Aggrecan binding immunoglobulins

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

[0002] background Osteoarthritis is one of the most common causes of disability worldwide. It affects 30 million Americans and is the most common joint disorder. By 2025, it is predicted to affect over 20% of the U.S. population. The disease can occur in any joint, most frequently in the knee, hip, fingers, and spine. Osteoarthritis (OA) can be defined as a diverse group of conditions characterized by a combination of joint symptoms, signs resulting from loss of articular cartilage, and changes in adjacent tissues, including bone, tendons, and muscles. OA is characterized by the progressive erosion of articular cartilage (the cartilage that covers bone). Ultimately, the disease leads to the total destruction of articular cartilage, sclerosis of the underlying bone, and osteophyte formation, all of which result in loss of movement and pain. Pain is the most prominent symptom of OA and is the most frequent reason patients seek medical help.

[0003] Aggrecan is the major proteoglycan in articular cartilage (Kiani et al. 2002 Cell Research 12:19-32). This molecule is important for the proper function of articular cartilage because it provides the hydrogel structure that gives cartilage its load-bearing properties. Aggrecan is a large, multimodular molecule (2317 amino acids) expressed by chondrocytes. Its core protein consists of three globular domains (G1, G2, and G3) and a large extended region between G2 and G3 for the addition of glycosaminoglycan chains. This extended region contains two domains, one substituted with keratan sulfate chains (KS domain) and one substituted with chondroitin sulfate chains (CS domain). The CS domain has 100–150 glycosaminoglycan (GAG) chains attached to it. Aggrecan forms large complexes with hyaluronan, in which 50–100 aggrecan molecules interact with one hyaluronan molecule via the G1 domain and link protein. Upon water uptake (due to GAG content), these complexes form reversibly deformable gels that resist compression. The structure, fluid retention, and function of articular cartilage are related to the aggrecan matrix content and the amount of chondroitin sulfate bound to the intact core protein.

[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 the G1 domain, and 2) the degradation of collagen, which irreversibly destroys cartilage structure.

[0005] Although aging, obesity, and joint injury have been identified as risk factors leading to osteoarthritis, the cause of OA is unknown, and currently there are no pharmacological treatments that can halt disease progression or heal joints. 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. Fasinumab, a non-opioid anti-NGF pain treatment, has been shown to improve significant pain scores during a Phase II / III clinical trial. Duloxetine has been approved for the treatment of chronic knee pain due to osteoarthritis and is conditionally recommended by the American College of Rheumatology. In a large, multicenter study, strontium ranelate was found to significantly reduce the rate of joint space width loss in patients with symptomatic knee osteoarthritis, as well as improve pain scores compared to placebo. However, at this time, biologic agents, interleukin-1 receptor antagonists and anti-tumor necrosis factor antibodies, have not been shown to be effective or to alter the course of osteoarthritis (Smelter Hochberg 2013 Current Opin. Rheumatol. 25:310). Thus, many such treatments are ineffective and / or have side effects. Ultimately, patients undergo total knee or hip replacement if pain cannot be controlled.

[0006] Pharmacological treatment begins with oral administration of paracetamol, which may be combined with NSAIDS or COX-2 inhibitors and weak opioids. The major drawbacks of oral administration of drugs are limited bioavailability at the target site and the risk of side effects such as liver damage, gastrointestinal (GI) ulcers, 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 invention relates to an ISV as described herein, wherein said ISV binds to the G1 domain of aggrecan, preferably said ISV has a pI greater than 8, and / or said ISV has a pI greater than 2. * 10 -2 s -1 Less than K off and / or the ISV comprises: * 10 -6 EC less than M 50 It has.

[0015] In one aspect, the 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 NO: 24, 20, or 21; or 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: 24, wherein 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; The ISV is selected from the group:

[0018] In one aspect, the 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 NO: 24 and 109; or b) an amino acid sequence having two or one amino acid difference(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 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; 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 NO: 60 and 111; or f) an amino acid sequence having two or one amino acid(s) difference(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, wherein said ISV is selected from the group of ISVs wherein CDR1 is selected from the group consisting of SEQ ID NOs: 24 and 109; CDR2 is selected from the group consisting of SEQ ID NOs: 42 and 110; and CDR3 is selected from the group consisting of SEQ ID NOs: 60 and 111.

[0020] In one aspect, the present invention relates to an ISV as described herein, wherein said ISV belongs to epitope bin 1 or epitope bin 4, preferably said ISV consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 36; and b) an amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 36, wherein 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 5, 4, 3, 2 or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 54, wherein 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 is changed to R; 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 NO: 60; and f) an amino acid sequence having 5, 4, 3, 2, or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 60, wherein at position 1, P is absent; at position 2, T is changed to R or absent; at position 3, T is changed to M or 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 is changed to R; and at position 10, Y is changed to N or E. and / or at position 13, Y is changed to L or 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 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.

[0025] In one aspect, the invention relates to an ISV as described herein, 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 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 present invention relates to an ISV as described herein, wherein the ISV is selected from the group consisting of an ISV having SEQ ID NO: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19, and an ISV having more than 80%, such as 90% or 95%, sequence identity to any one of SEQ ID NO: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19.

[0027] In one aspect, the present invention relates to an ISV as described herein, wherein said ISV binds to the G1-IGD-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.

[0028] 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 NOs: 32, 30, and 23; and b) an amino acid sequence having 3, 2, or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 32, wherein 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 NOs: 50, 41, 48, and 51; and d) an amino acid sequence having 2 or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 50, wherein at position 7, G is changed to S or and / or at position 8, R is changed to T; and / or iii) CDR3 is selected from the group consisting of: e) SEQ ID NOs: 68, 59, 66 and 69; 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: 68, wherein 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, the ISV is selected from the group of ISVs wherein 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 NOs: 50, 41, 48 and 51; and CDR3 is selected from the group consisting of SEQ ID NOs: 68, 59, 66 and 69, and even more preferably, the ISV is selected from the group of ISVs wherein 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.

[0029] In one aspect, the present invention relates to an ISV as described herein, wherein the ISV is selected from the group consisting of an ISV having SEQ ID NO: 13, 4, 11 and 14, and an ISV having more than 80%, such as 90% or 95%, sequence identity 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 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 are added after position 14 of SEQ ID NO: 64; preferably wherein said ISV has CDR1 selected from the group consisting of SEQ ID NOs: 28, 22, 26 and 33; CDR2 selected from the group consisting of SEQ ID NOs: 46, 40, 44 and 52; and CDR3 and CDR1 is SEQ ID NO:33, CDR2 is SEQ ID NO:52, and CDR3 is SEQ ID NO:70.

[0033] In one aspect, the present invention relates to an ISV as described herein, wherein the ISV is selected from the group consisting of an ISV having SEQ ID NO: 9, 3, 7 and 15, and an ISV having more than 80% sequence identity, 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 serum protein, preferably wherein said serum protein binding moiety binds to serum albumin; even more preferably wherein said serum protein binding moiety is an ISV that binds to serum albumin; even more preferably wherein said serum albumin binding ISV is an ISV that binds to serum albumin and has 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; even more preferably, the serum albumin-binding ISVs include 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 invention relates to a polypeptide as described herein, further comprising a serum protein-binding moiety or serum protein, wherein the serum protein-binding moiety is a non-antibody-based polypeptide. In one aspect, the invention relates to a polypeptide as described herein, further comprising PEG.

[0040] In one aspect, the invention relates to a polypeptide as described herein, wherein the ISVs are linked to each other directly or via a linker. In one aspect, the invention relates to a polypeptide as described herein, wherein the first ISV and / or the second ISV and / or optionally the third ISV and / or optionally the fourth ISV and / or optionally the serum albumin-binding ISV 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 a group of polypeptides and / or constructs comprising an ISV that binds to a target as shown in Table E-1 and Table E-2, respectively, and an ISV that binds to one or two aggrecans 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, optionally further comprising one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers; preferably said one or more other groups, residues, moieties or binding units are selected from the group consisting of a polyethylene glycol molecule, a serum protein or fragment thereof, a binding unit capable of binding to a serum protein, an Fc portion, and a small protein or peptide capable of binding to a serum protein.

[0043] In one aspect, the invention relates to a nucleic acid that encodes 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 invention relates to a method for producing 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 followed by: b) isolating and / or purifying the ISV as described herein, or the polypeptide as described herein.

[0045] In one aspect, the 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 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 pharmaceutical. Preferably, the composition, ISV, polypeptide, or construct as described herein is for use in preventing or treating arthropathy and chondrodystrophies, arthritic diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or detachments, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated discs, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis.

[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 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 arthropathies and chondrodystrophies, arthritic diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or detachments, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated discs, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis.

[0051] Other aspects, advantages, applications and uses of the polypeptides and compositions will become apparent from the further disclosure herein. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), both supra and infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. [Brief explanation of the drawings]

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

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

[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 began 3 days postoperatively by intravenous injection. Histopathology was performed 42 days postoperatively. The extent of medical and overall parenchymal cartilage degeneration, as well as the percentage reduction in cartilage degeneration, were determined. 20 animals per group were used.

[0055] [Figure 4] Serum concentrations (mean concentrations in ng / ml) over time after the first dose (h) of polypeptide in osteoarthritic 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 average concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0056] Detailed Description Unless otherwise stated or defined, all terms used have their ordinary meaning in the art, which will be apparent to those skilled in the art. For reference, see, 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, NY, 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"Amino acid sequence" or "protein sequence" (as in terms such as "a sequence" or "protein sequence") should generally be understood to encompass both the appropriate amino acid sequence as well as the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive interpretation.

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

[0060] Amino acids are L-amino acids commonly found in naturally occurring proteins. Amino acid residues may be designated according to the standard three-letter or one-letter amino acid code. See, for example, Table A-2 on page 48 of WO 08 / 020079. Amino acid sequences containing D-amino acids are not intended to be encompassed by this definition. Any amino acid sequence containing post-translationally modified amino acids may initially be described as the translated amino acid sequence using the symbols shown in this Table A-2, along with the modified positions, e.g., hydroxylation or glycosylation, although these modifications may not be explicitly indicated in the amino acid sequence. Any peptide or protein that can be represented as a sequence modified by linkages, bridges, 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 amino acid sequence is said to "comprise" or "consist essentially of" another nucleotide sequence or amino acid sequence, respectively, this can mean that the latter nucleotide sequence or amino acid sequence is incorporated into the former nucleotide sequence or amino acid sequence, but more usually this generally means that the former nucleotide sequence or amino acid sequence, respectively, comprises in its sequence a stretch of nucleotides or amino acid residues which has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, regardless of how the former sequence was actually produced or obtained (e.g. by any suitable method described herein). As a non-limiting example, when a polypeptide of the invention is said to comprise an immunoglobulin single variable domain ("ISV"), this can mean that the immunoglobulin single variable domain sequence is incorporated into the sequence of the polypeptide of the invention, but more usually this generally means that the polypeptide of the invention comprises the sequence of the ISV in its sequence, regardless of how the polypeptide of the invention was produced or obtained. Also, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, the former recited nucleic acid or nucleotide sequence preferably means that when it is expressed as an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, the latter nucleotide sequence is in the same reading frame as the former recited larger nucleic acid or nucleotide sequence).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 nucleotide at the corresponding position in the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence and multiplying by 100%. A deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered a single nucleotide (position) difference. Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings using a known computer algorithm for sequence alignment, such as NCBI's Blast v2.0. Several other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768. Generally, for the purposes of determining the percentage of "sequence identity" between two nucleotide sequences according to the calculation methods outlined above, the nucleotide sequence with the greatest number of nucleotides will be referred to as the "first" nucleotide sequence and the other nucleotide sequence will be referred to as the "second" nucleotide sequence.

[0066] For purposes 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%. A deletion, insertion, substitution, or addition of an amino acid residue in a second amino acid sequence compared to a first amino acid sequence is considered a difference of a single amino acid residue (position), i.e., an "amino acid difference," 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, also using standard settings. Generally, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the greatest number of amino acid residues will be referred to as the "first" amino acid sequence, and the other amino acid sequence will be referred to as the "second" amino acid sequence.

[0067] In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art may take into account so-called "conservative" amino acid substitutions, i.e., substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure and which can generally be described as amino acid substitutions that have little or no effect on the function, activity or other biological properties of a polypeptide. Such conservative amino acid substitutions are well known in the art, for example, from WO 04 / 037999, GB 335768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300, and (preferred) types and / or combinations of such substitutions may be selected based on the relevant teachings of WO 04 / 037999, or, for example, WO 98 / 49185, and the 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 "amino acid difference(s)" refers to the insertion, deletion or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence, i.e., it is understood that the two amino acid sequences may contain one, two or more such amino acid differences. Furthermore, in the amino acid sequences and / or polypeptides of the present invention, the term "amino acid difference(s)" refers to the insertion, deletion or substitution of a single amino acid residue at the position of the CDR sequence specified in b), d) or f) compared to the CDR sequence of a), c) or e), respectively; it is understood that the CDR sequences of b), d) and f) may contain 1, 2, 3, 4 or up to 5 such amino acid differences compared to the CDR sequence of a), c) or e), respectively.

[0072] The "amino acid difference(s)" may be one, two, three, four, or up to five substitutions, deletions, insertions, or any combination thereof, which improve the properties of the aggrecan-binding agent of the invention, e.g., the polypeptide of the invention, or at least do not impair the desired property, or balance or combination of desired properties, of the aggrecan-binding agent of the invention, e.g., the polypeptide of the invention. In this regard, the resulting aggrecan-binding agent of the invention, e.g., the polypeptide of the invention, should bind to aggrecan with the same, approximately the same, or higher affinity, as measured by surface plasmon resonance (SPR), compared to a polypeptide comprising one or more CDR sequences that does not have at least one, two, three, four, or up to five substitutions, deletions, or insertions.

[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 somehow affects the intrinsic binding affinity of the molecule being measured, for example, due to artifacts associated with coating one molecule on the biosensor, the measured K D However, the apparent K D It will be apparent to those skilled in the art that the apparent K D can be measured. In such a situation, the measured affinity can be influenced by the avidity of the interaction by the two molecules. In particular, K D Accurate measurement of K can be very labor intensive and therefore often results in an apparent K D A value is determined to assess the binding strength of two molecules. As long as all measurements are made in a consistent manner (e.g., keeping assay conditions constant), the apparent K D Measurement, true K D It should be noted that K D and apparent K D should be treated with equal importance or relevance.

[0083] The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (e.g., ISVD and / or polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity, for example, as described on pages 53-56 of WO 08 / 020079 (incorporated herein by reference), which also describes several preferred techniques for measuring binding between an antigen-binding molecule (such as a polypeptide or ISVD of the invention) and the antigen of interest. Typically, an antigen-binding protein (e.g., an ISVD and / or polypeptide of the invention) will bind to more than 10 different types of antigens or antigenic determinants. -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 at least 10-fold, e.g., at least 100-fold, and preferably at least 1000-fold, or better affinity (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 DPreferably, when an ISV and / or polypeptide is "specific for" a first target or antigen compared to a second target or antigen, it is directed against said first target or antigen (as defined herein), but not against said second target or antigen.

[0085] The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, saturation and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and their various variants known in the art; and 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 a measurement of binding equilibrium in the solution phase and avoids possible artifacts associated with the adsorption of one of the molecules onto a support such as plastic. As will be clear to those skilled in the art, the dissociation constant can be either a real or apparent dissociation constant. Methods for determining dissociation constants will be clear to those skilled in the art and include, for example, the techniques described in WO 08 / 020079, pages 53-56.

[0087] Finally, it should be noted that in many situations, experienced scientists may find it convenient to determine binding affinity relative to several reference molecules. For example, to assess the binding strength between molecules A and B, a reference molecule C is used that is known to bind to B and is suitably labeled with a fluorophore or chromophore group or other chemical moiety such as biotin for easy detection in ELISA or FACS (fluorescence-activated cell sorting) or other formats (fluorophore for fluorescence detection, chromophore for light absorption detection, biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration, and the concentration of A is varied for a given concentration or amount of B. As a result, the IC 50 The value is obtained corresponding to the concentration of A at which the signal measured for C in the absence of A is halved. D K Dref and the total concentration of the reference molecule, c ref Assuming that is known, the apparent K for the interaction AB D is expressed by the following formula: D =IC 50 / (1+c ref / K Dref ) can be obtained from ref < <K Dref If K D ≒IC 50 Note that IC 50 in a consistent manner for the binder to which the measurements are compared (e.g., fixed c ref Differences in the strength or stability of molecular interactions can affect IC 50 This measurement will be referred to as K throughout this document. D and, or apparent K D is considered to be equal to

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

[0089] Half-maximal effective concentration (EC 50 The term EC (Eq.) refers to the concentration of a compound that induces a response halfway between baseline and maximum after a specified exposure time. In the context of the present invention, it is used as a measure of the potency of a polypeptide, ISV (e.g., nanobody). EC (Eq.) of a quantitative dose-response curve. 50 represents the concentration of a compound at which 50% of its maximal effect is observed. Concentrations are preferably expressed in molar units.

[0090] In biological systems, small changes in ligand concentration typically result in a rapid change in response followed by a sigmoidal function. The inflection point at which the increase in response with increasing ligand concentration slows down is known as the EC 50 This can be determined mathematically by the deviation of the best-fit line. It is often convenient to rely on a graph for the estimation. EC 50 is provided in the examples section, experiments are performed to find the most accurate 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" for two different antigens or antigenic determinants (e.g., 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) if it is specific for these different antigens or antigenic determinants (as defined herein).

[0097] In the context of the present invention, "modulating" or "to modulate" generally means reducing or inhibiting 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), ADAMTS11 and / or a pro-inflammatory cytokine (e.g., interleukin-1α and -β, interleukin-6 and TNF-α, etc.) by an ISV, polypeptide or construct of the invention, as measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those described herein). In particular, "modulating" or "modulate" may mean reducing or inhibiting the activity of said member by 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, when measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those described herein) compared to the activity of said member in the same assay under the same conditions but without the presence of an immunoglobulin or polypeptide of the invention.

