosage is administered once a week or less frequently, for example, once every two weeks, once every three weeks, once a month, or even once every two months.

[0310] The dosing regimen may include long-term treatment. "Long-term" means a period of at least two weeks, and preferably several weeks, months, or years. Modifications necessary within this dosing range can be determined by one of ordinary skill in the art using only routine experimentation, subject to the teachings provided herein. See, for example, Remington’s Pharmaceutical Sciences (Martin, E.W., ed., 4th ed.), Mack Publishing Co., Easton, PA. Dosages can also be adjusted by the individual physician in the event of any complication.

[0311] In the art, there is a need for more effective treatments for disorders in which cartilage is affected in joints, such as osteoarthritis. Even when administered intra-articularly, the residence time of most drugs is insufficient to treat the affected cartilage. The inventors hypothesized that coupling a therapeutic agent to a moiety (also referred to as a "cartilage anchor protein" or "CAP") that "anchors" the drug in the joint, thereby increasing retention of the drug, should not impair the effectiveness of the therapeutic agent. This concept of anchoring increases not only the effectiveness of the drug, but also the operational specificity for the affected joint by reducing toxicity and side effects, thereby expanding the number of useful drug candidates. The inventors further hypothesized that aggrecan binders could potentially function as anchors, but that aggrecan is highly glycosylated and degraded in the various disorders in which cartilage is affected in joints. Further, considering the cost and large-scale testing in the various animal models required before a drug can enter the clinic, such aggrecan binders should preferably have broad cross-reactivity; for example, an aggrecan binder should bind to aggrecans of various species. Using a variety of sophisticated immunization, screening, and characterization methods, the inventors were able to identify various aggrecan binders that have the characteristics of excellent selectivity, stability, and specificity, enabling long-term retention and activity in joints.

[0312] In one aspect, the present invention relates to a composition according to the present invention, an ISV according to the present invention, a polypeptide according to the present invention, and / or a construct according to the present invention for use as a medicament. In one aspect, the present invention relates to a method for reducing and / or inhibiting the outflow of a composition, polypeptide or construct from a joint, wherein the method comprises administering to a person in need thereof a pharmaceutically active amount of at least one polypeptide according to the present invention, a construct according to the present invention, or a composition according to the present invention.

[0313] In the present invention, the term "reducing and / or inhibiting the outflow" means reducing and / or inhibiting the outward flow of a composition, polypeptide or construct from the inside to the outside in a joint. Preferably, the outflow is at least 10%, such as at least 20%, 30%, 40%, or 50%, or even for example at least 60%, 70%, 80%, 90%, or even 100% lower and / or inhibited compared to the outflow of the aforementioned composition, polypeptide or construct in a joint under the same conditions except in the presence of an aggrecan binder of the present invention, such as an ISV (s) that binds to aggrecan.

[0314] It is understood that the aggrecan binders of the present invention can be used in a variety of diseases that affect cartilage, such as osteoarthritis and chondrodystrophy, arthritis diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondrogenesis, costochondritis, spondyloepiphyseal dysplasia, intervertebral disc hernia, lumbar intervertebral disc degenerative disease, degenerative joint disease, and relapsing polychondritis, etc. (generally referred to herein as "diseases related to aggrecan").

[0315] In one aspect, the present invention relates to the use of the compositions, ISVs, polypeptides, and / or constructs according to the present invention for the prevention or treatment of Agrican-related diseases, such as arthrosis and chondrodystrophy, arthritis diseases, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondrogenesis, costochondritis, spondyloepiphyseal dysplasia, intervertebral disc hernia, lumbar intervertebral disc degeneration disease, degenerative joint disease, and relapsing polychondritis, etc.

[0316] In one aspect, the present invention relates to a method for preventing or treating arthrosis and chondrodystrophy, arthritis diseases, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondrogenesis, costochondritis, spondyloepiphyseal dysplasia, intervertebral disc hernia, lumbar intervertebral disc degeneration disease, degenerative joint disease, and relapsing polychondritis, etc., wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of at least a composition, ISV, polypeptide, or construct according to the present invention for a person in need thereof.

[0317] In one aspect, the present invention relates to the use of an ISV, polypeptide, composition or construct according to the present invention in the preparation of a pharmaceutical composition for treating or preventing arthrosis and chondrodystrophy, arthritis diseases, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondrogenesis, costochondritis, spondyloepiphyseal dysplasia, intervertebral disc hernia, lumbar intervertebral disc degeneration disease, degenerative joint disease, and relapsing polychondritis, etc.

[0318] By binding to aggrecan, the aggrecan binding agent of the present invention is expected to be able to reduce or inhibit the activity of a member of the serine protease family, cathepsin, matrix metalloproteinase (MMP) / matrilysin or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and / or ADAMTS11, in the degradation of aggrecan.

[0319] Accordingly, in one aspect, the present invention relates to a method of reducing or inhibiting the activity of a member of the serine protease family, cathepsin, matrix metalloproteinase (MMP) / matrilysin or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and / or ADAMTS11, in the degradation of aggrecan, wherein the method comprises administering a pharmaceutically active amount of at least an ISV, polypeptide, construct or composition according to the present invention to a person in need thereof.

