Aggrecan Binding Immunoglobulins

Abstract

The present invention relates to immunoglobulins that specifically bind Aggrecan and more in particular to polypeptides, nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to compositions and in particular to pharmaceutical compositions that comprise such polypeptides, for prophylactic, therapeutic or diagnostic purposes. In particular, the immunoglobulins of the present invention inhibit the activity of Aggrecan.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to immunoglobulins that bind Aggrecan and more in particular to polypeptides, that comprise or essentially consist of one or more such immunoglobulins (also referred to herein as "immunoglobulin(s) of the invention", and "polypeptides of the invention", respectively). The invention also relates to constructs comprising such immunoglobulins or polypeptides as well as 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 to pharmaceutical compositions, that comprise such immunoglobulins, polypeptides, constructs, nucleic acids and/or host cells; and to uses of immunoglobulins, polypeptides, constructs, nucleic acids, host cells and/or compositions, in particular for prophylactic and/or therapeutic purposes, such as the prophylactic and/or therapeutic purposes mentioned herein. Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

BACKGROUND

[0002] Osteoarthritis is one of the most common causes of disability worldwide. It affects 30 million Americans and is the most common joint disorder. It is projected to affect more than 20 percent of the U.S. population by 2025. The disease can occur in all joints, most often the knees, hips, hands and spine. Osteoarthritis (OA) can be defined as a diverse group of conditions characterised by a combination of joint symptoms, signs stemming from defects in the articular cartilage and changes in adjacent tissues including bone, tendons and muscle. OA is characterized by progressive erosion of articular cartilage (cartilage that covers the bones). Eventually, the disease leads to the total destruction of the articular cartilage, sclerosis of underlying bone, osteophyte formation etc., all leading to loss of movement and pain. Pain is the most prominent symptom of OA and this is most often the reason patients seek medical help.

[0003] Aggrecan is the major proteoglycan in the articular cartilage (Kiani et al. 2002 Cell Research 12:19-32). This molecule is important in the proper functioning of the articular cartilage because it provides a hydrated gel structure that endows the cartilage with load-bearing properties. Aggrecan is a large, multimodular molecule (2317 amino acids) expressed by chondrocytes. Its core protein is composed of three globular domains (G1, G2 and G3) and a large extended region between G2 and G3 for glycosaminoglycan chain attachment. This extended region comprises two domains, one substituted with keratan sulfate chains (KS domain) and one with chondroitin sulfate chains (CS domain). The CS domain has 100-150 glycosaminoglycan (GAG) chains attached to it. Aggrecan forms large complexes with Hyaluronan in which 50-100 Aggrecan molecules interact via the G1 domain and Link Protein with one Hyaluronan molecule. Upon uptake of water (due to the GAG content) these complexes form a reversibly deformable gel that resists compression. The structure, fluid retention and function of joint cartilage is linked to the matrix content of Aggrecan, and the amount of chondroitin sulfate bound to the intact core protein.

[0004] OA is characterized by 1) degradation of Aggrecan, progressively releasing domains G3 and G2 (resulting in `deflation` of the cartilage) and eventually release of the G1 domain and 2) degradation of Collagen, irreversibly destroying the 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 there are currently no pharmacological treatments that halt the disease progression or cure the joints. For large joints, a drug could be injected into the joint to help to limit potential side effects, like pain. Therapeutic strategies are primarily aimed at reducing pain and improving joint function. Fasinumab, a non-opioid anti-NGF pain treatment has been shown to give improvements on a key pain score during phase II/III trials. Duloxetine was approved for the treatment of chronic knee pain due to osteoarthritis and has been conditionally recommended by the American College of Rheumatology. Strontium ranelate was found to significantly decrease the rate of decline in joint space width as well as improve pain scores compared with placebo in a large multicenter study in patients with symptomatic knee osteoarthritis. However, at this moment the biologic agents interleukin-1 receptor antagonists and antitumor necrosis factor antibodies have neither been shown to be efficacious nor to alter the course of osteoarthritis (Smelter Hochberg 2013 Current Opin. Rheumatol. 25:310). Hence, many such therapies are ineffective and/or are associated with side effects. Ultimately patients will undergo total knee or hip replacement therapy if pain cannot be controlled.

[0006] Pharmacological therapy begins with oral administration of paracetamol either combined with NSAIDS or COX-2 inhibitors and a weak opioid. Major disadvantages of oral administration of drugs are the limited bio-availability at the site of interest and the risk of side effects, such as liver damage, Gastro-intestinal (GI)-ulcers, GI-bleeding and constipation. As OA has a localized nature, intra-articular administration of drugs provides an excellent opportunity to improve treatment. However, most of the 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 space and lack of retention within cartilage. Synovial residence time of a drug in the joint is often less than 24 h. Due to the rapid clearance of most IA injected drugs, frequent injections would be needed to maintain an effective concentration (Owen et al. 1994 Br. J, Clin Pharmacol. 38349-355). However, frequent IA-injections are undesired due to the pain and discomfort they may cause challenging patient compliance, as well as the risk of introducing joint infections.

[0007] Loffredo et at tested whether targeted delivery to cartilage by fusion with a heparin-binding domain would be sufficient to prolong the in viva function of the insulin-like growth factor 1 (IGF-1). Heparin is present in mast cells. However, the natural role of Heparin is unknown, but it is widely used as a blood-thinner (Loffredo et al. 2014 Arthritis Rheumatol. 66:1247-1255).

[0008] There remains a need for further cartilage anchoring proteins (CAP).

SUMMARY OF THE INVENTION

[0009] The present inventors hypothesized that the efficacy of a therapeutic drug could be increased significantly by coupling the therapeutic drug to a moiety which would "anchor" the drug in the joint and consequently increase retention of the drug, but which should not disrupt the efficacy of said therapeutic drug (also indicated herein as "cartilage anchoring protein" or "CAP"). This anchoring concept would not only increase the efficacy of drug, but also the operational specificity for a diseased joint by decreasing toxicity and side-effects, thus widening the number of possible useful drugs. The present inventors further hypothesized that Aggrecan binders might potentially function as such an anchor, although Aggrecan is heavily glycosylated and degraded in various disorders affecting cartilage in joints. Moreover, in view of the costs and extensive testing in various animal models required before a drug can enter the clinic, such Aggrecan binders should preferentially have a broad cross-reactivity, e.g. the Aggrecan binders should bind to Aggrecan of various species.