[0098] In the context of the present invention, "enhancing" or "to enhance" generally means increasing, potentiating or stimulating the activity of a polypeptide or construct of the invention as measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those described herein). In particular, it means that the activity of a polypeptide or construct of the invention, when measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those described herein), 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%, compared to the activity of the construct or polypeptide in the same assay under the same conditions but without the presence of an aggrecan-binding agent (e.g., an ISV that binds to aggrecan of the invention).

[0099] A "synergistic effect" of two compounds is one in which 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 invention, as used herein, is a function of the amount of ISV or polypeptide of the invention required to produce its specific effect, e.g., cartilage penetration, specific binding to aggrecan, and / or cartilage retention, etc., and as used in the Examples section, can be easily measured, e.g., by methods known to those of skill in the art.

[0101] In contrast, the "efficacy" of an ISV or polypeptide of the invention measures the maximum strength of its effect at saturating concentrations of the ISV or polypeptide. Efficacy indicates the maximum response that can be achieved from an ISV or polypeptide of the 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 MMP family member.

[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 encompassing the domain.

[0104] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that has the ability to retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for distinct functional properties of the protein and can often be added to or transferred to, or removed from, other proteins without loss of function of the remainder of the protein and / or domain.

[0105] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (such as a chain of a conventional four-chain antibody or of a heavy-chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized in that they consist of a bilayer sandwich of approximately seven antiparallel beta strands arranged as two beta sheets, characteristic of antibody molecules, retaining the immunoglobulin fold stabilized by optional conserved disulfide bonds.

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

[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 resides in and is formed by a single immunoglobulin domain. This distinguishes ISVs from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0108] In view of the above definition, the antigen-binding domain of a conventional four-chain antibody (e.g., an Ig, M, A, D or E molecule known in the art), or an Fv fragment such as a Fab fragment, a F(ab')2 fragment, a disulfide-linked Fv or scFv fragment, or a diabody derived from such a conventional four-chain antibody (all of which are known), is not generally considered to be an ISV, because in these cases, binding to each epitope of an antigen typically does not occur by one (single) immunoglobulin domain alone, but rather by a pair of (associated) immunoglobulin domains, e.g., a light chain and a heavy chain variable domain, i.e., the VH-VL immunoglobulin domains, which jointly bind to each epitope of the antigen.

[0109] In contrast, ISVs can specifically bind to an epitope of an antigen without combining with an additional 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 (e.g. a VL sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g. a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it is capable of forming a single antigen-binding unit (i.e. a functional antigen-binding unit that consists essentially of a single variable domain, where the single antigen-binding domain does not need to interact with other variable domains to form a functional antigen-binding unit).

[0111] In one embodiment of the invention, the ISV is a heavy chain variable domain sequence (e.g., a VH sequence), 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; LThese were chosen to distinguish them from the VL domains (also called "VL domains" or "VL domains"). For further description of VHHs and nanobodies, see, for example, the review by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as the following patent applications, which are also mentioned in the background: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 to 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 to Unilever; WO 02 / 48193 to Vlaams Instituut voor Biotechnologie (VIB); 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 from Algonomics NV and Ablynx NV; WO 03 / 050531 from Algonomics NV and Ablynx NV; WO 01 / 90190 from the National Research Council of Canada; WO 03 / 025020 (=EP 1433793) from the Institute of Antibodies, and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, and Further published patent applications by Ablynx NV may be cited. Reference may be made to further prior art described in these applications, in particular the list of references set out on pages 41-43 of WO 06 / 040153, which 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", in particular in one or more framework sequences. Further description of ISVs, Nanobodies, in particular 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 several linker sequences), and different modifications that increase the half-life of ISVs, Nanobodies and their preparation can be found, for example, in WO 08 / 101985 and WO 08 / 142164. For a further general description of nanobodies, reference is made to the prior art set out in WO 08 / 020079 (page 16), which is cited herein.

[0115] "Domain antibodies" (also known as "Dab"(s), "domain antibodies" and "dAb", the terms "domain antibody" and "dAb" being trademarks of the GlaxoSmithKline group of companies) are described, for example, in EP 0368684, Ward et al. (Nature 341: 544-546, 1989), Holt et al. (Tends in Biotechnology 21: 484-490, 2003), and WO 04 / 068820, as well as WO 06 / 030220, WO 06 / 003388 and WO 03 / 002609 and other published patent applications by Domantis. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammalian, in particular human, four-chain antibodies. To bind to an epitope as a single antigen-binding domain (i.e., not as a pair with each VL or VH domain), specific selection for such antigen-binding activity must be performed, for example, using a library of human single VH or VL domain sequences. Like VHHs, domain antibodies have a molecular weight of about 13 to 16 kDa, and if derived from entirely human sequences, they do not require humanization for, for example, human therapeutic use.

[0116] It should be noted that the single variable domains may also be derived from certain shark species, although these are less preferred in the context of the present invention as they are not of mammalian origin (e.g. the so-called "IgNAR domains", see WO05 / 18629).

[0117] Thus, within the meaning of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from camelid heavy chain antibodies of non-human origin, preferably from camelid sources. As mentioned above, they may be humanized. Furthermore, the term may include derived polypeptides of non-camelid (e.g. murine or human) origin that have been "camelized", for example, as described by 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 determined by the VHH sequence shown by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as well as by application to VHH domains derived from camelids as shown in Figure 2 of Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999). H Numbered according to the general numbering scheme for domains. V H Alternative methods for numbering the amino acid residues of a domain are known in the art, which are equally applicable to VHH domains. However, in the present specification, claims and figures, unless otherwise specified, we will follow the Kabat numbering system, as applied to VHH domains as described above.

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

[0120] CDR regions can be determined according to various methods. In determining the CDRs according to Kabat, FR1 of the VHH comprises amino acid residues at positions 1 to 30, CDR1 of the VHH comprises amino acid residues at positions 31 to 35, FR2 of the VHH comprises amino acid residues at positions 36 to 49, CDR2 of the VHH comprises amino acid residues at positions 50 to 65, FR3 of the VHH comprises amino acid residues at positions 66 to 94, CDR3 of the VHH comprises amino acid residues at positions 95 to 102, and FR4 of the VHH comprises amino acid residues at positions 103 to 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), where FR1 comprises amino acid residues at positions 1 to 25, CDR1 comprises amino acid residues at positions 26 to 35, FR2 comprises amino acid residues at positions 36 to 49, CDR2 comprises amino acid residues at positions 50 to 58, FR3 comprises amino acid residues at positions 59 to 94, CDR3 comprises amino acid residues at positions 95 to 102, and FR4 comprises amino acid residues at positions 103 to 113 (according to Kabat numbering).

[0122] ISVs, such as domain antibodies and nanobodies (including VHH domains), can be humanized. In particular, a humanized immunoglobulin single variable domain, such as a nanobody (including a VHH domain), may be an immunoglobulin single variable domain as generally defined in the previous paragraph, but with at least one amino acid residue (particularly at least one framework residue) that is a humanizing substitution (as defined herein) and / or corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions include substitutions of at least one amino acid residue (particularly at least one framework residue) that corresponds to a naturally occurring VHH domain. HH The framework region sequences of the sequences are compared with one or more closely related human V H The V sequences are compared to the corresponding framework sequences of the V sequences, and then one or more potentially useful humanizing substitutions (or combinations thereof) thus determined are added to the V sequences according to any of the known methods described herein. HH The resulting humanized V HHThe sequences can be confirmed by testing for affinity to the target, for stability, for ease and level of expression, and / or for other desired properties. In this way, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by those skilled in the art based on the disclosure herein, with reduced trial and error. Also based on the above, (the framework regions of) immunoglobulin single variable domains, such as nanobodies (including VHH domains), can be partially 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, which improve the affinity of the resulting immunoglobulin single variable domain for its respective antigen compared to the respective parent molecule. The affinity-matured immunoglobulin single variable domain molecules of the invention can be produced by methods known in the art, for example, as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas, et al. (Proc. Nat. Acad. Sci, USA 91: 3809-3813, 1994), Shier et al. (Gene 169: 147-155, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226: 889-896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, Oxford, 1995) and others. Press, 1996).

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

[0125] For example, and without limitation, one or more ISVs can be used as "binding units," "binding domains," or "building blocks" (these terms are used interchangeably) for the preparation of polypeptides, which may optionally contain one or more additional ISVs useful as binding units (i.e., for the same or other epitopes on aggrecan, and / or for one or more other antigens, proteins, or targets other than aggrecan).

[0126] The present invention provides aggrecan-binding agents, such as ISVs (also referred to herein as "ISVs of the invention") and / or polypeptides (also referred to herein as "polypeptides of the invention") that have specificity for and / or bind to 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). In humans, aggrecan is encoded by the ACAN gene, which is located on chromosome Chr15:q26.1.

[0127] Aggrecan is a large, multimodular molecule (2317 amino acids). Its core protein consists of three globular domains (G1, G2, and G3) and a large extended region between G2 and G3 where 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 hydrogel structure through its interaction with hyaluronan and linking proteins, which give cartilage its load-bearing properties. The G1 domain interacts with hyaluronan and linking proteins to form a stable ternary complex in the extracellular matrix (ECM). The G2 domain is homologous to the tandem repeats of G1 and linking proteins and is involved in product processing. G3 constitutes the carboxyl terminus of the core protein and enhances glycosaminoglycan modification and product secretion. The G3 domain also links proteoglycan aggregates to ECM proteins (fibulin and tenascin). Aggrecan degradation is thought to begin at the C-terminus. The population of aggrecan molecules without the G3 domain increases with age. Aggrecan interacts with laminin, fibronectin, tenascin, and collagen, but is also an enzymatic substrate for various A Disintegrin And Metalloprotease with Thrombospondin Motifs (ADAMTS), such as ADAMTS4, ADAMTS5, and ADAMTS11, and matrix metalloproteinases (MMPs), such as MMP8, MMP13, MMP19, and MMP20.

[0128] In one aspect, the invention relates to aggrecan-binding agents, such as ISVs and polypeptides, that specifically bind to aggrecan. The aggrecan-binding agents of the present invention are ultimately intended for use as pharmaceuticals in humans. Thus, in one aspect, the present invention relates to aggrecan-binding agents, such as ISVs and polypeptides, that specifically bind to human aggrecan (SEQ ID NO: 125). The present inventors have identified aggrecan-binding agents with highly improved species cross-reactivity and exquisite selectivity profiles.

[0129] Thus, in one aspect, the invention relates to an aggrecan binding agent, such as an ISV or polypeptide, which specifically binds to human aggrecan (P16112; SEQ ID NO: 125), dog 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 binding agents of the invention, such as the ISVs and / or polypeptides of the invention, have advantageous characteristics over prior art molecules; they are stable in joints, are retained in cartilage for long periods of time, are specific for cartilaginous tissue, and do not substantially bind, for example, to neurocan (O14594, SEQ ID NO: 134) and / or brevican (Q96GW7, SEQ ID NO: 135) (see Table B).

[0131] Thus, in one aspect, the present invention relates to an aggrecan binding agent, such as an ISV or polypeptide, wherein said aggrecan binding agent does not substantially bind to neurocan (O14594, SEQ ID NO: 134) and / or brevican (Q96GW7, SEQ ID NO: 135), preferably wherein said aggrecan is -5 higher than moles / liter, e.g., 10 -4 moles / liter K D values, and binds to neurocan and / or brevican.

[0132] In one aspect, the present invention relates to aggrecan binding agents, such as ISVs, that have greater than 10-fold, greater than 100-fold, preferably greater than 1000-fold selectivity for binding to aggrecan over neurocan and / or brevican.

[0133] Preferred aggrecan binding agents of the 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, such as disulfide-linked Fv or scFv fragments, or diabodies derived from such conventional four-chain antibodies, or individual chains thereof, as well as any portion, domain, or fragment thereof (including, but not limited to, antigen-binding domains or fragments, such as immunoglobulin single variable domains), monovalent polypeptides of the invention, or other binding agents).

[0134] Aggrecan-binding agents of the invention have been observed to have a pI of greater than 8, with only one exception (see Table 2.2). Without being bound by theory, the inventors hypothesize that the high positive charge of aggrecan may affect retention and cartilage penetration in its entirety, i.e., even when coupled to another entity such as in a multispecific polypeptide. Thus, the present invention relates to aggrecan-binding agents, such as ISVs, polypeptides or constructs of the invention, preferably ISVs of the invention, having a pI of 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 even greater, such as 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 or even 9.8.

[0135] The binding of the aggrecan-binding agents of the present invention, such as the ISVs and / or polypeptides of the present invention, to aggrecan can be measured in a variety of binding assays commonly known in the art. Exemplary assays include (but are not limited to) fluorescent ligand binding assays, fluorescence-activated cell sorting (FACS), radioligand binding assays, surface plasmon resonance (SPR), plasmon waveguide resonance (PWR), SPR imaging for affinity-based biosensors, whispering gallery microcavities (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 an aggrecan-binding agent of the invention, e.g., an ISV and / or polypeptide of the invention, to aggrecan is SPR, such as SPR as described in the Examples, where binding of an aggrecan-binding agent of the invention, e.g., an ISV and / or polypeptide of the invention, to aggrecan was determined. Some preferred K values ​​for binding of an aggrecan-binding agent of the invention, e.g., an ISV and / or polypeptide of the invention, to aggrecan are: D The value will become apparent from the further description and examples herein. Another particularly preferred assay is an ELISA as detailed in the examples (see Examples 1.2 and 2.4).

[0136] Binding of the aggrecan-binding agents of the present invention to aggrecan can also be measured in binding assays that preferably preserve the conformation of the aggrecan target. Exemplary 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 invention, the aggrecan-binding agent of the invention, such as an ISV and / or polypeptide of the invention, has a binding activity to said aggrecan of at least about 10, preferably as measured by surface plasmon resonance. 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 On-rate constants (K on )

[0138] In an embodiment of the invention, the aggrecan-binding agent of the invention, such as an ISV and / or polypeptide of the invention, exhibits a binding affinity to said aggrecan of about 10, preferably as measured by surface plasmon resonance. -3 s -1 Below, about 10 -4 s -1 Below, about 10 -5 s -1 Below, about 10 -6 s -1 Below, about 10 -7 s -1 Below, about 10 -8 s -1 Below, about 10 -9 s -1 Below, 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, 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 a binding activity of 10 to 15% of cynomolgus monkey (cyno) aggrecan. -7 M or less, preferably 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 polypeptides of the present invention may have a binding activity of 10 or more times higher than that of cynoaggrecan. -10 M~10 -7 M, e.g. 10 -10 M~10 -8 M, 10 -10 M~10 -9 M, EC 50 It may have a value.

[0142] In such 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 rat aggrecan of 10 -6 M or less, preferably 10 -7 M or less, preferably 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 polypeptides of the present invention may have a binding activity of 10 or more times higher than that of rat aggrecan. -10 M~10 -6 M, e.g. 10 -10 M~10 -7 M, 10 -10 M~10 -8 M, 10 -10 M~10 -9 M's EC 50 It may have a value.

[0143] In such an ELISA binding assay, an aggrecan binding agent of the invention, e.g., an ISV and / or polypeptide of the invention, which preferably binds to the G1 domain and / or the G1-IGD-G2 domain, exhibits a binding activity of 10 -6 M or less, preferably 10 -7 M or less, preferably 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 polypeptides of the present invention may have a binding activity of 10 or more to canine aggrecan. -10 M~10 -6 M, e.g. 10 -10 M~10 -7 M, 10 -10 M~10 -8 M, 10 -10 M~10 -9 M's EC 50 It may have a value.

[0144] In such 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 bovine aggrecan of 10 -6 M or less, preferably 10 -7 M or less, preferably 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 polypeptides of the invention may have a binding activity of 10 -10 M~10 -6 M, e.g. 10 -10 M~10 -7 M, 10 -10 M~10 -8 M, 10 -10 M~10 -9 M's EC 50It may have a value.

[0145] The term "cartilaginous tissue," as used herein, refers to cartilage, including elastic cartilage, hyaline cartilage, and fibrocartilage, which are defined by the ratio of cells (chondrocytes) to intercellular spaces and the relative amounts of collagen and proteoglycans. "Articular cartilage" is cartilage found on the articular surfaces of bones and is exclusively hyaline cartilage. The meniscus is composed entirely of fibrocartilage. Aggrecan is the major proteoglycan in the extracellular matrix (ECM), accounting for approximately 50% of the total protein content (the other approximately 50% is collagen II and several minor proteins, such as collagen IX).

[0146] The aggrecan binders of the invention have shown a preference for cartilaginous tissues in joints, such as cartilage and meniscus, over non-cartilaginous tissues, such as synovium, tendon, and / or epimysium. Accordingly, the invention relates to aggrecan binders, such as ISVs or polypeptides, wherein the aggrecan binders preferably bind to cartilaginous tissue, such as cartilage and / or meniscus, preferably at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or even more, compared to non-cartilaginous tissue.

[0147] A joint is understood to be the area where two or more bones meet. Most joints are mobile, allowing the bones to move. Joints consist of cartilage, synovial membrane, ligaments, tendons, bursae, and synovial fluid. Some joints also have menisci.