[0320] In the context of the present invention, the term "prevention or treatment" includes not only preventing and / or treating a disease, but also generally preventing, decelerating, or reversing the onset of a disease, preventing or delaying the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of any disease and / or symptom associated therewith, and / or preventing an increase in the severity of any disease and / or symptom associated therewith, and preventing, reducing or reversing any physical damage caused by the disease, and also generally includes any pharmacological action beneficial to the patient being treated.

[0321] The subject to be treated may be any warm-blooded animal, but is particularly a mammal, specifically a human. As will be apparent to those skilled in the art, the subject to be treated is specifically a person suffering from or at risk of the diseases, disorders and symptoms described herein.

[0322] Generally, a treatment regimen will include administration in one or more pharmaceutically effective amounts or dosages of one or more ISVs, polypeptides, compounds and / or constructs of the present invention, or one or more compositions containing the same. The specific amount(s) or dosage to be administered can also be determined by a physician based on the factors cited above.

[0323] Generally, depending on the particular disease, disorder or condition to be treated, the efficacy of the particular ISV, polypeptide, compound and / or construct of the present invention to be used, the particular route of administration, and the particular pharmaceutical formulation or composition to be used, a physician will be able to determine a suitable daily dosage.

[0324] Normally, in the above methods, the ISVs, polypeptides, compounds and / or constructs of the present invention will be used. However, it is within the scope of the present invention to use two or more ISVs, polypeptides and / or constructs of the present invention in combination.

[0325] The ISV, polypeptide and / or construct of the present invention can be used as a combination treatment regimen in combination with one or more further pharmaceutically active compounds or principles, i.e., with or without resulting in a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combination treatment regimen, based on the factors cited above and their expert judgment.

[0326] In particular, the ISV, polypeptide and / or construct of the present invention can be used in combination with, or for the prevention and / or treatment of the diseases, disorders and conditions cited herein, other pharmaceutically active compounds or principles, with or without resulting in a synergistic effect as a result. Examples of such compounds and principles, as well as the routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

[0327] When two or more substances or principles are used as part of a combined treatment regimen, they may be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g., essentially simultaneously, sequentially, or in an alternating regime). If substances or principles are to be administered simultaneously via the same route of administration, they can be administered as part of different pharmaceutical formulations or compositions, or a combined pharmaceutical formulation or composition, which will be apparent to those skilled in the art.

[0328] Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amounts and according to the same regimen as when the compound or principle is used alone, and such combined use may or may not result in a synergistic effect. However, when the combined use of two or more active substances or principles results in a synergistic effect, it may be possible to reduce the amount of one, more or all of the substances or principles to be administered while still achieving the desired therapeutic effect. This can be useful, for example, to avoid, limit or reduce any undesirable side effects associated with the use of one or more of the substances or principles when used in their normal amounts while still obtaining the desired pharmaceutical or therapeutic effect.

[0329] The effectiveness of the treatment regimen used according to the invention can be determined and / or monitored for the disease, disorder or condition involved in any manner known per se, which will be apparent to the clinician. The clinician can also, where appropriate and on a case-by-case basis, modify or adapt the particular treatment regimen to achieve the desired therapeutic effect, to avoid, limit or reduce any undesirable side effects and / or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing any undesirable side effects on the other hand.

[0330] Generally, the treatment regimen will be continued until the desired therapeutic effect is achieved and / or as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

[0331] In another aspect, the present invention relates to the use of the ISV, polypeptide, compound and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least a disease associated with aggrecan; and / or for use in one or more of the treatment methods described herein. The present invention also relates to the use of the ISV, polypeptide, compound and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least one disease or disorder that can be prevented and / or treated by modulating aggrecan, such as inhibiting aggrecan degradation.

[0332] The present invention also relates to the use of the ISV, polypeptide, compound and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least one disease, disorder or condition that can be prevented and / or treated by administering the ISV, polypeptide, compound and / or construct of the present invention to a patient. The present invention further relates to the ISV, polypeptide, compound and / or construct of the present invention, or a pharmaceutical composition comprising the same, for use in the prevention and / or treatment of a disease associated with at least one aggrecan.

[0333] The subject to be treated may be any warm-blooded animal, but in particular a mammal, and even more particularly a human. In veterinary applications, the subject to be treated includes any animal raised for commercial purposes or kept as a pet. As will be apparent to those skilled in the art, the subject to be treated will in particular be a person suffering from or at risk of the diseases, disorders and / or conditions described herein. Also, in such pharmaceutical compositions, one or more of the ISVs, polypeptides, compounds and / or constructs of the present invention, or the nucleotides encoding them, and / or pharmaceutical compositions containing them may be suitably combined with one or more other effective principles such as those described herein.

[0334] The present invention also relates to compositions (including, but not limited to, pharmaceutical compositions or preparations as further described herein) for use in vitro (e.g., in in vitro or cell assays) or in vivo (e.g., in unicellular or multicellular organisms, and particularly in mammals, and even more particularly in humans, such as in humans having or suffering from a risk of a disease, disorder or condition of the present invention). References to treatment should be understood to include both the treatment of established symptoms and prophylactic treatment, unless otherwise explicitly stated.