[0010] Using various ingenious immunization, screening and characterization methods, the present inventors were able to identify a number of Aggrecan binders with superior selectivity, stability and/or specificity features, which enabled prolonged retention and activity in the joint.

[0011] Accordingly, the present invention relates to an immunoglobulin single variable domain (ISV) that specifically binds to Aggrecan, preferably 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), pig (core) Aggrecan (SEQ ID NO: 129), mouse Aggrecan (SEQ ID NO: 130), rabbit Aggrecan (SEQ ID NO: 131), cynomolgus Aggrecan (SEQ ID NO: 132) and/or rhesus Aggrecan (SEQ ID NO: 133), even more preferably, wherein said ISV does not bind substantially to Neurocan (SEQ ID NO: 134) and/or Brevican (SEQ ID NO: 135).

[0012] In an aspect, the present invention relates to an ISV as described herein, wherein the ISV has more than 10 fold, more than 100 fold, preferably more than 1000 fold selectivity over Neurocan and/or Brevican for binding to Aggrecan, 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 in synovial fluid (SF) at 37.degree. C., and/or said ISV has a cartilage retention of at least 2, such as at least, 3, 4, 5 or 6 RU in a cartilage retention assay, and/or said ISV penetrates into the cartilage by at least 5 pa), such as at least 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m or even more, and/or said ISV essentially consists of a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation.

[0013] In an aspect, the present invention relates to an ISV as described herein, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: CDR1 is chosen 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 chosen 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 chosen 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 an aspect, the present invention relates to an ISV as described herein, wherein said ISV binds to the G1 domain of Aggrecan, preferably said ISV has a pl of more than 8, and/or said ISV has a Koff of less than 2*10.sup.-2 s.sup.-1, and/or said ISV has an EC.sub.50 of less than 1*10.sup.-6M.

[0015] In an aspect, the present invention relates to an ISV as described herein, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0016] i) CDR1 is chosen from the group consisting of: a) SEQ ID NOs: 24, 20, or 21; or b) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein at position 2 the S has been changed into R, F, I, or T; at position 3 the T has been changed into I; at position 5 the I has been changed into 5; at position 6 the I has been changed into S, T, or M; at position 7 the N has been changed into Y, or R; at position 8 the V has been changed into A, Y, T, or G; at position 9 the V has been changed into M; and/or at position 10 the R has been changed into G, K, or A; and/or [0017] ii) CDR2 is chosen from the group consisting of: c) SEQ ID NOs: 42, 38, or 39; or d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein at position 1 the T has been changed into A, or G; an S or N is inserted between position 3 and position 4 (position 2a Table 1.3B); at position 3 the S has been changed into R, W, N, or T; at position 4 the S has been changed into T or G; at position 5 the G has been changed into S; at position 6 the G has been changed into 5, or R; at position 7 the N has been changed into S, T, or R; at position 8 the A has been changed into T; and/or at position 9 the N has been changed into D or Y; and/or [0018] iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 60, 56 or 57; or f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, wherein at position 1 the P has been changed into G, R, D, or E, or is absent; at position 2 the T has been changed into R, L, P, or V, or is absent; at position 3 the T has been changed into M, 5, or R, or is absent; at position 4 the H has been changed into D, Y, G, or T; at position 5 the Y has been changed into F, V, T or G; at position 6 the G has been changed into L, D, S, Y, or W; an R, T, Y or V is inserted between position 6 and position 7 (position 6a Table 1.3C); at position 7 the G has been changed into P, or S; at position 8 the V has been changed into G, T, H, R, L, or Y; at position 9 the Y has been changed into R, A, 5, D or G; at position 10 the Y has been changed into N, E, G, W, or 5; a W is inserted between position 10 and position 11 (position 10a Table 1.3C); at position 11 the G has been changed into S, K, or V; at position 12 the P has been changed into E, or D, or is absent; and/or at position 13 the Y has been changed into L, or is absent.

[0019] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, 37 and 109; CDR2 is chosen from the group consisting of SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, 55, and 110; and CDR3 is chosen from the group consisting of SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73, 74, and 111.

[0020] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group of ISVs, wherein: [0021] CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; [0022] CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; [0023] CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; [0024] CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; [0025] CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; [0026] CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; [0027] CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; [0028] CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 71; [0029] CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 72; [0030] COR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 73; and [0031] CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 74.

[0032] In an aspect, the present invention relates to an ISV as described herein, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0033] i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 24 and 109; or b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein at position 7 the N has been changed into 5; and/or at position 9 the V has been changed into M; and/or [0034] ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 42 and 110; or d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein at position 1 the T has been changed into A; at position 3 the S has been changed into R; at position 4 the S has been changed into T; at position 8 the A has been changed into T; and/or at position 9 the N has been changed into D; and/or [0035] iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 60 and 111; or f) amino acid sequences that have 2, or 1 amino acids) difference with the amino acid sequence of SEQ ID NO: 60, wherein at position 4 the H has been changed into R; and/or at position 8 the V has been changed into D.

[0036] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group of ISVs, wherein CDR1 is chosen from the group consisting of SEQ ID NOs: 24 and 109; CDR2 is chosen from the group consisting of SEQ ID NOs: 42 and 110; and CDR3 is chosen from the group consisting of SEQ ID NOs: 60 and 111.