[0148] As shown in the examples, aggrecan binders of the invention possess diverse cartilage retention characteristics, allowing for 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 extended periods of time, even after a relatively short exposure of the aggrecan binder to cartilage, such as might be expected from intra-articular injection. Cartilage retention can be measured via an ex vivo cartilage retention assay, as described in the Examples section. The degree of retention can be measured by visual inspection of Western blots or via densitometric quantification. The scale used to determine the degree of retention can be defined in the art, for example, a scale of 0 to 6 RU (Retention Units), where 0 is no retention and 6 is complete retention in the assay. If necessary, the scale can be quantified using aggrecan binders of the invention, in which each aggrecan binder is assigned a score, e.g., complete retention and no retention are fixed. Alternatively, the scale can be set by assigning various intermediate scores via the aggrecan binders of the present invention, for example, an aggrecan binder containing two 114F08s (6RU) and a dummy aggrecan binder, such as ALB26-ALB26 (0RU); or an aggrecan binder containing two 114F08s (6RU); an aggrecan binder containing 608A05 (5RU); aggrecan binder 604G01 (4RU); an aggrecan binder containing two 601D02s (3RU); an aggrecan binder containing two 606A07s (2RU); aggrecan binder 112A01 (1RU); and a dummy aggrecan binder, such as ALB26-ALB26 (0RU). (See Table 2.2.) Thus, the present invention relates to an aggrecan binding agent, such as an ISV and / or a polypeptide according to the present invention, wherein said aggrecan binding agent 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 binders of the present invention should preferably be stable. As a first prerequisite, the biophysical properties of the aggrecan binders 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. The aggrecan binders were then tested for their long-term activity in the joint by incubation in synovial fluid at 37°C (see Example 6). No degradation of any of the constructs could be detected, indicating that the constructs were stable under conditions mimicking the in vivo situation.

[0150] In one aspect, the present invention relates to an aggrecan binding agent, e.g., an ISV, wherein the aggrecan binding agent has a stability in synovial fluid (SF) at 37°C for 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.

[0151] The present invention provides stretches of amino acid residues that are particularly suitable for binding to aggrecan (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). In particular, the present invention provides stretches of amino acid residues that bind to human aggrecan, wherein the binding of said stretches to said 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 constructs or polypeptides of the invention, in particular so that they form (part of) the antigen-binding site of the polypeptide of the invention. These stretches of amino acid residues may be present in and / or incorporated into the CDR sequences or VDR sequences of heavy chain antibodies raised against aggrecan. HHThese stretches of amino acid residues are also referred to herein as "CDR sequence(s) of the invention" (respectively "CDR1 sequence(s) of the invention", "CDR2 sequence(s) of the invention" and "CDR3 sequence(s) of the invention").

[0152] However, it should be noted that the present invention, in its broadest sense, is not limited to the specific structural role or function that these stretches of amino acid residues have in the polypeptides of the present invention, as long as these stretches of amino acid residues enable the polypeptides of the present invention to bind to aggrecan with the desired affinity and potency. Thus, in general, the present invention, in its broadest sense, provides polypeptides (herein also referred to as "polypeptides of the present invention") that are capable of binding to aggrecan with a specific affinity, avidity, efficacy, and / or potency and that comprise one or more CDR sequences described herein, in particular a suitable combination of two or more such CDR sequences, appropriately linked to each other via one or more additional amino acid sequences in such a manner that the entire polypeptide forms a binding domain and / or binding unit capable of binding to aggrecan. However, it should also be noted that the presence of only one such CDR sequence in the polypeptides of the present invention is sufficient to provide the binding ability of the polypeptides of the present invention to aggrecan, for example, the so-called "expedite fragments" described in WO 03 / 050531, again by reference thereto.

[0153] In a specific, but non-limiting aspect, the aggrecan-binding agents of the invention, such as the ISVs and / or polypeptides of the invention, may consist essentially of or comprise 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 4, 3, 2 or 1 amino acid(s) difference(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 4, 3, 2 or 1 amino acid(s) difference(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 4, 3, 2 or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 60; Preferably, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

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

[0155] In particular, the aggrecan-binding agents of the present invention, e.g., polypeptides and / or ISVs of the present invention, may be aggrecan-binding agents comprising one binding site, wherein the antigen-binding site comprises at least one stretch of amino acid residues selected from the group consisting of the CDR1 sequences, CDR2 sequences, and CDR3 sequences (or any suitable combination thereof) as described above. In a preferred aspect, however, the aggrecan-binding agents of the present invention, e.g., polypeptides and / or ISVs of the present invention, comprise more than one, e.g., two or more, stretches of amino acid residues selected from the group consisting of the CDR1 sequences of the present invention, the CDR2 sequences of the present invention, and / or the CDR3 sequences of the present invention. Preferably, the aggrecan-binding agents of the present invention, e.g., polypeptides and / or ISVs of the present invention, each comprise a stretch of three amino acid residues selected from the group consisting of the CDR1 sequences of the present invention, the CDR2 sequences of the present invention, and the CDR3 sequences of the present invention. The CDR combinations described herein as preferred for the aggrecan-binding agents of the present invention, e.g., polypeptides and / or ISVs of the present invention, are listed in Table A-2, i.e., preferably, are CDR combinations 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 to 19 and 114 to 118).

[0157] In a preferred embodiment, the present invention relates to an aggrecan-binding agent of the invention, such as an ISV and / or polypeptide of the 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 - 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, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0158] In a preferred embodiment, the present invention relates to an aggrecan-binding agent of the invention, such as an ISV and / or polypeptide of the 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, the aggrecan binding agent, such as an ISV and / or 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 present invention relates to an aggrecan binding agent, such as an ISV, wherein the ISV is selected from the group consisting of SEQ ID NOs: 117, 5, 118, 13, 114-116, 1-4, 6-12 and 14-19.

[0160] It should be further noted that the present invention is not limited by the origin of the aggrecan-binding agents of the invention, e.g., ISVs and / or polypeptides of the invention (or of the nucleic acids of the invention used to express them), nor by whether the aggrecan-binding agents of the invention, e.g., ISVs and / or polypeptides of the invention, or nucleic acids of the invention, are generated (or produced) or obtained (or obtained). Thus, the aggrecan-binding agents of the invention, e.g., ISVs and / or polypeptides of the invention, can be naturally occurring ISVs (from any suitable species) or synthetic or semi-synthetic ISVs and / or polypeptides.

[0161] Furthermore, it will be apparent to one skilled in the art that one or more of the CDRs described above can be "grafted" onto other "scaffolds", including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be apparent to those skilled in the art and are described, for example, in U.S. Pat. No. 7,180,370, WO 01 / 27160, EP 0605522, EP 0460167, U.S. Pat. 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 references therein. For example, the technique of grafting mouse or rat CDRs onto human frameworks and scaffolds is also known per se and can be used similarly to provide chimeric proteins comprising one or more of the CDR sequences defined herein for the monovalent polypeptides of the 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 herein above), 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), and 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 binding agents of the invention, such as the ISVs 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, which are preferably framework sequences or amino acid sequences that act as framework sequences or together form a scaffold for presenting the CDRs.

[0163] In a preferred embodiment, the aggrecan-binding agent of the invention, e.g., an ISV and / or 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 CDR1 sequence and one CDR2 sequence. In a particularly preferred, non-limiting embodiment, the aggrecan-binding agent of the invention, e.g., an ISV and / or polypeptide of the invention, has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein CDR1, CDR2, and CDR3 are as defined herein for the aggrecan-binding agent of the invention, e.g., an ISV and / or polypeptide of the invention, and FR1, FR2, FR3, and FR4 are framework sequences. In the aggrecan binding agents of the invention, e.g., the ISVs and / or polypeptides of the invention, the framework sequences may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person, e.g., from standard handbooks and the further disclosure herein and based on the prior art.

[0164] Thus, an aggrecan-binding agent of the invention, such as an ISV and / or polypeptide of the invention, comprises three complementarity determining regions (CDR1-CDR3, respectively), wherein: (i) CDR1 is (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 4, 3, 2, or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 24 or from any of SEQ ID NOs: 20-23, 25-37, and 109; and / or (ii) CDR2 is (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 4, 3, 2, or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 42 and any of SEQ ID NOs: 38-41, 43-55, and 110; 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) an amino acid sequence having 4, 3, 2, or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 60, or from any of SEQ ID NOs: 56-59, 61-74, and 111; selected from the group consisting of Preferably, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0165] The aggrecan binding agents of the present invention can be mapped to the G1 region, the G1-IGD-G2 region, or the G2 region of aggrecan. Thus, the present invention relates to aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the present invention, which bind to the G2 domain of aggrecan. As described in the examples, these aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the present invention have various preferred characteristics. Preferably, the aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the present invention have 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.

[0166] Comparison of the CDRs of the aggrecan-binding agents of the invention, such as the ISVs and / or polypeptides of the invention, revealed a number of amino acid changes that can be tolerated in the CDRs while retaining binding to the G2 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDRs of 601D02, which was used as a reference, is shown in Tables 1.5A, 1.5B, and 1.5C.

[0167] In one aspect, the invention relates to an aggrecan-binding agent, e.g., an ISV and / or polypeptide, of the invention, wherein: i) CDR1 is a) SEQ ID NOs: 28, 22, 26 and 33; 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 the amino acid difference(s) is / are defined as follows: - at position 1, G is changed to R; - in position 2, P is changed to S or R; - in position 3, T is changed to I; - in position 5, S is changed to N; - in position 6, R is changed to N, M or S; - in position 7, Y is changed to R or is absent; - in 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 c) SEQ ID NOs: 46, 40, 44 and 52; 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: 46, wherein the amino acid difference(s) are defined as follows: - in position 1, A is changed to S or Y; - in position 4, W is changed to L; - in position 5, S is changed to N; - in position 6, S is absent; - in position 7, G is absent; - at position 8, G is changed to A; - in position 9, R is changed to S, D or T; and / or - in position 11, Y is changed to N or R; and / or iii) CDR3 is e) SEQ ID NOs: 64, 58, 62 and 70; and f) An amino acid sequence having 5, 4, 3, 2 or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 64, wherein the amino acid difference(s) is / are defined as follows: - 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; - at position 7, T is changed to R, W or Y; - in position 8, Y is changed to R or S or is absent; - in position 9, T is changed to S or is absent; - in position 10, S is changed to E, K or is absent; - in position 11, E is changed to N, A or is absent; - in position 12, W is changed to D or is absent; - in position 13, N is changed to D or is absent; - in 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, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0168] In one aspect, the present invention provides a method for producing a composition comprising: - 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; The aggrecan-binding agent, e.g., ISV and / or polypeptide, of the invention is selected from the group of aggrecan-binding agents, wherein: Preferably, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0169] In one aspect, the present invention provides a method for producing a composition comprising: - 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; aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention, selected from the group of aggrecan-binding agents: Preferably, the aggrecan binding agent, such as an 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 binding agent, e.g., an ISV and / or polypeptide, of the present invention selected from the group consisting of SEQ ID NOs: 9, 3, 7 and 15, and aggrecan binding agents having more than 80%, such as 90% or 95%, sequence identity with any one of SEQ ID NOs: 9, 3, 7 and 15.

[0171] In one aspect, the present invention relates to an aggrecan-binding agent, such as an ISV and / or polypeptide, of the invention that 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 for binding to the G2-domain of aggrecan with an aggrecan-binding agent of the present invention, such as an ISV and / or polypeptide of the present invention preferably represented by any one of SEQ ID NOs: 9, 3, 7 and 15.

[0173] The present invention also relates to aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the present invention that bind to the G1-IGD-G2 domain of aggrecan. As described in the examples, these aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the present invention have various preferred characteristics. Preferably, the aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the present invention have 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.

[0174] Comparison of the CDRs of the aggrecan-binding agents of the invention, such as the ISVs and / or polypeptides of the invention, revealed amino acid changes that can be tolerated in the CDRs while retaining binding to the G1-IGD-G2 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDRs of 604F02, which was used as a reference, is presented in Tables 1.4A, 1.4B, and 1.4C.

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

[0176] In one aspect, the present invention provides a method for producing a composition comprising: - 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 NOs: 50, 41, 48 and 51; and - CDR3 is selected from the group consisting of SEQ ID NOs: 68, 59, 66 and 69; With respect to the aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention, Preferably, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0177] In one aspect, the present invention provides a method for producing a composition comprising: - 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; aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention, selected from the group of aggrecan-binding agents: Preferably, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0178] In one aspect, the present invention relates to an aggrecan binding agent, e.g., an ISV and / or polypeptide, of the present invention selected from the group consisting of aggrecan binding agents having SEQ ID NOs: 118, 13, 4, 11 and 14, and aggrecan binding agents having more than 80%, such as 90% or 95%, sequence identity with any one of SEQ ID NOs: 118, 13, 4, 11 and 14.

[0179] In one aspect, the present invention relates to an aggrecan-binding agent, such as an ISV and / or polypeptide, of the invention that 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 with an aggrecan-binding agent of the present invention, such as an ISV and / or polypeptide of the present invention, preferably represented by any one of SEQ ID NOs: 118, 13, 4, 11 and 14, for binding to the G1-IGD-G2 domain of aggrecan.

[0181] In particularly preferred embodiments, the present invention relates to aggrecan-binding agents of the present invention, e.g., ISVs and / or polypeptides of the present invention, that bind to the G1 domain of aggrecan. As described in the examples, these aggrecan-binding agents of the present invention, e.g., ISVs and / or polypeptides of the present invention, have a variety of preferred characteristics. Preferably, the aggrecan-binding agents of the present invention, e.g., ISVs and / or polypeptides, have 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.

[0182] Comparison of the CDRs of the aggrecan-binding agents of the invention, such as the ISVs and / or polypeptides of the invention, revealed a number of amino acid changes that can be tolerated in the CDRs while retaining binding to the G1 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDRs of 114F08, which was used as a reference, is shown in Tables 1.3A, 1.3B, and 1.3C.

[0183] In a preferred aspect, the invention relates to an aggrecan-binding agent of the invention, such as an ISV and / or polypeptide of the invention, comprising three complementarity determining regions (CDR1 to CDR3, respectively), wherein: i) CDR1 is a) SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36 and 37; 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: 24, wherein the amino acid difference(s) is / are defined as follows: - in position 2, S is changed to R, F, I or T; - in position 3, T is changed to I; - in position 5, I is changed to S; - in position 6, I is changed to S, R or M; - in 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 - in position 10, R is changed to G, K or A; and / or ii) CDR2 is c) SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54 and 55; 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, 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); - in position 3, S is changed to R, W, N or T; - in 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; - in position 7, N is changed to S, T or R; - in position 8, A is changed to T; and / or - in position 9, N is changed to D or Y; 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 5, 4, 3, 2 or 1 amino acid difference(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; - in 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; - in 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 - in position 13, Y is changed to L or is absent; selected from the group consisting of Preferably, the aggrecan binding agent, such as an ISV and / or 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 present invention relates to an aggrecan binding agent, e.g., an ISV and / or polypeptide, of the present invention, selected from the group of aggrecan binding agents 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 binding agent, e.g., an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1, FR2, FR3 and FR4 are framework sequences.

[0185] In a preferred aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - 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; aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention, selected from the group of aggrecan-binding agents: Preferably, the aggrecan binding agent, such as an ISV and / or 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 represents a family or set of clones grouped based on similarity in CDRs (which reflects similarity in functional characteristics) that further includes clones 114A09 (SEQ ID NO: 114) and 114B04 (SEQ ID NO: 115) (see Table A-2 and Tables 3.3A, 3.3B, and 3.3C). Thus, in another particularly preferred aspect, the present invention relates to an aggrecan-binding agent, e.g., an ISV and / or polypeptide, of the invention, comprising three complementarity-determining regions (CDR1-CDR3, respectively), wherein: i) CDR1 is selected from the group consisting of: a) SEQ ID NOs: 24 and 109; and b) an amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 24, wherein the amino acid difference(s) is / are defined as follows: - in 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; 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, wherein the amino acid difference(s) is / are defined as follows: - in position 1, T is changed to A; - in position 3, S is changed to R; - in position 4, S is changed to T; - in position 8, A is changed to T; and / or - in 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; and f) An amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) is / are defined as follows: - in position 4, H is changed to R; and / or - in position 8, V is changed to D; Preferably, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0187] In one aspect, the present invention provides a method for producing a composition comprising: - CDR1 is selected from the group consisting of SEQ ID NO: 24 and 109; - CDR2 is selected from the group consisting of SEQ ID NOs: 42 and 110; and - CDR3 is selected from the group consisting of SEQ ID NOs: 60 and 111; aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention, selected from the group of aggrecan-binding agents: Preferably, the aggrecan binding agent, such as an ISV and / or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1, FR2, FR3 and FR4 are framework sequences.

[0188] Furthermore, in the Examples section, it is shown that aggrecan-binding agents that bind to the G1 region of aggrecan and belong to epitope bin 1 or epitope bin 4 are particularly effective in cartilage retention assays. In one aspect, the present invention relates to aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention that belong to epitope bin 1 or epitope bin 4.

[0189] Comparison of the CDRs of aggrecan-binding agents of the invention, such as ISVs and / or polypeptides of the invention, belonging to epitope bin 1 revealed a number of amino acid changes that can be tolerated in the CDRs while retaining binding to the G1 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDRs of 608A05, which was used as a reference, is presented in Tables 2.3D, 2.3E and 2.3F.

[0190] In a preferred aspect, the invention relates to an aggrecan-binding agent, e.g., an ISV and / or polypeptide, of the invention, comprising three complementarity determining regions (CDR1 to CDR3, respectively), wherein: i) CDR1 is a) SEQ ID NOs: 36, 20 and 29; and b) an amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 36, wherein the amino acid difference(s) is / are defined as follows: - in position 3, T is changed to S; - in position 6, T is changed to S; - at position 8, T is changed to A; and / or - in position 9, M is changed to V; and / or ii) CDR2 is c) SEQ ID NOs: 54, 38 and 37; and d) An amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 54, wherein the amino acid difference(s) is / are defined as follows: - In position 1, A is changed to I; - in position 4, W is changed to R; - at position 7, G is changed to R; and / or - in position 8, T is changed to S; and / or iii) CDR3 is e) SEQ ID NOs: 73, 56 and 65; and f) An amino acid sequence having 5, 4, 3, 2, or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 73, wherein the amino acid difference(s) is / are defined as follows: - in position 1, R is changed to G; - in position 2, P is changed to R or L; - in position 3, R is changed to L or S; - in position 5, Y is changed to R; - in position 6, Y is changed to S or A; - at position 7, Y is changed to T or is absent; - in position 8, S is changed to P; - in position 9, L is changed to H or R; - in position 10, Y is changed to P or A; - in position 11, S is changed to A or Y; - at position 12, Y is changed to D; - in position 13, D is changed to F; - at position 14, Y is changed to G or is absent; and / or - After position 14, S is inserted; selected from the group consisting of Preferably, the aggrecan binding agent, such as an ISV and / or 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 provides a method for producing a composition comprising: - 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; aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention, selected from the group of aggrecan-binding agents: Preferably, the aggrecan binding agent, such as an ISV and / or 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 invention relates to aggrecan-binding agents, such as ISVs and / or polypeptides of the invention, belonging to epitope bin 1, which cross-block 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 binding agent of the present invention, such as an ISV and / or polypeptide belonging to epitope bin 1, preferably represented by any one of SEQ ID NOs: 1, 10 and 18.