[0335] Sequences are disclosed in the main body of the description and in a separate sequence listing in accordance with WIPO Standard ST.25. The sequence numbers identified by specific numbers should be the same in the main body of the description and in the separate sequence listing. By way of example, SEQ ID NO: 1 should define the same sequence in both the main body of the description and the separate sequence listing. If there is a conflict between the definition of a sequence in the main body of the description and the separate sequence listing (e.g., if SEQ ID NO: 1 in the main body of the description erroneously corresponds to SEQ ID NO: 2 in the separate sequence listing), references in the application, particularly to the specific sequences of particular embodiments, should be understood as references to the sequences in the main body of the application and not as references to the separate sequence listing. In other words, conflicts between the definition / naming of sequences in the main body of the description and the separate sequence listing should be resolved by correcting the separate sequence listing to the sequences and their naming disclosed in the main body of the application, including the description, examples, drawings and claims.

[0336] The present invention will now be further described by way of non-limiting preferred aspects, examples and drawings. The entire contents of all references cited throughout this application (including academic articles, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated by reference herein, particularly for the teachings mentioned above herein.

[0337] Examples Example 1 Immunization of llamas with Aglycan, cloning of an antibody fragment repertoire of only heavy chains, and preparation of phage The inventors realized that the goal of an animal model of OA is to reproducibly control the scale and progression of joint damage so that opportunities to detect symptoms and disease progression can be identified and new treatments can be developed. An ideal animal model is one that is relatively low-cost and presents a reproducible disease progression with a large enough effect size to detect differences within a short time. If the model progresses too rapidly to end-stage degeneration, it may not be possible to obtain intermediate time points that are representative of the pathophysiology of OA, and in the absence of this information, subtle effects of potential interventions may be missed. Understanding that OA is an end-stage phenotype that results from the interaction of mechanical and biochemical processes, animal models allow these factors to be studied in a controlled environment (see Teeple et al. 2013 AAPS J. 15: 438-446).

[0338] The ultimate goal of animal models is to reproduce human diseases (see Cohen-Solal et al. 2013 Bonekey Rep. 2: 422). Given the heterogeneity of profiles in human OA, multiple models are required. They can be either spontaneous or induced. Most of them focus on one factor that favors the development of OA, such as aging, mechanical stress (surgery), chemical deficiency (enzymes), or genetic factors. All of them differ with respect to severity, lesion location, and etiology. However, there is no animal model that addresses all aspects of OA during its development.

[0339] Therefore, for being useful in various animal models and ultimately in human patients, the CAP binder preferably has broad cross-reactivity, e.g., binds to aggrecan of more than one species. Preferably, the aggrecan binder binds to human aggrecan as well as to one or more of dog aggrecan, bovine aggrecan, rat aggrecan, porcine aggrecan, mouse aggrecan, rabbit aggrecan, cynomolgus monkey aggrecan, and / or rhesus monkey aggrecan.

[0340] Furthermore, the inventors noticed that the degradation of aggrecan seems to be initiated in the C-terminal region. The pool of aggrecan molecules lacking the G3 domain also increases with aging. The major feature of cartilage degeneration associated with arthritis is the decrease of aggrecan due to proteolytic cleavage in the interglobular region between the G1 domain and the G2 domain. Therefore, preferably, the aggrecan binder binds to the N-terminal region of aggrecan, i.e., the region other than the CS or G3 domain, such as the G1-IGD-G2 region, or the G1-domain, IGD, or G2 domain. Most preferably, the aggrecan binder will bind to the G1 domain that persists in chondrocytes and the ECM.

[0341] 1.1 Immunity Five llamas were immunized with recombinant (rec) human aggrecan (G1-IGD-G2 domain, R&D Systems, #1220-PG) (see Example 1.2). Serum samples were obtained after antigen administration and titers were determined by ELISA against human recombinant aggrecan G1-IGD-G2. All llamas showed specific serum titers.

[0342] 1.2 Primary screening RNA was extracted from PBL (primary blood lymphocytes) and used as a template for RT-PCR to amplify ISVs encoding gene fragments. These fragments were cloned into the phagemid vector pAX212, enabling the production of phage particles presenting ISVs fused with His6 tag and FLAG3 tag. Phages were prepared and stored according to standard protocols (see Phage Display of Peptides and Proteins: A Laboratory Manual, 1st edition, Brian K. Kay, Jill Winter, John McCafferty, Academic Press, 1996).