[0037] In an 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 essentially consists of 4 framework regions (ER1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0038] i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 36; and b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 36, wherein at position 3 the T has been changed into 5; at position 6 the T has been changed into 5; at position 8 the T has been changed into A; and/or at position 9 the M has been changed into V; and/or [0039] ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 54; and d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 54, wherein at position 1 the A has been changed into I; at position 4 the W has been changed into R; at position 7 the G has been changed into R; and/or at position 8 the T has been changed into 5; and/or [0040] iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 73; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 73, wherein at position 1 the R has been changed into G; at position 2 the P has been changed into R or L; at position 3 the R has been changed into L or S; at position 5 the Y has been changed into R; at position 6 the Y has been changed into S or A; at position 7 the Y has been changed into T, or is absent; at position 8 the S has been changed into P; at position 9 the L has been changed into H or R; at position 10 the Y has been changed into P or A; at position 11 the S has been changed into A or Y; at position 12 the Y has been changed into D; at position 13 the D has been changed into F; at position 14 the Y has been changed into G, or is absent; and/or after position 14 an S is inserted.

[0041] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 20, 29, and 36; CDR2 is chosen from the group consisting of SEQ ID NOs: 38, 47, and 54; and CDR3 is chosen from the group consisting of SEQ ID NOs: 56, 65, and 73.

[0042] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G1 domain of Aggrecan.

[0043] In an aspect, the present invention relates to an ISV, a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Na nobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to epitope bin 1 of the G1-domain of Aggrecan, and which competes for binding to the G1 domain of Aggrecan with the ISV as described herein.

[0044] In an aspect, the present invention relates to an ISV as described herein, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 24; and b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein at position 2 the S has been changed into I or F; at position 5 the I has been changed into 5; at position 6 the I has been changed into S or M; at position 7 the N has been changed into R or Y; at position 8 the V has been changed into A or Y; at position 9 the V has been changed into M; and/or at position 10 the R has been changed into K; and/or ii) CDR2 is chosen from the group consisting of: c) [0045] SEQ ID NO: 42; and d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein at position 1 the T has been changed into A or G; an N is inserted between position 2 and position 3 (position 2a Table 2.3B); at position 7 the N has been changed into R; at position 8 the A has been changed into T; and/or at position 9 the N has been changed into D; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 60; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, wherein at position 1 the P is absent; at position 2 the T has been changed into R or is absent; at position 3 the T has been changed into M or is absent; at position 4 the H has been changed into D or Y; at position 5 the Y has been changed into F or V; at position 6 the G has been changed into L or D; at position 8 the V has been changed into G or T; at position 9 the Y has been changed into R; at position 10 the Y has been changed into N or E; at position 11 the G has been changed into S or K; at position 12 the P has been changed into E or is absent; and/or at position 13 the Y has been changed into L or is absent; preferably CDR1 is chosen from the group consisting of SEQ ID NOs: 24, 25, and 27; CDR2 is chosen from the group consisting of SEQ ID NOs: 42, 43, and 45; and CDR3 is chosen from the group consisting of SEQ ID NOs: 60, 61, and 63; even more preferably, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G1 domain of Aggrecan.

[0046] In an aspect, the present invention relates to an ISV as described herein, a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to epitope bin 4 of the G1-domain of Aggrecan, and which competes for binding to the G1 domain of Aggrecan with the ISV as described herein.

[0047] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group consisting of ISVs with SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19, and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19.

[0048] In an 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 of more than 8 and/or has a Koff of less than 2*10.sup.-2 s.sup.-1 and/or has an EC50 of less than 1*10.sup.-6M.

[0049] In an aspect, the present invention relates to an ISV as described herein, in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 32, 30 and 23; and b) amino acid sequences that have 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 32, wherein at position 2 the R has been changed into L; at position 6 the S has been changed into T; and/or at position 8 the T has been changed into A; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 50, 41, 48 and 51; and d) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 50, wherein at position 7 the G has been changed into S or R; and/or at position 8 the R has been changed into T; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 68, 59, 66 and 69; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 68, wherein at position 4 the R has been changed into V, or P; at position 6 the A has been changed into Y; at position 7 the S has been changed into T; at position 8 the S is absent; at position 9 the N has been changed into P; at position 10 the R has been changed into T or L; at position 11 the G has been changed into E; and/or at position 12 the L has been changed into T or V, preferably, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 32, 30 and 23; CDR2 is chosen from the group consisting of SEQ ID NOs: 50, 41, 48 and 51; and CDR3 is chosen from the group consisting of SEQ ID NOs: 68, 59, 66 and 69, even more preferably, wherein said ISV is chosen 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.

[0050] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group consisting of ISVs with SEQ ID NOs: 13, 4, 11 and 14, and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 13, 4, 11 and 14.

[0051] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G1-IGD-G2 domain of Aggrecan. In an aspect, the present invention relates to an ISV, a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to the G1-IGD-G2 domain of Aggrecan, and which competes for binding to the G1-IGD-G2 domain of Aggrecan with the ISV as described herein.

[0052] In an 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 pl of more than 8, and/or has a Koff of less than 2*10.sup.-2 s.sup.-1 and/or has an EC50 of less than 1*10.sup.-6M

[0053] In an aspect, the present invention relates to an ISV as described herein, in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 28; and b) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 28, wherein at position 1 the G has been changed into R; at position 2 the P has been changed into S or R; at position 3 the T has been changed into I; at position 5 the S has been changed into N; at position 6 the R has been changed into N, M, or 5; at position 7 the Y has been changed into R or is absent; at position 8 the A has been changed into F or is absent; and/or at position 10 the G has been changed into Y; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 46; and d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 46, wherein at position 1 the A has been changed into 5, or Y; at position 4 the W has been changed into L; at position 5 the S has been changed into N; at position 6 the S is absent; at position 7 the G is absent; at position 8 the G has been changed into A; at position 9 the R has been changed into 5, D, or T; and/or at position 11 the Y has been changed into N or R; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 64; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 64, wherein at position 1 the A has been changed into R, or F; at position 2 the R has been changed into I, or L; at position 3 the I has been changed into H, or Q; at position 4 the P has been changed into G, or N; at position 5 the V has been changed into 5; at position 6 the R has been changed into G, N, or F; at position 7 the T has been changed into R, W, or Y; at position 8 the Y has been changed into R, or S, or is absent; at position 9 the T has been changed into S, or is absent; at position 10 the S has been changed into E, K or is absent; at position 11 the E has been changed into N, A, or is absent; at position 12 the W has been changed into D, or is absent; at position 13 the N has been changed into D, or is absent; at position 14 the Y is absent; and/or D and/or N are added after position 14 of SEQ ID NO: 64; preferably wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 28, 22, 26, and 33; CDR2 is chosen from the group consisting of SEQ ID NOs: 46, 40, 44, and 52; and CDR3 is chosen from the group consisting of SEQ ID NOs: 64, 58, 62, and 70; even more preferably, wherein said ISV is chosen from the group of ISVs, wherein: 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.