[0194] Comparison of the CDRs of aggrecan-binding agents of the invention, such as ISVs and / or polypeptides of the invention, belonging to epitope bin 4 revealed a number of amino acid changes that can be tolerated in the CDRs while retaining binding to the G1 domain of aggrecan. The sequence variability in the CDRs of all clones relative to the CDRs of 114F08, which was used as a reference, is presented in Tables 2.3A, 2.3B and 2.3C.

[0195] In one aspect, the invention relates to an aggrecan-binding agent, e.g., an ISV and / or polypeptide, of the invention, comprising three complementarity determining regions (CDR1-CDR3, respectively), wherein: i) CDR1 is a) SEQ ID NOs: 24, 25 and 27; and b) an amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 24, wherein the amino acid difference(s) is / are defined as follows: - in position 2, S is changed to I or F; - in position 5, I is changed to S; - in position 6, I is changed to S or M; - in position 7, N is changed to R or Y; - in position 8, V is changed to A or Y; - at position 9, V is changed to M; and / or - in position 10, R is changed to K; and / or ii) CDR2 is c) SEQ ID NOs: 42, 43 and 45; 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, wherein the amino acid difference(s) is / are defined as follows: - at position 1, T is changed to A or G; - N is inserted between positions 2 and 3 (position 2a, Table 2.3B); - in position 7, N is changed to R; - in position 8, A is changed to T; and / or - in position 9, N is changed to D; and / or iii) CDR3 is e) SEQ ID NOs: 60, 61 and 63; and f) An amino acid sequence having 5, 4, 3, 2 or 1 amino acid difference(s) from the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) is / are defined as follows: - in position 1, P is absent; - in position 2, T is changed to R or is absent; - in position 3, T is changed to M or is absent; - in position 4, H is changed to D or Y; - in 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; - in position 9, Y is changed to R; - in position 10, Y is changed to N or E; - at position 11, G is changed to S or K; - at position 12, P is changed to E or is absent; and / or - in position 13, Y is changed to L or is absent; selected from the group consisting of Preferably, the aggrecan binding agent, such as an ISV and / or 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 provides a method for producing a composition comprising: - 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; aggrecan-binding agents, e.g., ISVs and / or polypeptides, of the invention, selected from the group of aggrecan-binding agents: Preferably, the aggrecan binding agent, such as an ISV and / or 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 aggrecan-binding agents, such as ISVs and / or polypeptides of the present invention, belonging to epitope bin 4, which cross-block 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.

[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 epitope bin 4 of the G1-domain of aggrecan and competes for binding to the G1-domain of aggrecan with an aggrecan-binding agent 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 invention relates to aggrecan-binding agents, e.g., ISVs and / or polypeptides of the invention, selected from the group consisting of aggrecan-binding agents represented by SEQ ID NOs: 117, 118, 116, 114, 115, 5, 13, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19, and ISVs having more than 80%, such as 90%, or 95%, or even more, sequence identity to 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, non-limiting aspect, the aggrecan-binding agent of the present invention may be a stretch of amino acid residues comprising an immunoglobulin fold, or an aggrecan-binding agent capable of forming an immunoglobulin fold (i.e., by folding) under appropriate conditions (such as physiological conditions). Reference is made, inter alia, to the review by Halaby et al. (J. Protein Eng. 12: 563-71, 1999). Preferably, when properly folded to form an immunoglobulin fold, the stretch of amino acid residues is capable of properly forming an antigen-binding site for binding to aggrecan. Thus, in a preferred aspect, the aggrecan-binding agent of the present invention is an immunoglobulin, such as an immunoglobulin single variable domain.

[0201] Thus, the framework sequences are preferably (suitable combinations of) immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences (e.g., by sequence optimization (e.g., humanization or camelization)). For example, the framework sequences may be those of the light chain variable domain (e.g., V L immunoglobulin single variable domains, such as V H In a particularly preferred aspect, the framework sequences may be derived from V HH a framework sequence derived from a camelized conventional V (as defined herein) sequence (which framework sequence may optionally be partial or complete) H It can be either an array.

[0202] The framework sequences are preferably selected so that the monovalent polypeptides of the invention are capable of being used as ISVs, such as domain antibodies (or amino acid sequences 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®, V HH Sequence, humanized V HHSequence, camelization V H V obtained by sequence or affinity maturation HH and suitable framework sequences will be clear to the skilled person, for example from standard handbooks and based on the further disclosure herein and the prior art.

[0203] Another particularly preferred class of ISVs of the present invention are naturally occurring V H The amino acid sequence of the V domain corresponds to that of the V domain, but is "camelized", i.e., the naturally occurring V domain from a conventional four-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain are HH The term "camelizing" includes ISVs having an amino acid sequence in which one or more amino acid residues occurring at the corresponding position(s) in the V domain are replaced by one or more of the amino acid residues occurring at the corresponding position(s) in the V domain. This can be done in a manner known per se, which will be clear to the skilled person, for example, based on the description herein. Such "camelizing" substitutions are preferably H -V L The insertions are made at amino acid positions that form and / or are present in the interface and / or at the so-called Camelidae 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 Vs used as starting material or starting points for generating or designing camelized ISVs are inserted at positions of amino acids that form and / or are present in the interface and / or at the so-called Camelidae 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). H The sequence is preferably a V from a mammal H sequence, more preferably human V H Array, e.g. V H However, such camelized ISVs of the invention can be obtained in any suitable manner known per se and therefore, strictly speaking, can be obtained from naturally occurring V H It should be noted that the invention is not limited to polypeptides obtained using a polypeptide containing the domain as a starting material.

[0204] For example, again as further described herein, both "humanized" and "camelized" refer to naturally occurring V HH Domain or V H This can be done by providing a nucleotide sequence encoding the ISV of the invention, and then altering one or more codons in said nucleotide sequence in a manner known per se, so that the new nucleotide sequence encodes a "humanized" or "camelized" 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, the respective naturally occurring V HH Domain or V H Based on the amino acid sequence of the domain, the amino acid sequence of the desired humanized or camelized ISV of the present invention can be designed and then synthesized de novo using techniques for peptide synthesis known per se. HH Domain or V H Based on the amino acid or nucleotide sequence of the domain, a nucleotide sequence encoding the desired humanized or camelized ISV of the invention, respectively, can then be designed and synthesized de novo using techniques for nucleic acid synthesis known per se, and the nucleic acid so obtained can then be expressed in a manner known per se to provide the desired ISV of the invention.

[0205] In particular, framework sequences present in the aggrecan binding agents of the invention, e.g., the ISVs and / or polypeptides of the invention, may comprise one or more of the hallmark residues, e.g., as defined in WO 08 / 020079 (Tables A-3 to A-8), such that the aggrecan binding agents of the invention are Nanobodies. Some preferred, but non-limiting, examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein (see, e.g., Table A-2). Generally, Nanobodies (especially V HHNanobodies (e.g., partially humanized Nanobodies) can be characterized in particular by the presence of one or more "hallmark residues" in one or more of the framework sequences (e.g., as further described in WO 08 / 020079, page 61, line 24 to page 98, line 3). As used herein, "represented by" in relation to any SEQ ID NO: is equivalent to "comprising or consisting of" said SEQ ID NO:, and preferably is equivalent to "consisting of" said SEQ ID NO:.

[0206] More particularly, the present invention provides compounds having the (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and providing an aggrecan-binding agent comprising at least one ISV having an amino acid sequence of wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and which are: i) have at least 80%, more preferably 90%, even more preferably 95% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 117, 116, 118, 116, 115, 114 and 1-19 (see Table A-2), where, for the purposes of determining the degree of amino acid identity, the amino acid residues forming the CDR sequences are disregarded. In this regard, reference is also made to Table A-2 which lists 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; As well as 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 the Kabat numbering are selected from hallmark residues, e.g., as set forth in Tables A-3 to A-8 of WO 08 / 020079.

[0207] Thus, the present invention relates to ISVs and / or polypeptides, wherein said ISVs essentially consist of four framework regions (FR1 to FR4, respectively) and the three complementarity determining regions CDR1 to CDR3, e.g., an ISV that specifically binds to aggrecan consists of four framework regions (FR1 to FR4, respectively) and the three complementarity determining regions CDR1 to CDR3, and therapeutic ISVs, e.g., a member of the serine protease family, a cathepsin, a matrix metalloproteinase (MMP) / matrixin, or a disintegrin and metalloproteinase with thrombospondin, ISVs that bind to ADAMTS motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1), and / or ADAMTS11, consist of four framework regions (FR1 to FR4, respectively) and the three complementarity-determining regions (CDR1 to CDR3, respectively); ISVs that bind to serum albumin essentially consist of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively).

[0208] Aggrecan binding agents of the invention, such as ISVs and / or polypeptides of the invention, may also include specific mutations / amino acid residues described in the following co-pending U.S. provisional applications, all entitled "Improved immunoglobulin variable domains": US 61 / 994552, filed May 16, 2014; US 61 / 014,015, filed June 18, 2014; US 62 / 040,167, filed August 21, 2014; and US 62 / 047,560, filed September 8, 2014 (all assigned to Ablynx NV).

[0209] In particular, aggrecan binding agents of the invention, such as ISVs and / or polypeptides of the invention, may suitably comprise: (i) K or Q at position 112; or (ii) K or Q at position 110 combined 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 aforementioned co-pending U.S. provisional application, when an aggrecan-binding agent of the invention, e.g., an ISV and / or polypeptide of the invention, comprises one of the mutations (or a suitable combination thereof) in accordance with (i)-(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 selected from A or P; and - the amino acid residue at position 41 is preferably selected from A or P; and - the amino acid residue at position 89 is preferably selected from T, V or L; and - the amino acid residue at position 108 is preferably selected from Q or L; and - the amino acid residue at position 110 is preferably 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 aforementioned co-pending U.S. provisional application, the aforementioned mutations are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to the ISVs, polypeptides, and constructs of the invention. To this end, the aggrecan-binding agents of the invention, such as the ISVs and / or polypeptides of the invention, may also (optionally in combination with the aforementioned mutations) contain a C-terminal extension (X) n wherein n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), in which regards again reference is made to said U.S. provisional application and to WO 12 / 175741. In particular, the aggrecan-binding agents of the invention, such as the ISVs and / or polypeptides of the invention, may comprise such a C-terminal extension when it forms the C-terminal end of a protein, polypeptide, or other compound or construct comprising it (again, as further described in said U.S. provisional application and WO 12 / 175741).

[0212] The aggrecan binding agents of the present invention may be immunoglobulins, such as ISVs, derived in any suitable manner and from any suitable source, including naturally occurring V HHThe amino acid sequences may be humanized (i.e., from a suitable species of Camelidae), synthetic or semi-synthetic, including but not limited to "humanized" (as defined herein) Nanobodies or VHH sequences, "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), and Nanobodies obtained by affinity maturation (e.g., starting 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 engineering immunoglobulin sequences well known to those skilled in the art; or any suitable combination of any of the foregoing, as further described herein. Also, the immunoglobulin may be a VHH sequence. HH If the immunoglobulin comprises a synthetic or semi-synthetic sequence, such as a partially humanized sequence, then said immunoglobulin may optionally be further suitably humanized to provide one or more additional (partially or fully) humanized immunoglobulins of the invention, as further described herein. Similarly, if the immunoglobulin comprises a synthetic or semi-synthetic sequence, such as a partially humanized sequence, then said immunoglobulin may optionally be further suitably humanized, again as described herein, to provide one or more additional (partially or fully) humanized immunoglobulins of the invention.

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

[0214] ISVs can be used as "building blocks" for the preparation of polypeptides, which may optionally contain one or more further "building blocks", e.g. ISVs, e.g. building blocks with a therapeutic mechanism of action, e.g. therapeutic ISVs, directed against the same or another epitope on aggrecan and / or against one or more other antigens, proteins or targets other than aggrecan.

[0215] Generally, a protein or polypeptide or construct comprising or consisting essentially of a single component, a single ISV, or a single Nanobody will be referred to herein as a "monovalent" protein or polypeptide, or a "monovalent construct," respectively. A polypeptide or construct comprising two or more components or binding units (such as ISVs) will also be referred to herein as a "multivalent" polypeptide or construct, and the components / ISVs present in such a polypeptide or construct will also be referred to herein as being in a "multivalent format." For example, a "bivalent" polypeptide may comprise two ISVs, optionally linked via linker sequences, while a "trivalent" polypeptide may comprise three ISVs, optionally linked via two linker sequences; while a "tetravalent" polypeptide may comprise four ISVs, optionally linked via three linker sequences, etc.

[0216] In a multivalent polypeptide or construct, the two or more ISVs, e.g., Nanobodies, may be the same or different and may be directed against the same antigen or antigenic determinant (e.g., against the same moiety(s) 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), where at least one component is directed against a first antigen (i.e., aggrecan) and at least one component is directed against a second antigen (i.e., one different from aggrecan, such as a therapeutic target), will also be referred to as a "multispecific" polypeptide or multispecific construct, respectively, and the components (e.g., ISVs) present in such a polypeptide or construct will also be referred to herein as being in a "multispecific format". Thus, for example, a "bispecific" polypeptide of the invention is one that comprises at least one ISV directed against a first antigen (i.e., aggrecan) and at least one further ISV directed against a second antigen (i.e., one different from aggrecan, such as a therapeutic target), whereas a "trispecific" polypeptide of the invention is one that comprises at least one ISV directed against a first antigen (i.e., aggrecan), at least one further ISV directed against a second antigen (i.e., one different from aggrecan, such as a therapeutic target), and at least one further ISV directed against a third antigen (i.e., one different from both aggrecan and the second antigen); etc.

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

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

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

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

[0221] In one aspect, in its simplest form, a multivalent polypeptide or construct of the invention is a bivalent polypeptide or construct of the invention comprising a first ISV, e.g., a Nanobody, directed against aggrecan, and an identical second ISV, e.g., a Nanobody, directed against aggrecan, wherein said first and said second ISV, e.g., a Nanobody, are optionally linked via a linker sequence (as defined herein). In another form, a multivalent polypeptide or construct of the invention may be a trivalent polypeptide or construct of the invention comprising a first ISV, e.g., a Nanobody, directed against aggrecan, an identical second ISV, e.g., a Nanobody, directed against aggrecan, and a third ISV, e.g., a Nanobody, directed against an antigen other than aggrecan, such as a therapeutic target, wherein said first, second, and third ISVs, e.g., Nanobodies, are optionally linked via one or more, in particular two, linker sequences.

[0222] In another aspect, the multivalent polypeptide or construct of the invention may be a bispecific polypeptide or construct of the invention comprising a first ISV, e.g., a Nanobody, directed against aggrecan, and a second ISV, e.g., a Nanobody, directed against a second antigen, such as a therapeutic target, wherein said first and second ISV, e.g., a Nanobody, may optionally be linked via a linker sequence (as defined herein); on the other hand, the multivalent polypeptide or construct of the invention may also be a trispecific polypeptide or construct of the invention comprising a first ISV, e.g., a Nanobody, directed against aggrecan, a second ISV, e.g., a Nanobody, directed against a second antigen, such as a therapeutic target, and a third ISV, e.g., a Nanobody, directed against a third antigen, e.g., also a therapeutic target but different from said second antigen, wherein said first, second, and third ISV, e.g., a Nanobody, may optionally be linked via one or more, in particular two, linker sequences.

[0223] In a preferred aspect, the polypeptide or construct of the invention is a trivalent bispecific polypeptide or construct, respectively. In its simplest form, a trivalent bispecific polypeptide or construct of the invention may be a trivalent polypeptide or construct of the invention (as defined herein) comprising two identical ISVs, such as nanobodies, against aggrecan and a third ISV, such as nanobody, directed against another antigen, such as a therapeutic target, wherein said 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 a trivalent bispecific polypeptide or construct, respectively. A trivalent bispecific polypeptide or construct of the invention may be a trivalent polypeptide or construct (as defined herein) of the invention comprising two ISVs against aggrecan, such as nanobodies (wherein said ISVs against aggrecan may be the same or different), and a third ISV, such as nanobody, directed against another antigen, such as a therapeutic target, wherein said 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 shown in the examples described herein and in Tables E-1 and E-2.

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

[0226] In a preferred aspect, a multivalent polypeptide or construct of the invention comprises or essentially consists of two or more ISVs directed against aggrecan. In one aspect, the invention relates to a polypeptide or construct comprising or essentially consists of at least two aggrecan-binding ISVs according to the invention, e.g., two, three, or four ISVs (or suitable fragments thereof). The two or more ISVs may optionally be linked via one or more peptidic linkers.

[0227] The two or more ISVs present in a multivalent polypeptide or construct of the invention may be light chain variable domain sequences (e.g., V L sequence), or a heavy chain variable domain sequence (e.g., V H They may consist of heavy chain variable domain sequences derived from conventional four-chain antibodies, or they may consist of heavy chain variable domain sequences derived from heavy chain antibodies. In preferred aspects, 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), or Nanobodies® (including but not limited to V HH Humanized V HH Even if it consists of a sequence, camelization V H or V obtained by affinity maturationHH The two or more ISVs may consist of partially or fully humanized nanobodies or partially or fully humanized VHHs.