[0343] Phage display selections were performed using five immune libraries and two synthetic ISV libraries. The libraries were subjected to two to three rounds of enrichment against various combinations of recombinant human and (biotin-)rat aggrecan G1-IGD-G2 domains, full-length extracted bovine aggrecan, or whole bovine cartilage. Individual clones from the selection outputs were screened for binding to the human G1-IGD-G2 domain in ELISA (using periplasmic extracts from E. coli cells expressing the ISV). Sequencing of 542 ELISA-positive clones identified 144 unique ISV sequences. The ISVs were evaluated for cross-species reactivity and mapped for binding to individual human G1, IGD, and G2 domains by ELISA. Only a few ISVs showed similar binding levels to recombinant human, rat, dog, and bovine aggrecan G1-IGD-G2. Limited cross-species reactivity was particularly prominent for G1 domain binders, for which binding to bovine and dog aggrecan was particularly low. To identify G1 domain-binding ISVs with higher cross-species reactivity, phage display selections against bovine G1-IGD-G2, dog G1-IGD-G2, and the human G1 domain were performed. Of 1245 clones screened for binding to human, cynomolgus monkey, rat, dog, and bovine G1-IGD-G2 in ELISA, only 15 novel cross-species reactive ISVs were identified, 9 of which could be mapped to the G1-domain.

[0344] A total of 19 unique clones were selected as a "lead panel" for further characterization. An overview of the domain mapping and cross-species reactivity data for this lead panel is provided in Table 1.2.

Table 1

[0345] 1.3 G1 binder The sequence variability in the CDRs of the G1 binder was determined for clone 114F08. The amino acid sequences of the CDRs of clone 114F08 were used as a reference to compare the CDRs of all other clones (G1 binders) and are presented in Tables 1.3A, 1.3B, and 1.3C below (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95). [Table 2-1] [Table 2-2] [Table 2-3]

[0346] 1.4 G1-IGD-G2 binder The sequence variability in the CDRs of the G1-IGD-G2 (GIG) binders was determined for clone 604F02. The amino acid sequences of the CDRs of clone 604F02 were used as a reference to compare the CDRs of all other clones (GIG binders) and are presented in Tables 1.4A, 1.4B, and 1.4C below (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95). [Table 3-1] [Table 3-2] [Table 3-3]

[0347] 1.5 G2 binder The sequence variability in the CDRs of the G2 - binder was determined for clone 601D02. The amino acid sequences of the CDRs of clone 601D02 were used as a reference for comparison with the CDRs of all other clones (G2 - binders) and are shown in Tables 1.5A, 1.5B, and 1.5C below (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

Table 4 - 1

Table 4 - 2

Table 4 - 3

[0348] 1.6 Sequence optimization of ISV Diverse ISVs were subjected to a sequence optimization process. Sequence optimization is a process of mutating the parental ISV sequence. This process involves (i) humanizing the ISV, (ii) post - translational modification, and (iii) knocking out potential epitopes for existing antibodies that may occur. (i) For the purpose of humanization, the parental ISV sequence was mutated to obtain an ISV sequence with higher identity to the human IGHV3 - IGHJ germline consensus sequence. Specific amino acids that differ between the ISV and the human IGHV3 - IGHJ germline consensus in the framework region (with the exception of so - called hallmark residues) were changed to their human counterparts while keeping the protein's structure, activity, and stability unchanged. A handful of hallmark residues are known to be important for the stability, activity, and affinity of the ISV and are therefore not mutated. (ii) Amino acids that are present in the CDRs and for which there is experimental evidence of being sensitive to post - translational modification (PTM) were changed such that the PTM sites are inactivated while keeping the protein's structure, activity, and stability unchanged. (iii) Optimize the sequence of the ISV to minimize binding to any naturally occurring existing antibodies and reduce the potential to elicit a treatment-naïve immunogenic response without affecting the protein's structure, activity, and stability.

[0349] For the production of the optimized form of the ISV, all components of the ISV were produced as untagged proteins in Pichia pastoris according to standard protocols, purified via protein A affinity chromatography, and then desalted. Optimized forms of the ISV with diverse sequences are shown in Tables A-1 and A-2.

[0350] Example 2 Characterization of the Lead Panel (Purified ISV) - Aggrecan After primary screening, initial evaluation of binding via ELISA, determination of off-rates, and species cross-reactivity, the lead panel of ISVs was subjected to further characterization.

[0351] 2.1 Format the Aggrecan Lead Panel with ALB26 (n = 19) The final form of the molecule for clinical use is expected to contain one or two aggrecan-binding ISVs ("anchors"), and also one, two, or more ISVs or other moieties with a mode of therapeutic action. Thus, 19 selected clones were fused to ALB26 in monovalent or divalent form (CAP-ALB26 or ALB26-CAP-CAP) and expressed in P. pastoris. ALB26 is a variant of ALB11 with two mutations in CDR1 that completely abolish binding to albumin from different species (albumin-binding ISV). The fusion to ALB26 was performed to mimic the size of the final polypeptide form containing the aggrecan binder. Without wishing to be bound by any theory, the inventors hypothesized that the pI could affect cartilage penetration and retention. As a negative control or "dummy", divalent ALB26 (C01010030) was used.

[0352] 2.2 Ex vivo bovine cartilage retention Since there is no established assay to evaluate cartilage retention, the inventors developed a reliable and reproducible ex vivo cartilage retention assay using bovine cartilage. Bovine bones were typically retrieved from a local abattoir. Cartilage was cut away from the bone into slices approximately 1 mm thick and further punched into discs with a 3 mm diameter using a biopsy cutter. Cartilage discs were preferentially harvested from fresh cartilage.