[0054] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group consisting of ISVs with SEQ ID NOs: 9, 3, 7 and 15, and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 9, 3, 7 and 15.

[0055] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G2 domain of Aggrecan. In an aspect, the present invention relates to an ISV, a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to the G2-domain of Aggrecan, and which competes for binding to the G2 domain of Aggrecan with the ISV as described herein.

[0056] In an aspect, the present invention relates to an ISV as described herein, wherein said ISV is chosen from the group consisting of SEQ ID NO:s 1-19 and 114-118 and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 1-19 and 114-118.

[0057] In an aspect, the present invention relates to a polypeptide comprising at least one ISV as described herein, preferably said comprises at least two ISVs as described herein, wherein said at least two ISVs can be the same or different. Preferably, said at least two ISVs are independently chosen from the group consisting of SEQ ID NOs: 1-19 and 114-118, more preferably wherein said at least two ISVs are chosen from the group consisting of SEQ ID NOs: 5, 6, 8 and 114-117 or wherein said at least two ISVs are chosen from the group consisting of SEQ ID NOs: 13 and 118.

[0058] Preferably, in an aspect, the polypeptide of the invention comprises at least one further ISV, e.g. a therapeutic ISV. Preferably, said at least one further ISV binds to a member 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; wherein said at least one further ISV, e.g. a therapeutic ISV, preferably retains activity. Even more preferably, said at least one further ISV, such as an therapeutic ISV, inhibits an activity of a member 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.

[0059] In an aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide has a stability of at least 7 days, such as at least 14 days, 21 days, 1 month, 2 months or even 3 months in synovial fluid (SF) at 37.degree. C., and/or has a cartilage retention of at least 2, such as at least, 3, 4, 5 or 6 RU in a cartilage retention assay, and/or penetrates into the cartilage by at least 5 .mu.m, such as at least 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m or even more.

[0060] In an aspect, the present invention relates to a polypeptide as described herein, further comprising a serum protein binding moiety or a serum protein, preferably said serum protein binding moiety binds serum albumin; even more preferably said serum protein binding moiety is an ISV binding serum albumin; even more preferably, said ISV binding serum albumin essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1, to CDR3 respectively), in which CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG and CDR3 is GGSLSR; even more preferably said ISV binding serum albumin comprises Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11 (5112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (cf. Table C). In an aspect, the present invention relates to a polypeptide as described herein, further comprising a serum protein binding moiety or a serum protein, wherein said serum protein binding moiety is a non-antibody based polypeptide. In an aspect, the present invention relates to a polypeptide as described herein, further comprising PEG.

[0061] In an aspect, the present invention relates to a polypeptide as described herein, wherein said ISVs are directly linked to each other or are linked via a linker. In an aspect, the present invention relates to a polypeptide as described herein, wherein a first ISV and/or a second ISV and/or possibly a third ISV and/or possibly fourth ISV and/or possibly said ISV binding serum albumin are linked via a linker(s); preferably said linker is chosen from the group consisting of linkers of 5GS, 7GS, 9G5, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS (cf. Table D).

[0062] In an aspect, the present invention relates to a polypeptide as described herein, wherein said polypeptide is chosen from the group of polypeptides and/or constructs comprising an ISV binding a target as indicated and one or two ISVs binding Aggrecan as indicated in Table E-1 and Table E-2, respectively.

[0063] In an aspect, the present invention relates to a construct that comprises or essentially consists of an ISV as described herein, or a polypeptide as described herein, and which optionally further comprises 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 is chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

[0064] In an aspect, the present invention relates to a nucleic acid encoding an ISV as described herein, a polypeptide as described herein, or a construct as described herein.

[0065] In an aspect, the present invention relates to an expression vector comprising a nucleic acid as described herein.

[0066] In an aspect, the present invention relates to a host or host cell comprising a nucleic acid as described herein, or an expression vector as described herein.

[0067] In an aspect, the present invention relates to a method for producing an ISV as described herein or a polypeptide as described herein, said method at least comprising the steps of: a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid as described herein; optionally followed by: b) isolating and/or purifying the ISV as described herein, or the polypeptide as described herein.

[0068] In an aspect, the present invention relates to a composition comprising at least one ISV as described herein, a polypeptide as described herein, a construct as described herein, or a nucleic acid as described herein; preferably said composition is a pharmaceutical composition, which preferably further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.

[0069] In an aspect, the present invention relates to a composition as described herein, an ISV as described herein, a polypeptide as described herein, or a construct as described herein, for use as a medicament. Preferably, the composition, the ISV, the polypeptide, or the construct as described herein, is for use in preventing or treating arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis.

[0070] In an aspect, the present invention relates to a method for preventing or treating arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of at least a composition, an ISV, a polypeptide, or a construct as described herein to a person in need thereof.

[0071] In an aspect, the present invention relates to a method for reducing and/or inhibiting the efflux of a compound, a polypeptide or construct from cartilaginous tissue, wherein said method comprises administering pharmaceutically active amount of at least one polypeptide as described herein, a compound or construct as described herein, or a composition as described herein to a person in need thereof.

[0072] In an aspect, the present invention relates to a method for inhibiting and/or blocking ADAMTS5 activity and/or MMP13 activity, wherein said method comprises administering a pharmaceutically active amount of at least one polypeptide as described herein, a construct as described herein, or a composition as described herein to a person in need thereof.