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

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

[0230] In a preferred aspect, the polypeptide or construct of the invention comprises or consists essentially of at least two ISVs, which may be the same or different and which are independently selected from the group consisting of SEQ ID NOs: 117, 118, 116, 115 and 1-19, more preferably, said at least two ISVs are selected from the group consisting of SEQ ID NOs: 117, 5, 6, 8, 114-116, and / or said at least two ISVs are selected from the group consisting of SEQ ID NOs: 118 and 13.

[0231] In a further aspect, the present invention relates to a multiparatopic (preferably biparatopic) polypeptide or construct comprising two or more immunoglobulin single variable domains directed against aggrecan that bind to the same epitope(s), such as those bound by any one of SEQ ID NOs: 117, 118, 114, 115, 116 and 1-19.

[0232] It is understood that the final form of the molecule for clinical use will contain one or two components that bind to aggrecan, e.g., ISVs, and one or more components that have a therapeutic mode of action, e.g., ISVs, and possibly additional moieties. In the Examples section, it will be shown that such a form retains both aggrecan binding and retention properties, as well as a therapeutic effect, e.g., enzymatic and / or inhibitory function. The one or more components that have a therapeutic mode of action, e.g., ISVs, may be any component ("therapeutic component" or "therapeutic ISV") that has a therapeutic effect in diseases involving aggrecan, such as arthritis, osteoarthritis, spondyloepiphyseal dysplasia, lumbar disc degenerative disease, osteoarthritis, rheumatoid arthritis, osteochondritis dissecans, aggrecanopathy, diseases in which aggrecan is used to direct, anchor, and / or retain other, e.g., therapeutic, components at a desired site (e.g., in a joint). The present invention therefore relates to a polypeptide or construct according to the invention, wherein one or more further components, such as further ISVs, retain activity.

[0233] The present invention relates to polypeptides or constructs comprising or essentially consisting 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 further ISV, in particular a therapeutic ISV, wherein said at least one further ISV preferably binds to a therapeutic target, for example 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.

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

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

[0236] The stability of a polypeptide, construct or ISV of the invention can be measured by conventional assays known to those skilled in the art. Exemplary assays include (but are not limited to) assays in which the activity of the polypeptide, construct or ISV is determined, followed by incubation in synovial fluid for a desired period of time and subsequent activity determination, for example as detailed again in the Examples section (see Example 6).

[0237] The desired activity of the therapeutic components of the multivalent polypeptides or constructs of the invention can be measured by conventional assays known to those skilled in the art, such as those in which GAG release is assayed, as detailed in the Examples section (see Example 8).

[0238] The relative affinity may depend on the position of the ISVDs in the polypeptide. The order (orientation) of the ISVDs in the polypeptides of the invention can be selected according to the needs of the skilled artisan. The order of the individual ISVDs, as well as whether the polypeptide includes a linker, are matters of design choice. Some orientations, with or without a linker, may provide preferred 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 polypeptides of the invention may be (from N-terminus to C-terminus): (i) ISV1 (e.g., Nanobody1)-[linker]-ISV2 (e.g., Nanobody2)-[C-terminal extension]; or (ii) ISV2 (e.g., Nanobody2)-[linker]-ISV1 (e.g., Nanobody1)-[C-terminal extension]; (where the portion between the square brackets, i.e., the linker and the C-terminal extension, are optional). All orientations are encompassed by the invention. Polypeptides containing ISV orientations that provide the desired binding characteristics can be readily identified by routine screening, for example, as illustrated in the Examples section. A preferred order, from N-terminus to C-terminus, is: therapeutic ISV-[linker]-ISV that binds to aggrecan-[C-terminal extension], where the portion between the brackets is optional. Another preferred order, from N-terminus to C-terminus, is: therapeutic ISV-[linker]-ISV that binds to aggrecan-[linker]-ISV that binds to aggrecan-[C-terminal extension], where the portion between the brackets is optional.

[0239] Aggrecan-binding agents of the invention, e.g., polypeptides and / or ISVs of the invention, may or may not further comprise one or more other groups, residues (e.g., amino acid residues), moieties, or binding units (these aggrecan-binding agents, e.g., polypeptides and / or ISVs, with or without the additional groups, residues, moieties, or binding units, are all referred to as "compound(s) of the invention," "construct(s) of the invention," and / or "polypeptide(s) of the invention"). If present, such additional groups, residues, moieties, or binding units may or may not provide additional functionality to the aggrecan-binding agent, such as a polypeptide and / or ISV, or may or may not modify the aggrecan-binding properties of the polypeptide and / or ISV.

[0240] For example, such further groups, residues, moieties or binding units may be one or more further amino acid sequences, such that the resulting polypeptide is a (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulins. Even more preferably, said one or more other groups, residues, moieties or binding 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 VHH, humanized VHH or camelized VH sequences).

[0241] As mentioned above, additional binding units, e.g., ISVs with different antigen specificities, can be linked to form multispecific polypeptides. By linking ISVs of two or more specificities, bispecific, trispecific, etc. polypeptides or constructs can be formed. For example, a polypeptide of the invention may comprise two or more ISVs directed against aggrecan and at least one ISV directed against another target. All such constructs and modifications thereof are encompassed (as would be readily envisioned by one skilled in the art) by the term "compounds of the invention, constructs of the invention, and / or polypeptides of the invention" as used herein.

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

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

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

[0245] For example, such an amino acid sequence may be one that increases half-life, solubility or absorption, decreases immunogenicity or toxicity, eliminates or reduces undesirable side effects, and / or confers other advantageous properties and / or reduces undesirable properties of the compounds, constructs or polypeptides of the invention compared to the polypeptides of the invention themselves. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see, e.g., WO 00 / 27435) or hapten molecules (e.g., haptens recognized by circulating antibodies, see, e.g., WO 98 / 22141).

[0246] In certain aspects of the invention, constructs or polypeptides of the invention may have a moiety that confers an extended half-life compared to a corresponding construct or polypeptide of the invention that does not contain that moiety. Some preferred, but non-limiting, examples of such constructs and polypeptides of the invention will be apparent to those skilled in the art based on the further disclosure herein, and include, for example: an ISV or polypeptide of the invention that has been chemically modified (e.g., by pegylation) to extend its half-life; an aggrecan binding agent of the invention, such as an ISV and / or polypeptide of the invention, that comprises at least one additional binding site for binding to a serum protein (such as serum albumin); or a polypeptide of the invention that comprises at least one amino acid sequence of the invention linked to at least one moiety (and particularly 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, e.g. polypeptides of the invention, comprising such half-life extending moieties or ISVs will be clear to the skilled person based on the further disclosure herein; and include, for example, 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 binding units capable of binding to one or more serum proteins (e.g. domain antibodies, ISVs suitable for use as domain antibodies, single domain antibodies, ISVs suitable for use as single domain antibodies, dAbs, ISVs suitable for use as dAbs, or nanobodies capable of binding to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin; reference is made to the further description and references set out herein); polypeptides in which the amino acid sequence of the invention is linked to an Fc portion (such as human Fc) or suitable portion or fragment thereof; or small proteins or peptides capable of binding to one or more serum proteins, e.g. 01 / 45746, WO 02 / 076489, WO2008 / 068280, WO2009 / 127691 and PCT / EP2011 / 051559.

[0247] In one aspect, the invention provides a construct, e.g., a polypeptide, of the invention, 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, a construct or polypeptide of the invention having an extended half-life preferably has a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times, or more than 20 times longer than the half-life of the corresponding construct or polypeptide of the invention per se, i.e., one that does not include the moiety that confers the extended half-life. For example, a construct or polypeptide of the invention having an extended half-life may have a half-life that is extended by 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48, or 72 hours, e.g., in humans, compared to the corresponding construct or polypeptide of the invention per se, i.e., one that does not include the moiety that confers the extended half-life.

[0249] In a preferred, but non-limiting aspect of the invention, the constructs of the invention, e.g., polypeptides of the invention, have a serum half-life, e.g., in humans, that is extended by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding construct or polypeptide of the invention itself, i.e., without the moiety that confers the extended half-life.

[0250] In another preferred, but non-limiting, aspect of the present invention, such a construct of the present invention, e.g., a polypeptide of the present invention, exhibits a serum half-life in humans of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 72 hours or longer. For example, a compound or polypeptide of the present invention may have a half-life of at least 5 days (e.g., about 5-10 days), preferably at least 9 days (e.g., about 9-14 days), more preferably at least about 10 days (e.g., about 10-15 days), or at least about 11 days (e.g., about 11-16 days), more preferably at least about 12 days (e.g., about 12-18 days or longer), or longer than 14 days (e.g., about 14-19 days).

[0251] In a particularly preferred, but non-limiting aspect of the invention, the invention provides a construct of the invention, e.g., a polypeptide of the invention, comprising, in addition to one or more components that bind to aggrecan and optionally one or more therapeutic components, at least one component that binds to serum albumin, e.g., an ISV that binds to serum albumin (such as human serum albumin), as described herein, wherein the serum albumin-binding ISV comprises or essentially consists 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 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, eg, a polypeptide, of the invention comprising a serum protein binding moiety, wherein said serum protein binding moiety is a non-antibody-based polypeptide.

[0253] In one aspect, the invention relates to compounds or constructs as described herein that comprise one or more other groups, residues, moieties or binding units, such as: Preferably, it is selected from the group consisting of a polyethylene glycol molecule, a serum protein or a fragment thereof, a binding unit capable of binding to a serum protein, an Fc portion, and a small protein or peptide capable of binding to a serum protein.

[0254] In one aspect, the invention relates to a construct, e.g., a polypeptide, of the invention comprising a moiety that confers half-life extension, wherein said moiety is PEG. Thus, the invention relates to a construct or polypeptide of the invention comprising PEG.

[0255] The 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) may contain an N-terminal Met residue, e.g., as a result of expression in a heterologous host cell or host organism; b) may form a signal or leader sequence that directs secretion of the polypeptide from a host cell after synthesis (e.g., to provide a pre-, pro-, or prepro-form of a polypeptide of the invention, depending on the host cell used to express the polypeptide). Suitable secretory leader peptides will be apparent to those skilled in the art and may be those further described herein. Typically, such leader sequences are linked to the N-terminus of the polypeptide, although the invention in its broadest sense is not limited thereto; c) For example, affinity techniques directed against said sequences or residues may be used to form "tags", e.g., amino acid sequences or residues that allow or facilitate purification of the polypeptide. Said sequences or residues can then 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 or polypeptide sequence via a cleavable linker sequence or may comprise a cleavable motif). Some preferred, but non-limiting, examples of such residues are multiple histidine residues, glutathione residues and myc tags (e.g., AAAEQKLISEEDLNGAA); d) one or more amino acid residues that may be functionalized and / or 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 the derivatives of the polypeptides of the invention described herein.

[0256] In the constructs of the present invention, e.g., polypeptides of the present invention, two or more components, such as ISVs, and optionally one or more other groups, drugs, agents, residues, moieties, or binding units, may be directly linked to each other (e.g., as described in WO 99 / 23221) and / or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof. 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 linkers or spacers used in the art to link amino acid sequences. Preferably, the linkers or spacers are 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, in particular two, linker sequences. The present invention also provides constructs or polypeptides of the present invention comprising a first ISV that binds to aggrecan and / or a second ISV 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 linkers.

[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 ScFv fragments, the linker sequences used are those of the relevant V H and V L It should have a length, degree of flexibility, and other properties that allow the domains to assemble to form the complete antigen-binding site; however, it should be noted that there are no particular limitations on the length or flexibility of the linkers used in the polypeptides of the invention, since each ISV (e.g., 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 a gly-ser linker, for example (gly x ser y ) z Linkers of the type (gly4ser) or (gly3ser2) as described, for example, in WO 99 / 42077, and the GS30, GS15, GS9, and GS7 linkers described in the applications by Ablynx mentioned herein (see, for example, WO 06 / 040153 and WO 06 / 122825), as well as hinge-like regions, such as the hinge region of a naturally occurring heavy chain antibody or similar sequences (such as those described in WO 94 / 04678). Preferred linkers are presented in Table D (SEQ ID NOs: 154-170).

[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, e.g., WO 04 / 081026.

[0261] It is within the scope of the present invention that the length, degree of flexibility, and / or other properties (typically, but not critically, as 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, e.g., polypeptide of the invention, including, but not limited to, affinity, specificity, or avidity for a chemokine or for one or more other antigens. Based on the disclosure herein, one of skill in the art will be able to determine, optionally after some limited routine experimentation, the optimal linker(s) for use in a specific construct of the invention, e.g., polypeptide of the invention.

[0262] For example, in a multivalent polypeptide of the invention comprising aggrecan and another target-directed component, ISV, or nanobody, the length and flexibility of the linker is preferably such that it allows each component of the invention (such as an ISV) to be present in the polypeptide and bind to its cognate target (e.g., an antigenic determinant on each of the targets). Again, based on the disclosure herein, one skilled in the art will be able to determine the optimal linker(s) for use in a specific construct of the invention, e.g., a polypeptide of the invention, optionally after some limited routine experimentation.

[0263] It is also within the scope of the present invention that the linker(s) used impart one or more other advantageous properties or functions to the constructs of the invention, e.g., the polypeptides of the invention, and / or 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 comprising one or more charged amino acid residues can provide improved hydrophilic properties, while a linker that forms or includes a small epitope or tag can be used for detection, identification, and / or purification purposes. Again, based on the disclosure herein, one of skill in the art will be able to determine the optimal linker for use in a specific 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 can be the same or different. Again, based on the disclosure herein, one of skill in the art will be able to determine, optionally after some limited routine experimentation, the optimal linker for use in a specific construct or polypeptide of the invention.

[0265] Typically, for ease of expression and production, constructs of the invention, e.g., polypeptides of the invention, will be linear polypeptides. However, the invention, in its broadest sense, is not limited thereto. For example, when constructs of the invention, e.g., polypeptides of the invention, include three or more components, ISVs, or nanobodies, they can be linked using a linker having three or more "arms," ​​each of which is linked to a component, ISV, or nanobody to provide a "star-shaped" construct. It is also possible to use circular constructs, although these are generally less preferred.

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

[0267] Thus, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein said linker is selected from the group consisting 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 linkers. Thus, the present invention relates to a construct of the invention, eg, a polypeptide of the invention, wherein said polypeptide is selected from the group consisting of the polypeptides of Table E-1 and Table E-2.

[0268] Also encompassed by the invention are compounds, constructs and / or polypeptides comprising an ISV or polypeptide of the invention that further comprise a tag or other functional moiety (eg, a toxin, label, radiochemical, etc.).

[0269] The other group, residue, moiety, or binding unit may be, for example, a chemical group, residue, moiety that may or may not be biologically and / or pharmacologically active by itself. For example, but not limited to, such a group may be linked to one or more ISVs or polypeptides of the invention to form a "derivative" of the polypeptide of the invention.

[0270] Thus, in its broadest sense, the present invention also encompasses compounds, constructs and / or polypeptides that are derivatives of the polypeptides of the invention, such derivatives being generally obtainable by modification of one or more amino acid residues in and / or forming the polypeptides of the invention, in particular by chemical and / or biological (e.g. enzymatic) modification.

[0271] Examples of such modifications, as well as examples of amino acid residues in a polypeptide sequence (i.e., on the protein backbone or preferably on the side chains) that can be modified by such modifications and the potential uses and advantages of such modifications, 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 (e.g., by covalent linkage or any other suitable method) of one or more functional groups, residues or moieties, particularly one or more (functional) groups, residues or moieties, into and on the polypeptides of the invention, thereby conferring one or more desired properties or functionalities on the polypeptides of the invention. Examples of such functional groups will be apparent to those skilled in the art.

[0273] For example, such modifications may involve the introduction (e.g., by covalent linkage or any other suitable method) of one or more functional groups that increase the half-life, solubility, and / or absorption of the polypeptides of the invention, reduce the immunogenicity and / or toxicity of the polypeptides of the invention, eliminate or reduce any undesirable side effects of the polypeptides of the invention, and / or confer other advantageous properties and / or reduce undesirable properties of the polypeptides of the invention, or any combination of two or more of the foregoing. Examples of such functional groups and techniques for introducing them will be apparent to those skilled in the art and will generally include any of the functional groups and techniques described in the general prior art cited above, as well as functional groups and known techniques per se for the modification of pharmaceutical proteins, in particular antibodies or antibody fragments (including ScFvs and single domain antibodies), see, for example, Remington (Pharmaceutical Sciences, 16 th ed., Mack Publishing Co., Easton, PA, 1980). Such functional groups may, for example, be directly linked (e.g., covalently) to the polypeptides of the invention, or may optionally be 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 involves the addition of a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or its derivative (e.g., methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as PEGylation used in the field of antibodies and antibody fragments, such as (single) domain antibodies and ScFv, for example, see Chapman (Nat. Biotechnol. 54: 531-545, 2002), Veronese and Harris (Adv. Drug Deliv. Rev. 54: 453-456, 2003), Harris and Chess (Nat. Rev. Drug. Discov. 2: 214-221, 2003) and WO 04 / 060965).Various reagents for PEGylation of proteins are also commercially available, for example from Nektar Therapeutics (USA).

[0275] Preferably, site-specific PEGylation, particularly via cysteine ​​residues, is used (see, for example, Yang et al. (Protein Engineering 16: 761-770, 2003)). For example, in this case, PEG can be added to cysteine ​​residues naturally present in the polypeptide of the present invention. Alternatively, the polypeptide of the present invention may be modified so that one or more cysteine ​​residues for PEG addition are optimally introduced therein. Alternatively, 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 either case, protein engineering techniques known to those skilled in the art can be used directly.

[0276] Preferably, in the case of the polypeptide of the present invention, PEG having a molecular weight of 5,000 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 involves N- or O-linked glycosylation, usually as part of co- and / or post-translational modification, depending on the host cell used to express the polypeptides of the invention.