[0353] After relatively short exposure of the nanobody to cartilage (which could be expected by intra-articular injection), the ability of ISV to be retained in cartilage over a longer period was determined. The assay consisted of incubating cartilage ex vivo, typically 3 mm bovine discs (about 10 mg wet weight), ON with 10 μg / mL nanobody (100 μL), and then washing for up to 5 days (PBS / 0.1% BSA / 0.1% NaN3 / 100 mM NaCl). Subsequently, the bound (retained) nanobody was released from the cartilage in SDS-containing SDS-PAGE sample buffer (LDS sample buffer, Invitrogen) and analyzed by Western blot (WB). The assay was typically performed using 4 cartilage discs per nanobody sample; 2 discs were analyzed immediately after nanobody incubation (t0) to determine the initial amount of bound nanobody; 2 discs were analyzed after washing (t 1-5days )). The degree of retention was defined as the ratio of the amount of nanobody detected at t 1-5days and t0. To increase the throughput of the assay, the determination of this ratio was performed by visual inspection of the Western blot, which provided a score from 0 - 6, where 0 is no retention and 6 is complete retention.

[0354] The results are summarized in Table 2.2.

Table 5

[0355] Nine constructs were found to be very well retained in cartilage (score 5 - 6). This "top 9" included both monovalent and divalent constructs for the aggrecan-binding moiety that binds to all of the recombinant G1, G2, or G1-IGD-G2 domains. In this assay, 14 constructs showed moderate retention (<5 - 2 score), and 5 constructs showed detectable but low retention (<2 - 1 score). It is notable that all but one of the aggrecan constructs had pI values in the range of 8 to over 9.

[0356] 2.3 Epitope binding For epitope binding, the purified ALB26 fusion nanobody constructs were screened in a competitive ELISA against the same set of nanobodies fused to the FLAG tag. Briefly, the assay setup was as follows. Monoclonal phage ELISA was incubated at a semi-saturation dilution of phage, with or without 1 μm of purified nanobody (or 5 μg / mL of mAb). The ratio between the absorbance at 450 nm in the presence of the purified nanobody (or mAb) and the absorbance at 450 nm in its absence was used to determine whether overlapping or non-overlapping epitopes were recognized.

[0357] The resulting epitope bins are shown in Table 2.2 (top). The constructs (on the G1-domain) in epitope bins 2 and 3 had low cartilage retention scores (0-1) in the ex vivo bovine cartilage retention assay. However, there did not appear to be a direct correlation between the binding to bovine aggrecan G1-IGD-G2 measured by ELISA and bovine cartilage retention. Without being bound by any theory, the inventors hypothesized that these epitopes may not be readily accessible in native cartilage tissue. The sequence variability of the CDRs of the clones belonging to the bin is represented below and above (i.e., bin 8 together with 604F02 as a reference compound; Tables 1.4A-C).

[0358] The sequence variability of the G1 binder of epitope bin 4 to 114F08 is represented in Tables 2.3A, 2.3B and 2.3C below. The amino acid sequence of the CDRs of clone 114F08 was used as a reference for comparing the CDRs of all other clones (epitope bin 4 binders) (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

Table 6-1

Table 6-2

Table 6-3

[0359] The sequence variability of the G1 binder of epitope bin 1 to 608A05 is represented in Tables 2.3D, 2.3E and 2.3F below. The amino acid sequence of the CDRs of clone 608A05 was used as a reference for comparing the CDRs of all other clones (epitope bin 1 binders) (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

Table 7-1

Table 7-2

Table 7-3

[0360] 2.4 Characteristics of Binding - ELISA and SPR Based on the ex vivo bovine cartilage retention and epitope binding data, several exemplary constructs from various epitope bins were selected for further characterization. For the reasons stated above, binders to the G2 domain were excluded from further characterization at this stage.

[0361] The selected constructs were characterized in ELISA against recombinant G1-IGD-G2 regions from human, cynomolgus monkey, rat, dog, and bovine aggrecan to determine their species cross-reactivity and characterized in ELISA against recombinant human neurocan and brevican to determine selectivity. The determined EC 50 values are listed in Table 2.4A.

[0362] SPR (ProteOn) experiments were performed to determine the off-rates for the "monovalent" aggrecan-ALB26 format. The interaction of the nanobody with the aggrecan surface was found to be heterogeneous. The heterogeneity could have been due to rebinding events, a heterogeneous population of immobilized aggrecan, and / or heterogeneous glycosylation patterns. As a result, the calculated off-rates are only indicative. Overall, the dissociation kinetics were rapid for the aggrecan containing the nanobody (Table 2.4B).

Table 8-1

Table 8-2

[0363] Example 3 Biophysical Characterization of Monovalent Lead Constructs-Aglicans Since all selected constructs, whether in combination or not, exhibited various advantageous features, ISV114F08 and 604F02 and their corresponding ALB26-forms (C010100054, -118 and 094) were used as exemplary constructs representing a lead panel for further characterization.

[0364] 3.1 Expression of Monovalent 114F08 and 604F02 in E. coli and P. pastoris For biophysical characterization, the monovalent nanobodies 114F08 and 604F02 were expressed in E. coli and / or P. pastoris using a FLAG3-His6 tag and purified according to standard protocols (e.g., Maussang et al. 2013 J Biol Chem 288(41): 29562-72).