[0073] In an aspect, the present invention relates to the use of an ISV as described herein, a polypeptide as described herein, a construct as described herein, or a composition as described herein, in the preparation of a pharmaceutical composition for treating or preventing arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis.

[0074] Other aspects, advantages, applications and uses of the polypeptides and compositions will become clear 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.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

FIGURE LEGENDS

[0075] FIG. 1: Examples of autoradiography images of sections of rat joints 2 or 4 weeks post injection with .sup.125I-labeled ALB26-CAP constructs. For each of the 2 weeks post injection results and 4 weeks post injection results: Left panel: histological section; Right panel: autoradiography.

[0076] FIG. 2: Representative MARG images. Specific MARG staining appears as black grains on the images and is indicated by the arrows.

[0077] FIG. 3: Inhibition of cartilage degradation by Nanobodies in a rat MMT model using anti-MMP13-CAP Na nobody (C010100754) or an anti-ADAMTS5-CAP Nanobody (C010100954). Treatment started 3 days post-surgery by IA injection. Histopathology was performed at day 42 post surgery. The medial and total substantial cartilage degeneration width was determined, as well as the percentage reduction of cartilage degeneration. 20 animals were used per group.

[0078] FIG. 4: Serum concentrations (mean concentration in ng/ml) versus time after first dose (h) of polypeptides in osteoarthritis rats and healthy rats, receiving a single intra-articular injection of 400 .mu.g Nanobody per joint (right knee). Dots represent individual concentrations in healthy animals; triangles represent individual concentrations in OA animals; and lines represent mean concentrations.

DETAILED DESCRIPTION

[0079] Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al. (Molecular Cloning: A Laboratory Manual (2.sup.nd Ed.) Vols. 1-3, Cold Spring Harbor Laboratory Press, 1989), F. Ausubel et al. (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 1987), Lewin (Genes II, John Wiley & Sons, New York, N.Y., 1985), Old et al. (Principles of Gene Manipulation: An Introduction to Genetic Engineering (2.sup.nd edition) University of California Press, Berkeley, Calif., 1981); Roitt et al. (Immunology (6.sup.th Ed.) Mosby/Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's Essential Immunology (10.sup.th Ed.) Blackwell Publishing, U K, 2001), and Janeway et al. (Immunobiology (6.sup.th Ed.) Garland Science Publishing/Churchill Livingstone, New York, 2005), as well as to the general background art cited herein.

[0080] Unless indicated otherwise, ail methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta (Adv. Drug Day. Rev. 58 (5-6): 640-56, 2006), Levin and Weiss (Mal. Biosyst. 2(1): 49-57, 2006), Irving et al. (J. Immunol. Methods 248(1-2): 31-45, 2001), Schmitz et al. (Placenta 21 Suppl. A: S106-12, 2000), Gonzales et al. (Tumour Biol. 26(1): 31-43, 2005), which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0081] The term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V.sub.HH sequence" or "protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.

[0082] Amino acid sequences are interpreted to mean a single amino acid or an unbranched sequence of two or more amino acids, depending of the context. Nucleotide sequences are interpreted to mean an unbranched sequence of 3 or more nucleotides.

[0083] Amino acids are those L-amino acids commonly found in naturally occurring proteins, Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. Reference is for instance made to Table A-2 on page 48 of WO 08/020079. Those amino acid sequences containing D-amino acids are not intended to be embraced by this definition. Any amino acid sequence that contains post-translationally modified amino acids may be described as the amino acid sequence that is initially translated using the symbols shown in this Table A-2 with the modified positions; e.g., hydroxylations or glycosylations, but these modifications shall not be shown explicitly in the amino acid sequence. Any peptide or protein that can be expressed as sequence modified linkages, cross links and end caps, non-peptidyl bonds, etc., is embraced by this definition.

[0084] The terms "protein", "peptide", "protein/peptide", and "polypeptide" are used interchangeably throughout the disclosure and each has the same meaning for purposes of this disclosure. Each term refers to an organic compound made of a linear chain of two or more amino acids. The compound may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. The skilled artisan will appreciate that polypeptides generally comprise fewer amino acids than proteins, although there is no art-recognized cut-off point of the number of amino acids that distinguish a polypeptide from a protein; that polypeptides may be made by chemical synthesis or recombinant methods; and that proteins are generally made in vitro or in vivo by recombinant methods, all as known in the art.

[0085] A nucleic acid or amino acid sequence is considered to be "(in) (essentially) isolated (form)"--for example, compared to the reaction medium or cultivation medium from which it has been obtained-when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a nucleic acid or amino acid sequence is considered "(essentially) isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more. A nucleic acid or amino acid that is "in (essentially) isolated form" is preferably essentially homogeneous, as determined by using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide-gel electrophoresis.

[0086] When a nucleotide sequence or amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of" another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein). By means of a non-limiting example, when a polypeptide of the invention is said to comprise an immunoglobulin single variable domain ("ISV"), this may mean that said immunoglobulin single variable domain sequence has been incorporated into the sequence of the polypeptide of the invention, but more usually this generally means that the polypeptide of the invention contains within its sequence the sequence of the ISVs irrespective of how said polypeptide of the invention has been generated or obtained. Also, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, the first mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed into 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, that the latter nucleotide sequence is in the same reading frame as the first mentioned, larger nucleic acid or nucleotide sequence). Also, when a construct of the invention is said to comprise a polypeptide or ISV, this may mean that said construct at least encompasses said polypeptide or ISV, respectively, but more usually this means that said construct encompasses groups, residues (e.g. amino acid residues), moieties and/or binding units in addition to said polypeptide or ISV, irrespective of how said polypeptide or ISV is connected to said groups, residues (e.g. amino acid residues), moieties and/or binding units and irrespective of how said construct has been generated or obtained.

[0087] By "essentially consist of" is meant that the ISV used in the method of the invention either is exactly the same as the ISV of the invention or corresponds to the ISV of the invention which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino-terminal end, at the carboxy-terminal end, or at both the amino terminal end and the carboxy-terminal end of the ISV.