[0278] Depending on the intended use of the marker polypeptides of the invention, a further modification may be the introduction of one or more detectable markers or other signal-generating groups or moieties. Suitable labels and techniques for attaching, using, and detecting them will be apparent to those of skill in the art and include, but are not limited to, fluorescent markers (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and fluorescent metals such as Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels, or bioluminescent labels (luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, dioxetane, or GFP and its analogs), radioisotopes (e.g., 3 H, 125 I, 32 P, 35 S, 14 C. 51 Cr, 36 Cl, 57 Co, 58 Co, 59 Fe and 75 Se), metals, metal chelates or metal cations (e.g. 99m Tc, 123 I, 111 In, 131 I, 97 Ru, 67 Cu, 67 Ga, 68Ga), or other metals or metal cations particularly suitable for in vivo, in vitro or in situ diagnostics and imaging (e.g. 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe), as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose sugar-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase). Other suitable labels will be apparent to those of skill in the art and include, for example, moieties that can be detected using NMR or ESR spectroscopy.

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

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

[0281] Yet another modification may be the introduction of a functional group that is part of a specific binding pair (e.g., biotin-(streptavidin) binding pair). Such a functional group allows the polypeptides of the present invention to be bound to other proteins, polypeptides, or chemicals to which the other half of the binding pair is attached, by forming a so-called binding pair. For example, the polypeptides of the present invention can be conjugated with biotin and linked to other proteins, polypeptides, compounds, or carriers that are conjugated with avidin or streptavidin. For example, such conjugated polypeptides of the present invention can be used as reporters in diagnostic systems, e.g., in which a detectable signal-generating agent is conjugated to avidin or streptavidin. Such binding pairs can also be used, for example, to bind the polypeptides of the present invention to carriers, such as carriers suitable for pharmaceutical use. See, for example, the liposome formulations described by Cao and Suresh (Journal of Drug Targeting 8: 257, 2000). Such binding pairs may also be used to link therapeutically active agents to the polypeptides of the present invention.

[0282] Other potential chemical and enzymatic modifications will be apparent to those skilled in the art. For research applications (e.g., testing function-activity relationships), such modifications may be introduced. See, for example, Lundblad and Bradshaw (Biotechnol. Appl. Biochem. 26: 143-151, 1997).

[0283] Preferably, said compounds, constructs, polypeptides and / or derivatives have an affinity (optimally, K D value (actual or apparent), K A value (actual or apparent), K on Velocity and / or K off speed, or IC as further described herein 50 The antibody binds to aggrecan via the ATP-dependent ATPase (measured and / or expressed as a value). Such compounds, constructs and / or polypeptides of the invention, and derivatives thereof, may be in essentially isolated form (as defined herein).

[0284] In one aspect, the invention relates to a construct of the invention comprising or consisting essentially of an ISV according to the invention or a polypeptide according to the invention, and which further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers.

[0285] In one aspect, the present invention relates to a construct of the present invention, wherein the one or more other groups, residues, moieties or binding units are selected from the group consisting of a polyethylene glycol molecule, a serum protein or fragment thereof, a binding unit capable of binding to a serum protein, an Fc portion, and a small protein or peptide capable of binding to a serum protein. The present invention further relates to methods for preparing the compounds, constructs, polypeptides, nucleic acids, host cells, and compositions described herein.

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

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

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

[0289] Thus, the present invention also relates to the use of an ISV polypeptide of the invention (as described herein) in preparing a multivalent polypeptide. The method for preparing a multivalent polypeptide will comprise linking an ISV of the invention to at least one further ISV of the 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 description herein. For example, the polypeptides of the present invention can be prepared using any known method for preparing antibodies, particularly antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some suitable, non-limiting methods for preparing polypeptides and nucleic acids include the methods and techniques described herein.

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

[0292] The nucleic acid of the present invention can be in the form of single-stranded or double-stranded DNA or RNA.According to one aspect of the present invention, the nucleic acid of the present invention is essentially isolated form as defined herein.The nucleic acid of the present invention can also be present in and / or be a part of a vector, such as an expression vector, for example, a plasmid, a cosmid or a YAC, and this can also be essentially isolated form.Therefore, the present invention also relates to an expression vector comprising the nucleic acid or nucleotide sequence of the present invention.

[0293] The nucleic acids of the present invention can be prepared or obtained directly using known methods based on the information regarding the polypeptides of the present invention described herein, and / or can be isolated from suitable natural sources. Furthermore, as will be apparent to those skilled in the art, to prepare the nucleic acids of the present invention, several nucleotide sequences, e.g., at least two nucleic acids encoding the ISVs of the present invention and, for example, nucleic acids encoding one or more linkers, can be linked by any suitable method. Techniques for generating the nucleic acids of the present invention will be apparent to those skilled in the art and include, but are not limited to, automated DNA synthesis, site-directed mutagenesis, joining two or more natural and / or synthetic sequences (or two or more portions thereof), introducing mutations that result in the expression of truncated expression products, introducing one or more restriction enzyme sites (e.g., creating cassettes and / or regions that can be easily cleaved and / or ligated using appropriate restriction enzymes), and / or mutagenesis by PCR reactions using one or more "mismatched" primers. These and other techniques will be apparent to those skilled in the art; references to standard handbooks, such as Sambrook et al. and Ausubel et al. (supra), as well as the examples below, are included.

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

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

[0296] The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform host cells or host organisms, i.e. for the expression and / or production of the polypeptides of the invention. Suitable hosts or host cells will be clear to the skilled person and may, for example, be any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or (non-human) eukaryotic organism for the expression and production of antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments), which will be clear to the skilled person, as well as all other host cells or (non-human) hosts known per se. Reference is also made to the general background art cited herein above, as well as to, 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 polypeptides of the present invention can also be expressed and / or produced in cell-free expression systems; suitable examples of such systems will be apparent to those skilled in the art. Suitable techniques for transforming the host or host cells of the invention will be clear to those skilled in the art and will depend on the intended host cell / host organism and the genetic construct to be used. Again, reference is made to the above-mentioned handbooks and patent applications. Transformed host cells (which may be in the form of a stable cell line) or host organisms (which may be stable mutant strains or lines) form a further aspect of the invention. Thus, the present invention relates to hosts or host cells comprising a nucleic acid according to the invention, or an expression vector according to the invention.Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions) a polypeptide of the invention (and in the case of a host organism, in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and / or descendants of a host cell or host organism of the invention, which may be obtained, for example, by cell division or by sexual or asexual reproduction.

[0297] To bring about / obtain expression of the polypeptide of the present invention, the transformed host cell or transformed host organism may generally be kept, maintained and / or cultured under conditions such that the (desired) polypeptide of the present invention is expressed / produced. Suitable conditions will be clear to those skilled in the art and will usually depend on the host cell / host organism used and on the (appropriate) regulatory elements controlling the expression of the nucleotide sequence of the present invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraph on the genetic construct of the present invention.

[0298] The polypeptide of the invention can then be isolated from the host cell / host organism and / or from the medium in which said host cell or host organism has been cultured using techniques for protein isolation and / or purification known per se, such as (preparative) chromatographic and / or electrophoretic techniques, differential precipitation techniques, affinity techniques (e.g. using specific cleavable amino acid sequences fused to the polypeptide of the invention), and / or preparative immunological techniques (i.e. using antibodies against the polypeptide to be isolated).

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

[0300] Generally, for pharmaceutical use, the constructs, polypeptides, and / or ISVDs of the present invention can be formulated as pharmaceutical preparations or compositions comprising at least one construct, polypeptide, and / or ISVD of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient, and / or adjuvant, and optionally one or more pharmaceutically active polypeptides and / or compounds. By way of non-limiting example, such formulations can be in a form suitable for oral administration, parenteral administration (such as by intravenous, intramuscular, or subcutaneous injection or intravenous infusion), topical administration (such as intra-articular administration), inhalation, skin patch, implant, suppository, etc., with intra-articular administration being preferred. Such suitable dosage forms, which may be solid, semi-solid, or liquid depending on the mode of administration, as well as methods and carriers for use in the preparations thereof, 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] Thus, in a further aspect, the present invention relates to a pharmaceutical composition comprising at least one construct of the invention, at least one polypeptide of the invention, at least one ISV of the invention, or at least one nucleic acid of the invention, and at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and optionally one or more additional 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 invention, preferably at least one of Table E-1 or Table E-2, and at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and optionally one or more additional 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 to WO 04 / 041862, WO 04 / 041863, WO 04 / 041865, WO 04 / 041867 and WO 08 / 020079), as well as to 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 (S. Dubel, ed.), Wiley, Weinheim, 2007 (see, for example, pages 252-255).

[0303] In a particular aspect, the present 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 ScFvs and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing them will be apparent to those skilled in the art and include, for example, preparations suitable for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intracavity, intraarterial, or intrathecal administration) or for local (e.g., intraarticular, transdermal, or intradermal) administration.

[0305] Preparations for parenteral administration may be, for example, sterile solutions, suspensions, dispersions, or emulsions suitable for infusion or injection. Suitable carriers or diluents for such preparations include, for example, those described on page 143 of WO 08 / 020079. Usually, aqueous solutions or suspensions 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, e.g., U.S. Patent No. 5,399,346, which is incorporated by reference for its gene therapy delivery methods. Using gene therapy delivery methods, primary cells transfected with genes encoding the constructs, polypeptides, and / or ISVs of the invention can be further transfected with tissue-specific promoters that target specific organs, tissues, grafts, tumors, joints, or cells, and can further be transfected with signal and stabilizing sequences for localized expression within the cells.

[0307] The constructs, polypeptides, and / or ISVs of the invention may also be administered intravenously, intraarticularly, or intraperitoneally by infusion or injection, particular examples of which are further described on pages 144 and 145 of WO 08 / 020079 or in PCT / EP2010 / 062975 (entire document). Useful dosages of the constructs, polypeptides, and / or ISVs of the invention can be determined by comparing their in vitro activity with in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known in the art; see, e.g., U.S. Pat. No. 4,938,949.

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

[0309] The desired dose can be conveniently presented as a divided dose administered at appropriate intervals in a single dose, for example, as two, three, four or more sub-doses per day.The sub-doses themselves can be further divided, for example, into a number of separate, loosely spaced administrations.Preferably, the dose is administered once a week, or even 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. Necessary modifications in this dosing range can be determined by one skilled in the art using only routine experimentation, given the teachings provided herein. See, for example, Remington's Pharmaceutical Sciences (Martin, EW, ed., 4th ed.), Mack Publishing Co., Easton, PA. Dosages can also be adjusted by individual physicians in the event of any complications.

[0311] There is a need in the art for more effective treatments for disorders affecting cartilage in joints, such as osteoarthritis. Even when administered intra-articularly, the residence time of most drugs is insufficient to treat affected cartilage. The inventors hypothesized that the efficacy of therapeutic agents could be significantly increased by coupling them to a moiety (also referred to as a "cartilage anchor protein" or "CAP") that "anchors" the drug in the joint, resulting in increased drug retention but without impairing the efficacy of the therapeutic agent. This anchoring concept not only increases drug efficacy but also 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-binding agents could potentially function as anchors, but aggrecan is heavily glycosylated and degraded in various disorders affecting cartilage in joints. Furthermore, given the cost and extensive testing in various animal models required before a drug can enter the clinic, such aggrecan binding agents should preferentially have broad cross-reactivity, e.g., they should bind to aggrecan from various species. Using a variety of sophisticated immunization, screening, and characterization methods, we have been able to identify a variety of aggrecan binding agents with excellent selectivity, stability, and specificity characteristics, allowing for long-term retention and activity in the joint.

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

[0313] In the present invention, the term "reducing and / or inhibiting outflow" means reducing and / or inhibiting the outward flow of a composition, polypeptide, or construct from within the joint to the outside. Preferably, the outflow is reduced and / or inhibited by at least 10%, for example, at least 20%, 30%, 40%, or 50%, or even by at least 60%, 70%, 80%, 90%, or even 100%, compared to the outflow of the composition, polypeptide, or construct in the joint under the same conditions but without the presence of an aggrecan-binding agent of the present invention, for example, an ISV(s) that binds to aggrecan.

[0314] It will be understood that the aggrecan binding agents of the present invention can be used in a variety of diseases affecting cartilage, such as arthropathy and chondrodystrophies, arthritic diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or detachments, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated discs, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis (generally referred to herein as "aggrecan-associated diseases").

[0315] In one aspect, the invention relates to compositions, ISVs, polypeptides, and / or constructs according to the invention for use in preventing or treating aggrecan-associated diseases, such as arthropathies and chondrodystrophies, arthritic diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic tears or detachments, achondroplasia, costochondritis, spondyloepiphyseal dysplasia, herniated discs, lumbar degenerative disc disease, degenerative joint disease, and relapsing polychondritis.

[0316] 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 tear or detachment, 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 the person in need thereof, of a composition, ISV, polypeptide, or construct according to the invention.

[0317] In one aspect, the invention relates to the use of an ISV, polypeptide, composition or construct according to the invention in the preparation of a pharmaceutical composition for treating or preventing arthropathy 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.

[0318] By binding to aggrecan, the aggrecan-binding agents of the present invention are expected to reduce or inhibit the activity of members of the serine protease family, cathepsins, matrix metalloproteinases (MMPs) / matrixins, or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1), and / or ADAMTS11, in degrading aggrecan.

[0319] Thus, in one aspect, the present invention relates to a method for reducing or inhibiting 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, in degrading aggrecan, said method comprising administering to a person in need thereof a pharmaceutically active amount of at least an ISV, polypeptide, construct or composition according to the invention.

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

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

[0322] Generally, a treatment regimen will involve the administration of one or more pharmaceutically effective amounts or doses of, or one or more compositions comprising, one or more ISVs, polypeptides, compounds and / or constructs of the invention. The specific amount(s) or doses to be administered can be determined by a physician, again based on the factors cited above.

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

[0324] Typically, in the above methods, an ISV, polypeptide, compound and / or construct of the invention will be used, however, it is within the scope of the invention to use two or more ISVs, polypeptides and / or constructs of the invention in combination.

[0325] The ISVs, polypeptides and / or constructs of the invention can be used in combination with one or more additional pharmaceutically active compounds or principles, i.e., as a combined treatment regimen that may or may not result in a synergistic effect. Again, the clinician will be able to select such additional compounds or principles, and a suitable combined treatment regimen, based on the factors cited above and their professional judgment.

[0326] In particular, the ISVs, polypeptides and / or constructs of the invention can be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and / or treatment of the diseases, disorders and conditions cited herein, which may or may not result in a synergistic effect. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering the same, 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, and may be administered at essentially the same time or at different times (e.g., essentially simultaneously, sequentially, or on an alternating regimen). When substances or principles are to be administered at the same time via the same route of administration, they may be administered as different pharmaceutical formulations or compositions, or as part of a combined pharmaceutical formulation or composition, as will be apparent to one of skill 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 amount 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 may 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 present 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, who will also be able to modify or alter a particular treatment regimen, where appropriate and for each individual case, to achieve the desired therapeutic effect and to avoid, limit, or reduce 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 undesirable side effects, on the other hand.

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

[0331] In another aspect, the present invention relates to the use of an 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 aggrecan-associated diseases; and / or for use in one or more of the methods of treatment described herein. The present invention also relates to the use of the ISVs, polypeptides, compounds and / or constructs 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, for example by inhibiting aggrecan degradation.

[0332] The present invention also relates to the use of an ISV, polypeptide, compound and / or construct of the 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 to a patient an ISV, polypeptide, compound and / or construct of the invention. The present invention further relates to the ISVs, polypeptides, compounds and / or constructs of the invention, or pharmaceutical compositions comprising same, for use in the prevention and / or treatment of at least one aggrecan-associated disease.

[0333] The subject to be treated can be any warm-blooded animal, but particularly mammals, and especially humans.In veterinary applications, the subject to be treated includes any animal that is raised for commercial purposes or kept as a pet.As will be clear to those skilled in the art, the subject to be treated will particularly be the person who suffers from or has the risk of the diseases, disorders and / or conditions described herein. Again, in such pharmaceutical compositions, one or more ISVs, polypeptides, compounds and / or constructs of the invention, or nucleotides encoding same, and / or pharmaceutical compositions containing same may be suitably combined with one or more other effective principles, such as those described herein.

[0334] The present invention also relates to compositions (such as, without limitation, pharmaceutical compositions or preparations as further described herein) for use in vitro (e.g., in in vitro or cellular assays) or in vivo (e.g., in unicellular or multicellular organisms, and particularly in mammals, more particularly in humans, e.g., in humans at risk of or suffering from a disease, disorder or condition of the invention). It should be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless expressly stated otherwise.

[0335] The sequences are disclosed in the body of the description and in a separate sequence listing in accordance with WIPO Standard ST.25. SEQ ID NO: 1, identified by a specific number, should be the same in the body of the description and in the separate sequence listing. For example, SEQ ID NO: 1 should define the same sequence in both the body of the description and the separate sequence listing. If there is a discrepancy between the sequence definition in the body of the description and the separate sequence listing (e.g., SEQ ID NO: 1 in the body of the description erroneously corresponds to SEQ ID NO: 2 in the separate sequence listing), the reference to a specific sequence in the application, particularly in a particular embodiment, should be understood as a reference to the sequence in the body of the application, not as a reference to the separate sequence listing. In other words, the discrepancy between the sequence definition / name in the body of the description and the separate sequence listing should be resolved by amending the separate sequence listing to the sequences and their names disclosed in the body of the application, including the description, examples, figures, and claims.

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

[0337] example Example 1. Immunization of llamas with aggrecan, cloning of heavy chain-only antibody fragment repertoires, and phage preparation The inventors have realized that the purpose of an animal model of OA is to controllably reproduce the scale and progression of joint damage, so that opportunities for detecting symptoms and disease progression can be identified and new treatments can be developed.The ideal animal model is relatively low-cost and exhibits reproducible disease progression, with a large enough effect size to detect differences within a short period of time.If the model progresses too quickly to end-stage degeneration, intermediate time points that are representative of the pathophysiology of OA may not be available, and in the absence of this information, the subtle effects of potential interventions may be missed.Knowing that OA is an end-stage phenotype that is the result of the interaction between 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 recapitulate human disease (see Cohen-Solal et al. 2013 Bonekey Rep. 2: 422). Given the heterogeneity of the profile in human OA, many models are needed. They are either spontaneous or induced. Most of them focus on a single factor that favors the development of OA, such as aging, mechanical stress (surgery), chemical deficiency (enzymes), or genetic factors. All of them differ in terms of severity, lesion localization, and pathogenesis. However, no animal model addresses all aspects of developing OA.