[0365] 3.2 pI, Tm and Analytical SEC of 114F08 and 604F02 For the thermal shift assay (TSA), 5 μL of purified monovalent nanobody (800 μg / ml) was incubated with 5 μL of the fluorescent probe Sypro Orange (Invitrogen, S6551) (final concentration 10×) in 10 μL of buffer (buffered at various pH values in the range of 3.5 - 9 with 100 mM phosphate, 100 mM borate, 100 mM citrate, 115 mM NaCl). The samples were heated from 37 °C to 99 °C at a rate of 4.4 °C / s in a LightCycler 480II instrument (Roche), and then cooled to 37 °C at a rate of 0.03 °C / s. Heat-induced unfolding exposes hydrophobic patches of the protein, to which Sypro Orange binds, resulting in an increase in fluorescence intensity (Ex / Em = 465 / 580 nm). The inflection point of the first derivative of the fluorescence intensity curve serves as a measure of the melting temperature (Tm), which essentially follows Ericsson et al., 2006 (Anals of Biochemistry, 357: 289 - 298).

[0366] Analytical size exclusion chromatography (analytical SEC) experiments were performed using a mobile phase of 10 mM phosphate, 300 mM Arg-HCl (pH 6.0) on an Ultimate 3000 instrument (Dionex) combined with a Biosep-SEC-3 (Agilent) column. An 8 μg nanobody sample (in d-PBS, 0.5 mg / mL) was injected.

[0367] The isoelectric points of the two aglycan ISVs were relatively basic. The sequences are shown in Table A-1. The melting temperatures were determined to be 61.0 °C for 114F08 and 70.0 °C for 604F02. None of the clones showed signs of aggregation or multimerization as determined by analytical SEC. Thus, following positive functional properties, the ISVs exhibit favorable biophysical properties.

[0368] 3.3 114F08 family members The sequence variability in the CDRs of the 114F08 family members is represented in Tables 3.3A, 3.3B and 3.3C below. The amino acid sequence of the CDRs of clone 114F08 was used as a reference to compare the CDRs of all other clones (114F08 family members) (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

Table 9-1

Table 9-2

Table 9-3

[0369] Example 4 Ex vivo binding to cartilage from diverse species Exemplary CAP-containing polypeptides (also herein named "CAP-containing constructs" or "constructs") have been shown to bind to recombinant / extracted human proteins and to bovine cartilage in a bovine ex vivo cartilage retention assay. To show that these exemplary CAP-containing constructs also bind to cartilage from other species, the above-described experiments using bovine cartilage were essentially repeated using human cartilage and rat cartilage.

[0370] 4.1 Ex vivo binding to human cartilage To confirm that the exemplary CAP-containing constructs also bind to human cartilage, selected constructs were tested using frozen human cartilage pieces in an ex vivo cartilage binding assay. After a 30-minute wash, binding was determined by Western blot. The results are summarized in Table 4.1.

Table 10

[0371] 4.2 Binding to rat cartilage ex vivo To facilitate testing of constructs in the rat in vivo model, binding to rat cartilage was evaluated. Thus, assays were set up using femurs from rats with intact cartilage. Exemplary constructs C010100054, -118, and -094 were incubated overnight with rat cartilage and then washed for 30 minutes, and release of the bound constructs was followed by Western blot analysis.

[0372] The results are shown in Table 4.2. We found that all tested constructs bound well to rat cartilage. [Table 11]

[0373] Example 5 Tissue specificity Above, it has been shown both in vitro and ex vivo that the constructs of the present invention bind specifically to aggrecan. In addition, these constructs should preferably bind to the cartilage of the joint while binding not at all or less to other tissues in the joint. Binding of exemplary CAP-containing constructs to synovium, tendon, epimysium and meniscus was evaluated using the same setup as for the ex vivo cartilage binding assay. After a short tissue wash (30 minutes) after incubation of the constructs, construct release and Western blot analysis were performed.

[0374] The results are summarized in Table 5. The results show that the CAP binder exhibits preferential binding to cartilaginous tissues, including meniscus, over other tissues found in the joint. [Table 12]

[0375] Example 6 Nanobody Stability in Bovine Synovial Fluid For a variety of reasons, including patient convenience and safety, it is preferable for the construct to remain stable in the synovium for a longer period of time. Therefore, the stability of an exemplary ALB26 fusion CAP construct in synovial fluid (SF) was evaluated by incubation of the construct in non - arthritic bovine SF at 37 °C for up to 7 days.

[0376] The results are summarized in Table 6. [Table 13] No degradation of the construct could be observed.

[0377] Example 7 Retention in IL - 1α - Stimulated Explant Cartilage At this point, all experiments addressing the binding and retention of CAPs containing nanobodies to cartilage were performed in healthy (non - arthritic) ex vivo cartilage. Arthritic cartilage is characterized by degraded collagen and aggrecan. Therefore, it is also reasonable to evaluate the binding and retention of an aggrecan binder to cartilage when degradation of these proteins is occurring. For this purpose, an exemplary ALB26 fusion CAP construct was tested in a cartilage explant assay where the cartilage was stimulated to induce degradation.