[0088] For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence--compared to the first nucleotide sequence--is considered as a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as, e.g. NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768. Usually, for the purpose of determining the percentage of "sequence identity" between two nucleotide sequences in accordance with the calculation method outlined hereinabove, the nucleotide sequence with the greatest number of nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the "second" nucleotide sequence.

[0089] For the purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence (also referred to herein as "amino acid identity") may 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%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence--compared to the first amino acid sequence--is considered as a difference at a single amino acid residue (position), i.e., as an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings. Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences in accordance with the calculation method outlined hereinabove, the amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.

[0090] Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the 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 on the basis of the pertinent teachings from, e.g. WO 04/037999 or e.g. WO 98/49185 and from the further references cited therein.

[0091] Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a)-(e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar 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, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into lie or into Leu.

[0092] Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species such as, for instance, developed by Schulz et al. ("Principles of Protein Structure", Springer-Verlag, 1978), on the analyses of structure forming potentials developed by, e.g. Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45-149, 1978), and on the analysis of hydrophobicity patterns in proteins developed by e.g. 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, 1986), all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a V.sub.HH domain from a llama is for example given by Desmyter et al. (Nature Structural Biology, 3: 803, 1996), Spinelli et al. (Natural Structural Biology, 3: 752-757, 1996) or Decanniere et al, (Structure, 7 (4): 361, 1999). Further information about some of the amino acid residues that in conventional V.sub.H domains form the V.sub.H/V.sub.L interface and potential camelizing substitutions on these positions can be found in the prior art cited above.

[0093] Amino acid sequences and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length.

[0094] When comparing two amino acid sequences, the term "amino acid(s) difference" refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences. More particularly, in the amino acid sequences and/or polypeptides of the present invention, the term "amino acid(s) difference" refers to an insertion, deletion or substitution of a single amino acid residue on a position of the CDR sequence specified in b), d) or f), compared to the CDR sequence of respectively a), c) or e); it being understood that the CDR sequence of b), d) and f) can contain one, two, three, four or maximal five such amino acid differences compared to the CDR sequence of respectively a), c) or e).

[0095] The "amino acid(s) difference" can be any one, two, three, four or maximal five substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Aggrecan binder of the invention, such as the polypeptide of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Aggrecan binder of the invention, such as the polypeptide of the invention. In this respect, the resulting Aggrecan binder of the invention, such as the polypeptide of the invention should at least bind Aggrecan with the same, about the same, or a higher affinity compared to the polypeptide comprising the one or more CDR sequences without the one, two, three, four or maximal five substitutions, deletions or insertions, said affinity as measured by surface plasmon resonance (SPR).

[0096] In this respect, the amino acid sequence of the CDRs according to b), d) and/or f) may be an amino acid sequence that is derived from an amino acid sequence according to a), c) and/or e) respectively by means of affinity maturation using one or more techniques of affinity maturation known per se.

[0097] For example, and depending on the host organism used to express the polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art.

[0098] A "Na nobody family", "V.sub.HH family" or "family" as used in the present specification refers to a group of Nanobodies and/or V.sub.HH sequences that have identical lengths (i.e. they have the same number of amino acids within their sequence) and of 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.

[0099] The terms "epitope" and "antigenic determinant", which can be used interchangeably, refer to the part of a macromolecule, such as a polypeptide or protein that is recognized by antigen-binding molecules, such as immunoglobulins, conventional antibodies, ISVs and/or polypeptides of the invention, and more particularly by the antigen-binding site of said molecules. Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an immunoglobulin.

[0100] The part of an antigen-binding molecule (such as an immunoglobulin, a conventional antibody, an ISV and/or a polypeptide of the invention) that recognizes the epitope is called a "paratope".

[0101] An amino acid sequence (such as an ISV, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can "bind to" or "specifically bind to", that "has affinity for" and/or that "has specificity for" a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against" or "directed against" said epitope, antigen or protein or is a "binding" molecule with respect to such epitope, antigen or protein, or is said to be "anti"-epitope, "anti"-antigen or "anti"-protein (e.g., "anti"-Aggrecan).

[0102] The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as the K.sub.D, or dissociation constant, which has units of mol/liter (or M). The affinity can also be expressed as an association constant, K.sub.A, which equals 1/K.sub.D and has units of (mol/liter).sup.-1 (or M.sup.-1). In the present specification, the stability of the interaction between two molecules will mainly be expressed in terms of the K.sub.D value of their interaction; it being clear to the skilled person that in view of the relation K.sub.A=1/K.sub.D, specifying the strength of molecular interaction by its K.sub.D value can also be used to calculate the corresponding K.sub.A value. The K-value characterizes the strength of a molecular interaction also in a thermodynamic sense as it is related to the change of free energy (DG) of binding by the well-known relation DG=RTIn(K.sub.D) (equivalently DG=-RTIn(K.sub.A)), where R equals the gas constant, T equals the absolute temperature and In denotes the natural logarithm.

[0103] The K.sub.D for biological interactions which are considered meaningful (e.g. specific) are typically in the range of 10.sup.-12 M (0.001 nM) to 10.sup.-5M (10000 nM). The stronger an interaction is, the lower is its K.sub.D.

[0104] The K.sub.D can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as k.sub.off, to the rate of its association, denoted k.sub.on (so that K.sub.D=k.sub.off/k.sub.on and K.sub.A=k.sub.on/k.sub.off), The off-rate k.sub.off has unit s.sup.-1 (where s is the SI unit notation of second). The on-rate k.sub.on has units M.sup.-1 s.sup.-1. The on-rate may vary between 10.sup.2 M.sup.-1 s.sup.-1 to about 10.sup.7 M.sup.-1 s.sup.-1, approaching the diffusion-limited association rate constant for bimolecular interactions. The off-rate is related to the half-life of a given molecular interaction by the relation t.sub.1/2=In(2)/k.sub.off. The off-rate may vary between 10.sup.-6 s.sup.-1 (near irreversible complex with a t.sub.in of multiple days) to 1 s.sup.-1 (t.sub.1/2=0.69 s).