[0339] Therefore, to be useful in various animal models and ultimately in human patients, the CAP binder preferably has broad cross-reactivity, for example, binds to aggrecan of more than one species. Preferably, the aggrecan binder binds to human aggrecan, as well as 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 have found that aggrecan degradation appears to begin in the C-terminal region. The aggregation of aggrecan molecules that do not contain the G3 domain also increases with age. A key feature of cartilage degeneration associated with arthritis is the loss of aggrecan due to proteolytic cleavage in the interglobular region between the G1 and G2 domains. Therefore, preferably, the aggrecan binder binds to the N-terminal region of aggrecan, i.e., a 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, which remains present in chondrocytes and the ECM.

[0341] 1.1 Immunization 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 challenge 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 PBLs (primary blood lymphocytes) and used as a template for RT-PCR to amplify ISV-encoding gene fragments. These fragments were cloned into the phagemid vector pAX212, allowing the generation of phage particles displaying ISVs fused to His6 and FLAG3 tags. Phages were prepared and stored according to standard protocols (see Phage Display of Peptides and Proteins: A Laboratory Manual, 1st ed., 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 intact bovine cartilage. Individual clones from the selection output were screened for binding to the human G1-IGD-G2 domain in ELISA (using periplasmic extracts from E. coli cells expressing the ISVs). Sequencing of 542 ELISA-positive clones identified 144 unique ISV sequences. ISVs were assessed for species cross-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, canine, and bovine aggrecan G1-IGD-G2. Limited species cross-reactivity was particularly evident for the G1 domain binders, which showed particularly low binding to bovine and canine aggrecan. To identify G1 domain-binding ISVs with higher species cross-reactivity, phage display selection was performed against bovine G1-IGD-G2, canine G1-IGD-G2, and the human G1 domain. Of 1245 clones screened for binding to human, cynomolgus monkey, rat, canine, and bovine G1-IGD-G2 in ELISA, only 15 novel species-cross-reactive ISVs were identified, of which 9 could be mapped to the G1 domain.

[0344] A total of 19 unique clones were selected as a "lead panel" for further characterization. A summary of domain mapping and species cross-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 G2-binders was determined for clone 601D02. The amino acid sequences of the CDRs of clone 601D02 were used as a reference to compare 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 Array optimization of ISV The various ISVs were subjected to a sequence optimization process, which involves mutating the parent ISV sequence. This process includes humanizing the ISV (i), post-translational modification (ii), and knocking out epitopes for potential pre-existing antibodies (iii). (i) For humanization purposes, the parent ISV sequence is mutated to obtain an ISV sequence with higher identity to the human IGHV3-IGHJ germline consensus sequence. Specific amino acids in the framework regions that differ between the ISV and the human IGHV3-IGHJ germline consensus (with the exception of so-called hallmark residues) are changed to their human counterparts, while leaving the protein structure, activity, and stability unchanged. A handful of hallmark residues are known to be important for ISV stability, activity, and affinity and are therefore not mutated. (ii) Amino acids present in the CDRs and with experimental evidence of susceptibility to post-translational modifications (PTMs) are altered such that the PTM sites are inactivated while the protein structure, activity, and stability remain unchanged. (iii) The sequence of the ISV is optimized to minimize binding of any naturally occurring pre-existing antibodies, reducing the likelihood of eliciting a treatment-emergent immunogenic response, without affecting the structure, activity, and stability of the protein.

[0349] For the generation of sequence-optimized ISVs, the ISV components were produced as untagged proteins in Pichia pastoris, purified via protein A affinity chromatography, and then desalted, all according to standard protocols. The ISVs in their various sequence-optimized forms are shown in Tables A-1 and A-2.

[0350] Example 2 Lead panel (purified ISVs) - Characterization of aggrecan Following primary screening, initial assessment of binding via ELISA, determination of off-rates and species cross-reactivity, ISVs from the lead panel were subjected to further characterization.

[0351] 2.1 Formatting the aggrecan lead panel in 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 therapeutic modes of action. Therefore, 19 selected clones were fused to ALB26 (CAP-ALB26 or ALB26-CAP-CAP) in a monovalent or bivalent format 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 ISVs). 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, we hypothesized that the pI may affect cartilage penetration and retention. Bivalent ALB26 (C01010030) was used as a negative control or "dummy."

[0352] 2.2 Ex vivo bovine cartilage retention Since there is no established assay to assess cartilage retention, we developed a reliable and reproducible ex vivo cartilage retention assay using bovine cartilage. Bovine bones were typically collected from local slaughterhouses. Cartilage was cut from the bone into strips approximately 1 mm thick and then cut into 3 mm diameter discs using a biopsy cutter. Cartilage discs were preferentially harvested from fresh cartilage.

[0353] The ability of ISVs to be retained in cartilage for longer periods after a relatively short exposure of nanobodies to cartilage (as might be expected with intra-articular injection) was determined. The assay consisted of incubating ex vivo cartilage, typically 3 mm bovine discs (approximately 10 mg wet weight), with 10 μg / mL nanobody (100 μL) in ON, followed by washing (PBS / 0.1% BSA / 0.1% NaN3 / 100 mM NaCl) for up to 5 days. Bound (retained) nanobodies were then released from the cartilage in SDS-containing SDS-PAGE sample buffer (LDS sample buffer, Invitrogen) and analyzed by Western blot (WB). Assays were typically performed with four cartilage discs per nanobody sample; two discs were analyzed immediately after nanobody incubation (t0) to determine the initial amount of bound nanobody; two discs were analyzed after washing (t1). 1-5days The degree of retention was analyzed by t 1-5days The t was defined as the ratio of the amount of nanobody detected at t and the amount of nanobody detected at t. To increase the throughput of the assay, determination of this ratio was performed by visual inspection of the Western blot, which provided a score of 0 to 6, where 0 is no retention and 6 is complete retention.

[0354] A summary of the results is shown in Table 2.2. [Table 5]

[0355] We found that nine constructs were very well retained in cartilage (scores of 5-6). This "top nine" included both monovalent and bivalent constructs with aggrecan-binding moieties that bound to all of the recombinant G1, G2, or G1-IGD-G2 domains. Fourteen constructs showed moderate retention (scores of <5-2) and five constructs showed detectable but poor retention (scores of <2-1) in this assay. It is noteworthy that all but one aggrecan construct had pI values ​​in the range of 8 to >9.

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

[0357] The resulting epitope bins are shown in Table 2.2 (above). Constructs in epitope bins 2 and 3 (on the G1-domain) 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 binding to bovine aggrecan G1-IGD-G2 measured by ELISA and bovine cartilage retention. Without being bound by any theory, we hypothesize that these epitopes may not be readily accessible in native cartilage tissue. The sequence variability of the CDRs of clones belonging to a bin is represented below and above (ie, bin 8 with 604F02 as the reference compound; Table 1.4AC).

[0358] The sequence variability of epitope bin 4 G1 binders relative to 114F08 is presented below in Tables 2.3A, 2.3B, and 2.3C. The amino acid sequence of the CDRs of clone 114F08 was used as a reference to compare 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 epitope bin 1 G1 binders relative to 608A05 is presented below in Tables 2.3D, 2.3E, and 2.3F. The amino acid sequence of the CDRs of clone 608A05 was used as a reference to compare 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 Binding Characteristics - ELISA and SPR Based on the ex vivo bovine cartilage retention and epitope binning 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] Selected constructs were characterized in ELISA against recombinant G1-IGD-G2 domains from human, cynomolgus monkey, rat, dog, and bovine aggrecan to determine their species cross-reactivity, and in ELISA against recombinant human neurocan and brevican to determine selectivity. 50 The values ​​are listed in Table 2.4A.

[0362] SPR (ProteOn) experiments were performed to determine the off-rates for the "monovalent" aggrecan-ALB26 format. We found that the interaction of nanobodies with the aggrecan surface was heterogeneous. The heterogeneity could be due to rebinding events, heterogeneous populations of immobilized aggrecan, and / or heterogeneous glycosylation patterns. As a result, the calculated off-rates are only indicative. Overall, dissociation kinetics were rapid for aggrecan containing nanobodies (Table 2.4B). [Table 8-1] [Table 8-2]

[0363] Example 3. Biophysical characterization of monovalent lead construct - aggrecan Since all selected constructs, 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 the lead panel for further characterization.

[0364] 3.1 Expression of monovalent 114F08 and 604F02 in E. coli and P. pastoris For biophysical characterization, monovalent nanobodies 114F08 and 604F02 were expressed in E. coli and / or P. pastoris with 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 (100 mM phosphate, 100 mM borate, 100 mM citrate, 115 mM NaCl, buffered at various pHs ranging from 3.5 to 9). Samples were heated from 37 to 99°C at a rate of 4.4°C / s in a LightCycler 480II instrument (Roche), after which they were 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), essentially according to Ericsson et al., 2006 (Anals of Biochemistry, 357: 289-298).

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

[0367] The isoelectric points of the two aggrecan 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. Therefore, next to their positive functional properties, ISVs exhibit advantageous biophysical properties.

[0368] 3.3 114F08 family members The sequence variability in the CDRs of 114F08 family members is presented below in Tables 3.3A, 3.3B, and 3.3C. The amino acid sequences of the CDRs of clone 114F08 were 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 various species Exemplary CAP-containing polypeptides (also designated herein as "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 demonstrate that these exemplary CAP-containing constructs also bind to cartilage from other species, experiments such as those described above with bovine cartilage were essentially repeated with human and rat cartilage.

[0370] 4.1 Ex vivo binding to human cartilage To confirm that exemplary CAP-containing constructs also bind to human cartilage, selected constructs were tested in an ex vivo cartilage binding assay using frozen human cartilage pieces. After a 30-minute wash, binding was determined by Western blot. The results are summarized in Table 4.1. [Table 10] All constructs were found to bind to human cartilage better than the dummy construct.

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

[0372] The results are shown in Table 4.2. All tested constructs were found to bind well to rat cartilage. [Table 11]

[0373] Example 5 Tissue specificity As shown above, the constructs of the present invention have been shown to specifically bind to aggrecan both in vitro and ex vivo. In addition, these constructs should also preferably bind to articular cartilage while not binding, or binding less, to other tissues in the joint. Binding of exemplary CAP-containing constructs to synovium, tendon, epimysium, and meniscus was assessed using the same setup as for the ex vivo cartilage binding assay. After ON incubation with the constructs and brief tissue wash (30 min), construct release and Western blot analysis were performed.

[0374] The results are summarized in Table 5. The results indicate that the CAP binder exhibits preferential binding to cartilaginous tissue, including the 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 longer periods of time. Therefore, the stability of exemplary ALB26-fused CAP constructs in synovial fluid (SF) was assessed by incubation of the constructs 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 any of the constructs could be observed.

[0377] Example 7 Retention in IL-1α-stimulated explant cartilage At this point, all experiments addressing cartilage binding and retention of nanobody-containing CAPs have been performed in healthy (non-arthritic) ex vivo cartilage. Arthritic cartilage is characterized by degraded collagen and aggrecan. Therefore, it is also relevant to evaluate the binding and retention of aggrecan binders in cartilage when degradation of these proteins is occurring. To this end, exemplary ALB26-fused CAP constructs were tested in a cartilage explant assay in which cartilage was stimulated to induce degradation.

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

[0379] The Western blot results are presented in Table 7.1. [Table 14]

[0380] The results of retention of CAP 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") had reduced retention in stimulated cartilage compared to unstimulated cartilage after 5 days of washing, while constructs C01010118 ("118") and C01010094 ("094" or "94") showed little sensitivity to stimulation. Furthermore, binding of the G2 aggrecan domain (exemplified by C01010045) appeared to be reduced compared to binding to the other domains, 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 possible influence of the cartilage anchoring moiety, CAP, on the efficacy of protease-inhibitory nanobodies in cartilage tissue, exemplary CAP constructs were fused to ADAMTS5 (ATS5)-blocking ISVs and tested in a GAG (GlycosAminoGlycan)-release cartilage explant assay.

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

[0384] In the GAG ​​release assay, bovine cartilage explants were cultured for 5 days in the presence of IL-1α and oncostatin M (for induction of ADAMTS5) and a dose range of the construct, followed by quantification of the released GAG content in the culture supernatant.

[0385] The constructs tested and the results of the GAG ​​release assay are summarized in Table 8. [Table 16] The results show that loading an anchoring arm (CAP-ISV construct) onto an ADAMTS5 inhibitor still allows for efficient inhibition of GAG release.

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

[0387] 9.1 Biodistribution studies of the ALB26-CAP construct Nanobodies, 125 I labeled ( 125 The constructs were injected into the knee joints of healthy rats. Autoradiographic images of the joints were taken at various time points up to 4 weeks after injection. These images allowed the evaluation of the retention and tissue (cartilage) specificity of the constructs in an in vivo setting. Representative images are shown in FIG.

[0388] From the results, it can be concluded that all constructs showed specific binding to cartilage. Even 4 weeks after injection, clear staining was observed for both the "monovalent" and "bivalent" aggrecan-binding agents.

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

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

[0391] A representative MARG image is shown in FIG. [Table 17]

[0392] All of the aggrecan binders generally showed penetration into healthy cartilage. Construct 626 occasionally also showed some more pronounced staining on the surface. Varying degrees of cartilage staining and penetration were observed in the operated knees: no staining was observed with monovalent Construct 054; staining was absent or mild with monovalent Construct 094, while bivalent Construct 626 produced somewhat more pronounced staining, albeit with variable depth of penetration (see Table 9.2B). [Table 18]

[0393] Example 10 In vivo rat MMT DMOAD showed statistically significant effects. To further demonstrate the in vivo efficacy of the CAP binder of the present invention, a surgically induced medial meniscus tear (MMT) model was used in rats. Briefly, the CAP binder of the present invention was coupled to an anti-MMP13 ISV (designated "0754" or "C010100754") or an anti-ADAMTS5 ISV (designated "0954" or "C010100954"). Rats underwent surgery on one knee to induce OA-like symptoms. Treatment began with intravenous injection on postoperative day 3. Histopathology was performed on postoperative day 42. Intermediate and final serum samples were obtained for exploratory biomarker analysis. The extent of medical and overall net cartilage degeneration, as well as the percentage of cartilage degeneration reduction, were determined. Twenty animals were used per group. Inhibition of cartilage degradation in the medial tibia by nanobodies is shown in FIG.

[0394] The results show that after 42 days, the ADAMTS5-CAP and MMP13-CAP constructs substantially reduced cartilage width compared to vehicle. These results suggest that the CAP moiety (a) does not negatively affect the activity of either anti-MMP13 ISV(0754) or anti-ADAMTS5 ISV(0954); and (b) allows these constructs to remain in the joint for a longer period of time.

[0395] Example 11 Retention of CAP binders in healthy and osteoarthritic rats is similar in vivo. In a cartilage retention study in healthy rats, it was shown that the polypeptides of the invention were measurable in the cartilage for up to 112 days after intra-articular (IA) injection (data not shown). Because cartilage composition can have an effect on cartilage binding and absorption in the systemic circulation, the pharmacokinetics of the polypeptides of the invention were compared in vivo in diseased osteoarthritic and healthy rats, followed by a comparison of serum levels of the polypeptides at short intervals.

[0396] In particular, 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 an anti-MMP13 ISV and an anti-ADAMTS5 ISV to generate the MMP13-ADAMTS5-CAP-CAP construct (designated "0949" or "C010100949" nanobody). Rats underwent surgery on one knee to induce OA-like symptoms (OA group). Each treatment group (healthy and OA) consisted of 15 animals and received a single intravenous injection of 400 μg / 30 μl of nanobody on day 7 (healthy) or day 7 after surgery (MMT). Serum samples were collected from anesthetized rats on days 0, 7 (0 h = pre-dose sample), 8 (at different time points up to 24 h after treatment), 9 (48 h after treatment), 10 (3 days after treatment), 14 (7 days after treatment), 21 (14 days after treatment), and 42 (35 days after treatment). The collected serum samples were used for determination of polypeptide concentrations in an electrochemoluminescence (ECL)-based integrated PK assay format and subsequent non-compartmental analysis. The retention of polypeptides in the serum of healthy and OA rats is shown in FIG.

[0397] The results show that no significant difference can be observed in the serum concentration of the polypeptide between healthy rats 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

1. An immunoglobulin single variable domain (ISV) that specifically binds to aggrecan.

2. 2. The ISV of claim 1, which specifically binds to human aggrecan [SEQ ID NO: 125].

3. 3. An ISV as described in claim 1 or 2, which specifically binds to dog aggrecan (sequence number 126), bovine aggrecan (sequence number 127), rat aggrecan (sequence number 128); porcine (core) aggrecan (sequence number 129); mouse aggrecan (sequence number 130), rabbit aggrecan (sequence number 131); cynomolgus monkey aggrecan (sequence number 132) and / or rhesus monkey aggrecan (sequence number 133).

4. An ISV according to any one of claims 1 to 3, which does not substantially bind to neurocan (SEQ ID NO: 134) and / or brevican (SEQ ID NO: 135).

5. 4. An ISV according to any one of claims 1 to 3, which has a selectivity for binding to aggrecan over neurocan and / or brevican that is more than 10-fold, more than 100-fold, preferably more than 1000-fold.

6. An ISV according to any one of claims 1 to 4, which preferably binds to cartilaginous tissue such as cartilage and / or meniscus.

7. 6. The ISV of any one of claims 1 to 5, which has a stability in synovial fluid (SF) at 37°C for at least 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months.

8. 7. The ISV of any one of claims 1 to 6, having cartilage retention of at least 2 RU, such as at least 3, 4, 5 or 6 RU in a cartilage retention assay.