[0378] Briefly, exemplary CAP-containing constructs were incubated overnight (ON) with bovine cartilage explants that were cultured with or without IL-1α and oncostatin M and then cultured for 5 days with daily medium exchange (washing). IL-1α and oncostatin M mainly induce aggrecan degradation during the 6-day experiment. The cartilage explants were analyzed for construct binding and retention by WB. Two independent experiments were performed (Exp A and Exp B).

[0379] The results of the Western blot are presented in Table 7.1.

Table 14

[0380] The results of CAP retention containing constructs in stimulated cartilage explants are summarized in Table 7.2.

Table 15

[0381] The results show that constructs C01010054 ("054" or "54") and C01010045 ("045" or "45") have reduced retention in stimulated cartilage after 5 days of washing compared to unstimulated cartilage, while constructs C01010118 ("118") and C01010094 ("094" or "94") showed little sensitivity to stimulation. Furthermore, the binding of the G2 aggrecan domain (exemplified by C01010045) appears to be reduced compared to the binding to the other domain, which would be consistent with the hypothesis that aggrecan degradation proceeds from the C-terminus.

[0382] Example 8 ADAMTS5-CAP GAG Release Assay To address the potential impact on the efficacy of protease - inhibitory nanobodies in cartilage tissue by the CAP, which is a cartilage anchor part, exemplary CAP constructs were fused to an ISV that blocks ADAMTS5 (ATS5) and tested in a GAG (GlycosAminoGlycan) - releasing cartilage explant assay.

[0383] Before testing the constructs in the GAG - releasing cartilage explant assay, their in vitro cartilage - binding and ADAMTS5 - inhibitory properties were confirmed. For the latter, an enzyme - peptide assay was performed, which showed that the enzyme - blocking function of the ADAMTS5 ISV was not impaired in any of the CAP - fusion constructs in vitro.

[0384] In the GAG - releasing assay, bovine cartilage explants were cultured for 5 days in the presence of IL - 1α and oncostatin M (for induction of ADAMTS5) and a range of doses of the constructs, and then the amount of released GAG in the culture supernatant was quantified.

[0385] The results of the tested constructs and the GAG - releasing assay are summarized in Table 8.

Table 16

[0386] Example 9 In vivo bioimaging of CAP - constructs Parallel to the in vitro and ex vivo characterization of exemplary aggrecan CAP constructs, to confirm the retention properties, the in vivo biodistribution was determined for some of the ALB26 - fusion constructs.

[0387] 9.1 In vivo biodistribution study of the ALB26-CAP construct The nanobody was 125 labeled with 125 I (via lysine coupling of I-SIB). The construct was injected into the knee joints of healthy rats. Autoradiography images of the joints were generated at various time points up to 4 weeks after injection. These images enable the evaluation of the retention of the construct and tissue (cartilage) specificity in an in vivo setting. Representative images are shown in Figure 1.

[0388] From the results, it can be concluded that all constructs showed specific binding to cartilage. Clear staining was observed for both the “monovalent” and “divalent” aggrecan binders even 4 weeks after injection.

[0389] 9.2 MARG of the ALB26-CAP construct The above biodistribution study (Example 9.1) showed specific retention of the ALB26-CAP construct in cartilage. However, the resolution of the images did not allow for an examination of the depth of penetration into cartilage. MARG (Micro-Auto-Radiography) was used to increase the imaging resolution and thereby enable the evaluation of penetration into cartilage.

[0390] Exemplary constructs used in the study are listed in Table 9.2A. For this study, the nanobody was 3 labeled with 3 H (via lysine coupling of H-NSP (N-succinimidyl propionate)) and injected into healthy rat joints and osteoarthritis (surgically induced via transection of the anterior cruciate ligament) rat joints; 8 rats per group. 7 - 14 days after injection, the rats were euthanized and the injected healthy joints and OA-induced joints were processed for MARG.

[0391] Representative MARG images are shown in Figure 2.

Table 17

[0392] All of the Agrican binders generally showed penetration into healthy cartilage. Construct 626 sometimes also showed some more prominent staining on the surface. In the operated knees, various degrees of cartilage staining and penetration were observed: for monovalent construct 054, no staining was observed; for monovalent construct 094, the staining was absent or mild, while the divalent construct 626 resulted in some more prominent staining with a varying depth of penetration (see Table 9.2B).

Table 18

[0393] Example 10 Rat MMT DMOAD in vivo showed a statistically significant effect. To further demonstrate the in vivo efficacy of the CAP binder of the present invention, a surgically induced medial meniscus tear (MMT) model in rats was used. Briefly, the CAP binder of the present invention was coupled to an anti-MMP13 ISV (named "0754" or "C010100754") or an anti-ADAMTS5 ISV (named "0954" or "C010100954"). Rats were surgically treated on one knee to induce OA-like symptoms. The treatment was started by IA injection on the 3rd day after surgery. Histopathology was performed 42 days after surgery. Intermediate and final serum samples were obtained for exploratory biomarker analysis. The medical and overall substantial cartilage degeneration width and the percentage reduction of cartilage degeneration were determined. 20 animals per group were used. Inhibition of cartilage degradation in the medial tibia by the nanobody is shown in Figure 3.