[0105] 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 radio-immunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.

[0106] The affinity of a molecular interaction between two molecules can be measured via different 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 the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k.sub.on, k.sub.off measurements and hence K.sub.D (or K.sub.A) values. This can for example be performed using the well-known BIACORE.RTM. instruments (Pharmacia Biosensor AB, Uppsala, Sweden). Kinetic Exclusion Assay (KINEXA.RTM.) (Drake et al. 2004, Analytical Biochemistry 328: 35-43) measures binding events in solution without labeling of the binding partners and is based upon kinetically excluding the dissociation of a complex. In-solution affinity analysis can also be performed using the GYROLAB.RTM. immunoassay system, which provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74), or ELISA.

[0107] It will also be clear to the skilled person that the measured K.sub.0 may correspond to the apparent K.sub.0 if the measuring process somehow influences the intrinsic binding affinity of the implied molecules for example by artifacts related to the coating on the biosensor of one molecule. Also, an apparent K.sub.D may be measured if one molecule contains more than one recognition site for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules. In particular, the accurate measurement of K.sub.D may be quite labor-intensive and as a consequence, often apparent K.sub.D values are determined to assess the binding strength of two molecules. It should be noted that as long as all measurements are made in a consistent way (e.g. keeping the assay conditions unchanged) apparent K.sub.D measurements can be used as an approximation of the true K.sub.D and hence in the present document K.sub.D and apparent K.sub.D should be treated with equal importance or relevance.

[0108] The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an ISVD or polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity, for instance as described on pages 53-56 of WO 08/020079 (incorporated herein by reference), which also describes some preferred techniques for measuring binding between an antigen-binding molecule (such as a polypeptide or ISVD of the invention) and the pertinent antigen. Typically, antigen-binding proteins (such as the ISVDs and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (K.sub.D) of 10.sup.-5 to 10.sup.-12 moles/liter or less, and preferably 10.sup.-7 to 10.sup.-12 moles/liter or less and more preferably 10.sup.-8 to 10.sup.-12 moles/liter (i.e., with an association constant (K.sub.A) of 10.sup.5 to 10.sup.12 liter/moles or more, and preferably 10.sup.7 to 10.sup.12 liter/moles or more and more preferably 10.sup.8 to 10.sup.12 liter/moles). Any K.sub.D value greater than 10.sup.-4 mol/liter (or any K.sub.a value lower than 10.sup.4 liter/mol) is generally considered to indicate non-specific binding. Preferably, a monovalent ISVD of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM, such as e.g., between 10 and 5 pM or less. Reference is also made to paragraph n) on pages 53-56 of WO 08/020079.

[0109] An ISV and/or polypeptide is said to be "specific for" a (first) target or antigen compared to another (second) target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed as a K.sub.D value, K.sub.A value, K.sub.off rate and/or K.sub.on rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times or more better than the affinity with which the ISVD and/or polypeptide binds to the second target or antigen. For example, the ISVD and/or polypeptide may bind to the first target or antigen with a K.sub.D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less or even less than that, than the K.sub.D with which said ISV and/or polypeptide binds to the second target or antigen. Preferably, 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 (as defined herein) said first target or antigen, but not directed against said second target or antigen.

[0110] 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 binding assays and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and the different variants thereof known in the art; as well as the other techniques mentioned herein.

[0111] A preferred approach that may be used to assess affinity is the 2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. 1985 (J. Immunol. Methods 77: 305-19). This method establishes a solution phase binding equilibrium measurement and avoids possible artifacts relating to adsorption of one of the molecules on a support such as plastic. As will be clear to the skilled person, the dissociation constant may be the actual or apparent dissociation constant. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned on pages 53-56 of WO 08/020079.

[0112] Finally, it should be noted that in many situations the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule. For example, to assess the binding strength between molecules A and B, one may e.g, use a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the 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 an IC.sub.50 value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved. Provided K.sub.D ref, the K.sub.D of the reference molecule, is known, as well as the total concentration c.sub.ref of the reference molecule, the apparent K.sub.D for the interaction A-B can be obtained from following formula: K.sub.D=IC.sub.50/(1+C.sub.ref/K.sub.Dref). Note that if c.sub.ref<<K.sub.D ref, K.sub.D.apprxeq.IC.sub.50. Provided the measurement of the IC.sub.50 is performed in a consistent way (e.g. keeping c.sub.ref fixed) for the binders that are compared, the difference in strength or stability of a molecular interaction can be assessed by comparing the IC.sub.50 and this measurement is judged as equivalent to K.sub.D or to apparent K.sub.D throughout this text.

[0113] The half maximal inhibitory concentration (IC.sub.50) can also be a measure of the effectiveness of a compound in inhibiting a biological or biochemical function, e.g. a pharmacological effect. This quantitative measure indicates how much of the polypeptide or ISV (e.g. a Nanobody) is needed to inhibit a given biological process (or component of a process, i.e. an 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.sub.50). IC.sub.50 values can be calculated for a given antagonist such as the polypeptide or ISV (e.g. a Nanobody) of the invention by determining the concentration needed to inhibit half of the maximum biological response of the agonist. The K.sub.D of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of antagonist such as the polypeptide or ISV (e.g. a Nanobody) of the invention on reversing agonist activity.

[0114] The term half maximal effective concentration (EC.sub.50) refers to the concentration of a compound which induces a response halfway between the baseline and maximum after a specified exposure time. In the present context it is used as a measure of a polypeptide, ISV (e.g. a Nanobody) its potency. The EC.sub.50 of a graded dose response curve represents the concentration of a compound where 50% of its maximal effect is observed. Concentration is preferably expressed in molar units.

[0115] In biological systems, small changes in ligand concentration typically result in rapid changes in response, following a sigmoidal function. The inflection point at which the increase in response with increasing ligand concentration begins to slow is the EC.sub.50. This can be determined mathematically by derivation of the best-fit line. Relying on a graph for estimation is convenient in most cases. In case the EC.sub.50 is provided in the examples section, the experiments were designed to reflect the K.sub.D as accurate as possible. In other words, the EC.sub.50 values may then be considered as K.sub.D values. The term "average K.sub.D" relates to the average K.sub.D value obtained in at least 1, but preferably more than 1, such as at least 2 experiments. The term "average" refers to the mathematical term "average" (sums of data divided by the number of items in the data).