9. 9. The ISV of any one of claims 1 to 8, which penetrates at least 5 μm into cartilage, such as at least 10 μm, 20 μm, 30 μm, 40 μm, 50 μm or even more.

10. 10. An ISV according to any one of claims 1 to 9, consisting 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.

11. 11. An ISV according to any one of claims 1 to 10, consisting 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; The ISV.

12. An ISV according to any one of claims 1 to 11, which binds to the G1 domain of aggrecan.

13. 13. An ISV as claimed in claim 12 having a pI greater than 8.

14. 2 ＊ 10 －２ s －１ Less than K ｏｆｆ 14. The ISV of claim 12 or 13, having:

15. 1 ＊ 10 －６ EC below M ５０ 15. The ISV of any one of claims 12 to 14, having:

16. 16. An ISV according to any one of claims 1 to 15, consisting essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), wherein: i) CDR1 is a) SEQ ID NO: 24, 20, or 21; or b) 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: 24, wherein: - in position 2, S is changed to R, F, I or T; - in position 3, T is changed to I; - in position 5, I is changed to S; - in position 6, I is changed to S, T or M; - in position 7, N is changed to Y or R; - in position 8, V is changed to A, Y, T or G; - in position 9, V is changed to M; and / or - in 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, or 39; or 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; - S or N is inserted between positions 3 and 4 (position 2a, Table 1.3B); - in position 3, S is changed to R, W, N or T; - in 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; - in position 7, N is changed to S, T or R; in position 8, A is changed to T; and / or - in position 9, N is changed to D or Y; selected from the group consisting of and / or iii) CDR3 is 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: - in position 1, P is changed to G, R, D or E or is absent; - in position 2, T is changed to R, L, P or V or is absent; - in position 3, T is changed to M, S or R or is absent; - in 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; - in position 9, Y is changed to R, A, S, D or G; - in 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; - in position 12, P is changed to E or D or is absent; and / or - in position 13, Y is changed to L or is absent, The ISV is selected from the group consisting of:

17. - 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; 17. An ISV according to any one of claims 1 to 16 selected from the group of ISVs:

18. - 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, 18. The ISV of claim 17 selected from the group of ISVs.

19. 17. An ISV according to any one of claims 1 to 16, consisting essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), wherein: i) CDR1 is a) SEQ ID NOs: 24 and 109; or b) an amino acid sequence having 2 or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 24, wherein: in position 7, N is changed to S; and / or - in position 9, V is changed to M; selected from the group consisting of and / or ii) CDR2 is c) SEQ ID NOs: 42 and 110; or 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: - in position 1, T is changed to A; - in position 3, S is changed to R; - in position 4, S is changed to T; in position 8, A is changed to T; and / or - in position 9, N is changed to D; selected from the group consisting of and / or iii) CDR3 is e) SEQ ID NOs: 60 and 111; or f) an amino acid sequence having 2 or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 60, wherein: in position 4, H is changed to R; and / or - in position 8, V is changed to D, The ISV is selected from the group consisting of:

20. - CDR1 is selected from the group consisting of SEQ ID NO: 24 and 109; - CDR2 is selected from the group consisting of SEQ ID NOs: 42 and 110; and - CDR3 is selected from the group consisting of SEQ ID NOs: 60 and 111; 17. An ISV according to any one of claims 1 to 16 selected from the group of ISVs:

21. An ISV according to any one of claims 12 to 20, which belongs to epitope bin 1 or epitope bin 4.

22. 22. An ISV according to claim 21, consisting essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), wherein: i) CDR1 is a) SEQ ID NO: 36; and b) an amino acid sequence having two or one amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 36, wherein: - in position 3, T is changed to S; - in position 6, T is changed to S; - in position 8, T is changed to A; and / or - in position 9, M is changed to V; selected from the group consisting of and / or ii) CDR2 is c) SEQ ID NO: 54; 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: 54, wherein: - in position 1, A is changed to I; - in position 4, W is changed to R; - in position 7, G is changed to R; and / or - in position 8, T is changed to S; selected from the group consisting of and / or iii) CDR3 is 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: - in position 1, R is changed to G; - in position 2, P is changed to R or L; - in position 3, R is changed to L or S; - in position 5, Y is changed to R; - in position 6, Y is changed to S or A; - at position 7, Y is changed to T or is absent; - in position 8, S is changed to P; - in position 9, L is changed to H or R; - in position 10, Y is changed to P or A; - in position 11, S is changed to A or Y; - in position 12, Y is changed to D; - in position 13, D is changed to F; - in position 14, Y is changed to G or is absent; and / or - after position 14, an S is inserted, The ISV is selected from the group consisting of:

23. - 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, 23. The ISV of claim 22 selected from the group of ISVs.

24. 24. An ISV according to claim 22 or 23, 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 humanised VHH sequence, a camelised VH sequence or a VHH sequence obtained by affinity maturation to the G1 domain of aggrecan.

25. An ISV, domain antibody, immunoglobulin suitable for use as a domain antibody, single domain antibody, immunoglobulin suitable for use as a single domain antibody, dAb, immunoglobulin suitable for use as a dAb, nanobody, VHH sequence, humanized VHH sequence, camelized VH sequence or VHH sequence obtained by affinity maturation which binds to epitope bin 1 of the G1-domain of aggrecan and competes with an ISV according to claim 22 or 23 for binding to the G1-domain of aggrecan.

26. 22. An ISV according to claim 21, consisting essentially of four framework regions (FR1 to FR4 respectively) and three complementarity determining regions (CDR1 to CDR3 respectively), wherein: i) CDR1 is a) SEQ ID NO: 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: - in position 2, S is changed to I or F; - in position 5, I is changed to S; - in position 6, I is changed to S or M; - in position 7, N is changed to R or Y; - in position 8, V is changed to A or Y; - in position 9, V is changed to M; and / or - in position 10, R is changed to K; selected from the group consisting of and / or ii) CDR2 is 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: - in position 1, T is changed to A or G; - N is inserted between positions 2 and 3 (position 2a, Table 2.3B); - in position 7, N is changed to R; in position 8, A is changed to T; and / or - in position 9, N is changed to D; selected from the group consisting of and / or iii) CDR3 is e) SEQ ID NO: 60; 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: 60, wherein: - in position 1, P is absent; - in position 2, T is changed to R or is absent; - in position 3, T is changed to M or is absent; - in position 4, H is changed to D or Y; - in 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; - in position 9, Y is changed to R; - in position 10, Y is changed to N or E; - at position 11, G is changed to S or K; - in position 12, P is changed to E or is absent; and / or - in position 13, Y is changed to L or is absent, The ISV is selected from the group consisting of:

27. - 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; 27. The ISV of claim 26 selected from the group of ISVs.

28. 28. An ISV according to claim 26 or 27, 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 humanised VHH sequence, a camelised VH sequence or a VHH sequence obtained by affinity maturation to the G1 domain of aggrecan.

29. An ISV, domain antibody, immunoglobulin suitable for use as a domain antibody, single domain antibody, immunoglobulin suitable for use as a single domain antibody, dAb, immunoglobulin suitable for use as a dAb, nanobody, VHH sequence, humanized VHH sequence, camelized VH sequence or VHH sequence obtained by affinity maturation which binds to epitope bin 4 of the G1-domain of aggrecan and competes with an ISV according to claim 26 or 27 for binding to the G1-domain of aggrecan.

30. 30. An ISV according to any one of claims 1 to 29 selected from the group consisting of an ISV having SEQ ID NO: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19 and an ISV having more than 80%, such as 90% or 95%, sequence identity to any one of SEQ ID NO: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19.

31. An ISV according to any one of claims 1 to 11, which binds to the G1-IGD-G2 domain of aggrecan.

32. 32. An ISV as claimed in claim 31 having a pI greater than 8.

33. 2 ＊ 10 －２ s －１ Less than K ｏｆｆ 33. The ISV of claim 31 or 32, having:

34. 1 ＊ 10 －６ EC below M ５０ 34. The ISV of any one of claims 31 to 33, having:

35. 35. An ISV according to any one of claims 31 to 34, wherein: i) CDR1 is a) SEQ ID NOs: 32, 30 and 23; and b) an amino acid sequence having 3, 2 or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 32, wherein: - in position 2, R is changed to L; in position 6, S is changed to T; and / or - in 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 two or one amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 50, wherein: at position 7, G is changed to S or R; and / or - in 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 5, 4, 3, 2 or 1 amino acid(s) difference(s) from the amino acid sequence of SEQ ID NO: 68, wherein: - in position 4, R is changed to V or P; - in position 6, A is changed to Y; - in position 7, S is changed to T; - in position 8, S is absent; - in position 9, N is changed to P; - in position 10, R is changed to T or L; at position 11, G is changed to E; and / or in position 12, L is changed to T or V, The ISV is selected from the group consisting of:

36. - 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 NOs: 50, 41, 48 and 51; and - CDR3 is selected from the group consisting of SEQ ID NOs: 68, 59, 66 and 69; 36. An ISV according to any one of claims 31 to 35 selected from the group of ISVs.

37. - 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, 37. The ISV of claim 36 selected from the group of ISVs.

38. 38. An ISV according to any one of claims 31 to 37 selected from the group consisting of an ISV having SEQ ID NO: 13, 4, 11 and 14 and an ISV having more than 80%, such as 90% or 95%, sequence identity with any one of SEQ ID NO: 13, 4, 11 and 14.

39. 39. An ISV according to any one of claims 31 to 38, 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 humanised VHH sequence, a camelised VH sequence or a VHH sequence obtained by affinity maturation to the G1-IGD-G2 domain of aggrecan.

40. 39. 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 humanised VHH sequence, a camelised VH sequence or a VHH sequence obtained by affinity maturation which binds to the G1-IGD-G2 domain of aggrecan and competes for binding to the G1-IGD-G2 domain of aggrecan with an ISV according to any one of claims 31 to 38.

41. An ISV according to any one of claims 1 to 11, which binds to the G2 domain of aggrecan.

42. 42. An ISV as claimed in claim 41 having a pI greater than 8.

43. 2 ＊ 10 －２ s －１ Less than K ｏｆｆ 43. The ISV of claim 41 or 42, having:

44. 1 ＊ 10 －６ EC below M ５０ 44. The ISV of any one of claims 41 to 43, having:

45. 45. An ISV according to any one of claims 41 to 44, wherein: i) CDR1 is a) SEQ ID NO: 28; and b) 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: 28, wherein: - at position 1, G is changed to R; - in position 2, P is changed to S or R; - in position 3, T is changed to I; - in position 5, S is changed to N; - in position 6, R is changed to N, M or S; - in position 7, Y is changed to R or is absent; in 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; 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: - in position 1, A is changed to S or Y; - in position 4, W is changed to L; - in position 5, S is changed to N; - in position 6, S is absent; - in position 7, G is absent; - at position 8, G is changed to A; in position 9, R is changed to S, D or T; and / or - in position 11, Y is changed to N or R; selected from the group consisting of and / or iii) CDR3 is 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; - in position 9, T is changed to S or is absent; - in position 10, S is changed to E, K or is absent; - in position 11, E is changed to N, A or is absent; - in position 12, W is changed to D or is absent; - in position 13, N is changed to D or is absent; in position 14, Y is absent; and / or - D and / or N is added after position 14 of SEQ ID NO: 64; The ISV is selected from the group consisting of:

46. - 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; 46. ​​An ISV according to any one of claims 41 to 45 selected from the group of ISVs:

47. - 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, 47. The ISV of claim 46 selected from the group of ISVs.

48. 48. An ISV according to any one of claims 41 to 47 selected from the group consisting of an ISV having SEQ ID NOs: 9, 3, 7 and 15 and an ISV having more than 80%, such as 90% or 95%, sequence identity with any one of SEQ ID NOs: 9, 3, 7 and 15.

49. 49. An ISV according to any one of claims 41 to 48, 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 humanised VHH sequence, a camelised VH sequence or a VHH sequence obtained by affinity maturation to the G2 domain of aggrecan.

50. 50. An ISV 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 which binds to the G2-domain of aggrecan and competes with an ISV according to any one of claims 41 to 48 for binding to the G2 domain of aggrecan.

51. 51. An ISV according to any one of claims 1 to 50 selected from the group consisting of SEQ ID NOs: 1 to 19 and 114 to 118 and ISVs having more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 1 to 19 and 114 to 118.

52. 52. A polypeptide comprising at least one ISV according to any one of claims 1 to 51, and optionally a second ISV, optionally a third ISV, and optionally a fourth ISV.

53. 53. A polypeptide according to claim 52, comprising at least two ISVs according to any one of claims 1 to 51, and optionally a third ISV, and optionally a fourth ISV.

54. 54. The polypeptide of claim 53, wherein at least two ISVs may be the same or different.

55. 55. The polypeptide of claim 54, wherein at least two ISVs are independently selected from the group consisting of SEQ ID NOs: 1-19 and 114-118.

56. 56. The polypeptide of claim 55, wherein at least two ISVs are selected from the group consisting of SEQ ID NOs: 5, 6, 8 and 114-117.

57. 56. The polypeptide of claim 55, wherein at least two ISVs are selected from the group consisting of SEQ ID NOs: 13 and 118.

58. 58. The polypeptide of any one of claims 52 to 57, comprising at least one further ISV.

59. 59. The polypeptide of claim 58, wherein at least one further ISV 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.

60. 60. The polypeptide of claim 58 or 59, wherein at least one further ISV retains activity.

61. 61. The polypeptide of any one of claims 58 to 60, wherein at least one further 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.

62. 62. The polypeptide of any one of claims 52 to 61, having a stability in synovial fluid (SF) at 37°C for at least 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months.

63. 63. The polypeptide of any one of claims 52 to 62, having cartilage retention of at least 2 RU, such as at least 3, 4, 5 or 6 RU in a cartilage retention assay.

64. 64. The polypeptide of any one of claims 52 to 63, which penetrates at least 5 μm into cartilage, such as at least 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, or even more.

65. 65. The polypeptide of any one of claims 52 to 64, further comprising a serum protein binding moiety or serum protein.

66. 66. The polypeptide of claim 65, wherein the serum protein binding moiety binds to serum albumin.

67. 67. A polypeptide according to claim 65 or 66, wherein the serum protein binding moiety is an ISV that binds to serum albumin.

68. 68. The polypeptide of claim 67, wherein the serum albumin-binding ISV 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.

69. 69. The polypeptide of claim 68, wherein the ISVs that bind serum albumin include Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, and Alb82-GGG.

70. 67. The polypeptide of claim 65 or 66, wherein the serum protein binding moiety is a non-antibody-based polypeptide.

71. 65. The polypeptide of any one of claims 52 to 64, further comprising PEG.

72. 72. A polypeptide according to any one of claims 52 to 71, wherein the ISVs are linked to each other directly or via a linker.

73. 70. A polypeptide according to any one of claims 52 to 69, wherein 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 serum albumin are linked via a linker.

74. 74. The polypeptide of claim 72 or 73, wherein the linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS linkers.

75. 75. The polypeptide of any one of claims 52 to 74, selected from the group of polypeptides and / or constructs comprising a target-binding ISV and one or two aggrecan-binding ISVs as shown in Table E-1 and Table E-2, respectively.

76. 10. A construct comprising or consisting essentially of an ISV according to any one of claims 1 to 51 or a polypeptide according to any one of claims 52 to 75, optionally further comprising one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers.

77. 77. The construct of claim 76, wherein the one or more other groups, residues, moieties or binding units are selected from the group consisting of a polyethylene glycol molecule, a serum protein or fragment thereof, a binding unit capable of binding to a serum protein, an Fc portion, and a small protein or peptide capable of binding to a serum protein.

78. A nucleic acid encoding an ISV according to any one of claims 1 to 51, a polypeptide according to any one of claims 52 to 75, or a construct according to claim 76 or 77.

79. 79. An expression vector comprising the nucleic acid of claim 78.

80. 80. A host or host cell comprising the nucleic acid of claim 78 or the expression vector of claim 79.

81. 76. A method for producing an ISV according to any one of claims 1 to 51 or a polypeptide according to any one of claims 52 to 75, comprising at least the following steps: a) expressing the nucleic acid of claim 78 in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b) isolating and / or purifying an ISV according to any one of claims 1 to 51 or a polypeptide according to any one of claims 52 to 75, The method comprising:

82. 79. A composition comprising at least one ISV according to any one of claims 1 to 51, a polypeptide according to any one of claims 52 to 75, a construct according to claim 76 or 77, or a nucleic acid according to claim 78.

83. 83. The composition of claim 82, which is a pharmaceutical composition.

84. 84. The composition of claim 83, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharmaceutically active polypeptides and / or compounds.

85. A composition according to any one of claims 82 to 84, an ISV according to any one of claims 1 to 51, a polypeptide according to any one of claims 52 to 75, or a construct according to claim 76 or 77 for use as a medicament.

86. 86. The composition, ISV, polypeptide or construct of claim 85 for use in 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.

87. 86. A method for preventing or treating arthropathy 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, comprising administering to a subject in need thereof at least a pharmaceutically active amount for a person in need thereof of the composition, ISV, polypeptide, or compound or construct of claim 85.

88. A method for reducing and / or inhibiting the efflux of compounds from cartilaginous tissue, comprising administering to a person in need thereof a pharmaceutically active amount of at least one polypeptide according to any one of claims 52 to 75, a construct according to claims 76 or 77, or a composition according to any one of claims 82 to 86.

89. 10. A method for inhibiting and / or blocking ADAMTS5 and / or MMP13 activity, comprising administering to a person in need thereof a pharmaceutically active amount of at least one polypeptide of any one of claims 52 to 75, a construct of claims 76 or 77, or a composition of any one of claims 82 to 86.

90. 86. Use of an ISV according to any one of claims 1 to 51, a polypeptide according to any one of claims 52 to 75, a construct according to claims 76 or 77, or a composition according to any one of claims 82 to 86 in the preparation of a pharmaceutical composition for the treatment or prevention of arthropathy 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.

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