[0394] The results show that after 42 days, the cartilage width was substantially reduced by the ADAMTS5-CAP construct and the MMP13-CAP construct compared to the vehicle. These results suggest that the CAP moiety has no negative impact on the activity of either (a) anti-MMP13 ISV (0754) or anti-ADAMTS5 ISV (0954); and (b) enables retention of these constructs in the joint for a longer period of time.

[0395] Example 11 Retention of the CAP binder in healthy and osteoarthritic rats is similar in vivo. In a cartilage retention study in healthy rats, it was shown that the polypeptide of the present invention was measurable in cartilage up to 112 days after intra-articular (I.A.) injection (data not shown). Since the composition of cartilage can affect the binding and absorption of cartilage in the systemic circulation, the pharmacokinetics of the polypeptide of the present invention were compared in vivo in diseased osteoarthritic rats and healthy rats, and then the serum levels of the polypeptide were compared without delay.

[0396] Specifically, a surgically induced medial meniscus tear (MMT) model in rats was used as described in Example 10 with some modifications. Briefly, the polypeptide of the present invention was coupled to anti-MMP13 ISV and anti-ADAMTS5 ISV to yield an MMP13-ADAMTS5-CAP-CAP construct (designated as "0949" or "C010100949" nanobody). Rats were surgically treated on one knee to induce OA-like symptoms (OA group). Each treatment group (healthy and OA) consisted of 15 animals and received a single I.A. injection of 400 μg / 30 μl of the nanobody on day 7 (healthy) or 7 days post-surgery (MMT). Serum samples were collected from anesthetized rats on day 0, on day 7 (at 0 h = pre-dose sample), on day 8 (at different time points up to 24 h post-treatment), on day 9 (48 hours after treatment), d10 (3 days after treatment), d14 (7 days after treatment), d21 (14 days after treatment), and d42 (35 days after treatment). The collected serum samples were used for the determination of polypeptide concentration in an electrochemoluminescence (ECL)-based comprehensive PK assay format and subsequent non-compartmental analysis. Retention of the polypeptide in the sera of healthy and OA rats is shown in Figure 4.

[0397] The results show that no clear differences can be observed in the serum concentrations of the polypeptide in healthy and OA rats. These results suggest that cartilage degradation has no effect on the pharmacokinetics of the polypeptide of the present invention.

[0398] [Table 19]

[0399] [Table 20-1] [Table 20-2]

[0400]

Table 21-1

Table 21-2

Table 21-3

Table 21-4

Table 21-5

Table 21-6

Table 21-7

Table 21-8

Table 21-9

[0401]

Table 22

[0402]

Table 23

[0403]

Table 24-1

[0404]

Table 24-2

Claims

**Claim 1** A composition for preventing or treating a disease related to aggrecan, comprising a polypeptide comprising an immunoglobulin single variable domain (ISV) that specifically binds to human aggrecan represented by SEQ ID NO: 125 and a therapeutic ISV, wherein the ISV that specifically binds to human aggrecan comprises four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), where; the ISV that specifically binds to human aggrecan is - CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; - CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; - CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; - CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; - CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; - CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; - CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; - CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 71; - CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 72; - CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 73; - CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 74; - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: ​ - CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59; - CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64; - CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; - CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62; and - selected from the group of ISVs where CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70, and the therapeutic ISV binds to a member of the serine protease family, cathepsin, matrix metalloproteinase (MMP) / matrilysin or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), said composition.

2. The composition according to claim 1, wherein the ISV that specifically binds to human aggrecan is selected from the group of ISVs having SEQ ID NOs: 1 to 19 and 114 to 118.

3. The composition according to claim 1 or 2, wherein the ISV that specifically binds to human aggrecan has a sequence identity higher than 80% with any one of SEQ ID NOs: 1 to 19 and 114 to 118.

4. The composition according to any one of claims 1 to 3, comprising a polypeptide comprising at least two ISVs that specifically bind to human aggrecan according to any one of claims 1 to 3.

5. The composition according to claim 4, wherein at least two ISVs are independently selected from the group consisting of SEQ ID NOs: 1 to 19 and 114 to 118.

6. The composition according to any one of claims 1 to 5, wherein the polypeptide further comprises a serum protein binding moiety or a serum protein.

7. The composition according to any one of claims 1 to 6, comprising a construct comprising the polypeptide according to any one of claims 1 to 6 and further comprising one or more other groups, residues, moieties or linking units.

8. The composition according to any one of claims 1 to 7, for use as a medicament.

9. The composition according to any one of claims 1 to 8, wherein the disease associated with an Agrican is arthritis and cartilage dystrophy; arthritis diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis; traumatic rupture or detachment, achondrogenesis, costochondritis, spondyloepiphyseal dysplasia, intervertebral disc hernia, lumbar intervertebral disc degeneration disease, degenerative joint disease, or relapsing polychondritis.

Images