[0116] It is also related to IC.sub.50 which is a measure of a compound its inhibition (50% inhibition). For competition binding assays and functional antagonist assays IC.sub.50 is the most common summary measure of the dose-response curve. For agonist/stimulator assays the most common summary measure is the EC.sub.50.

[0117] The inhibition constant (Ki) is an indication of how potent an inhibitor is; it is the concentration required to produce half maximum inhibition. Unlike IC.sub.50, which can change depending on the experimental conditions, Ki is an absolute value and is often referred to as the inhibition constant of a drug. The inhibition constant K.sub.i can be calculated by using the Cheng-Prusoff equation:

K i = IC .times. .times. 50 [ L ] K D + 1 ##EQU00001##

in which [L] is the fixed concentration of the ligand.

[0118] An ISV and/or polypeptide is said to be "specific for" a (first) target or antigen compared to another (second) target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed as a K.sub.D value, K.sub.A value, K.sub.off rate and/or K.sub.on rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times or more better than the affinity with which the ISV and/or polypeptide binds to the second target or antigen. For example, the ISV and/or polypeptide may bind to the first target or antigen with a K.sub.D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less or even less than that, than the K.sub.o with which said ISV and/or polypeptide binds to the second target or antigen. Preferably, 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 (as defined herein) said first target or antigen, but not directed against said second target or antigen.

[0119] The terms "(cross)-block", "(cross)-blocked", "(cross)-blocking", "competitive binding", "(cross)-compete", "(cross)-competing" and "(cross)-competition" are used interchangeably herein to mean the ability of an immunoglobulin, antibody, ISV, polypeptide or other binding agent to interfere with the binding of other immunoglobulins, antibodies, ISVs, polypeptides or binding agents to a given target. The extent to which an immunoglobulin, antibody, ISV, polypeptide or other binding agent is able to interfere with the binding of another to the target, and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays, which are common in the art. Particularly suitable quantitative cross-blocking assays include an ELISA and a fluorescence-activated cell sorting (FACS) binding assay with Aggrecan expressed on cells. In a FACS set up, the extent of (cross)-blocking can be measured by the (reduced) channel fluorescence.

[0120] Methods for determining whether an immunoglobulin, antibody, ISV, polypeptide or other binding agent directed against a target (cross)-blocks, is capable of (cross)-blocking, competitively binds or is (cross)-competitive as defined herein are described e.g. 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 the methods described herein (see e.g, Example 2.3).

[0121] An amino acid sequence is said to be "cross-reactive" for two different antigens or antigenic determinants (such as e.g., Aggrecan from different species of mammal, such as e.g., human Aggrecan, dog Aggrecan, bovine Aggrecan, rat Aggrecan, pig Aggrecan, mouse Aggrecan, rabbit Aggrecan, cynomolgus Aggrecan, and/or rhesus Aggrecan) if it is specific for (as defined herein) these different antigens or antigenic determinants.

[0122] In the context of the present invention, "modulating" or "to modulate" generally means reducing or inhibiting an activity of a member of the serine protease family, cathepsins, matrix metallo-proteinases (MMPs)/Matrixins or A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (Aggrecanase-2), ADAMTS4 (Aggrecanase-1), ADAMTS11 and/or pro-inflammatory cytokines, such as e.g. interleukin-1a, and -.beta., interleukin-6 and TNF-.alpha., 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 mentioned herein). In particular, "modulating" or "to modulate" may mean either reducing or inhibiting the activity of the aforementioned members as measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the activity of the aforementioned members in the same assay under the same conditions but without the presence of the immunoglobulin or polypeptide of the invention.

[0123] In the context of the present invention, "enhancing" or "to enhance" generally means increasing, potentiating or stimulating the activity of the polypeptides or constructs of the invention, as measured using a suitable in vitro, cellular, ex vivo or in vivo assay (such as those mentioned herein). In particular, increasing or enhancing the activity of a polypeptide or construct of the invention, as measured using a suitable in vitro, cellular, ex vivo or in viva assay (such as those mentioned herein), 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 the Aggrecan binder, e.g. ISV binding Aggrecan, of the invention.

[0124] 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 is preferably statistically different from the controls and the single drugs.

[0125] The term "potency" of an ISV or polypeptide of the invention, as used herein, is a function of the amount of the ISV or polypeptide of the invention required for its specific effect, such as, e.g. penetration into the cartilage, specific binding to Aggrecan and/or cartilage retention, to occur. It can be measured simply by the methods known to the person skilled in the art, and for instance as used in the examples section.

[0126] In contrast, the "efficacy" of the ISV or polypeptide of the invention measures the maximum strength of the effect itself, at saturating ISV or polypeptide concentrations. Efficacy indicates the maximum response achievable from the ISV or polypeptide of the invention. It refers to the ability of an ISV or polypeptide to produce the desired (therapeutic) effect, such as, e.g. binding to Aggrecan or retention to Aggrecan, and/or inhibiting an activity of an ADAMTS family member or MMP family member.

[0127] The "half-life" of a polypeptide or construct of the invention refers to the time taken for the serum concentration of the construct or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the construct or polypeptide and/or clearance or sequestration of the construct or polypeptide by natural mechanisms, see e.g. paragraph o) on page 57 of WO 08/020079. The in vivo half-life of a construct or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally be as described in paragraph o) on page 57 of WO 08/020079. As also mentioned in paragraph o) on page 57 of WO 08/020079, the half-life can be expressed using parameters such as the t1/2-alpha, t1/2-beta and the area under the curve (AUC). Reference is for example made to the 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, 2.sup.nd Rev. Edition, 1982). The terms "increase in half-life" or "increased half-life" refer to an increase in the t1/2-beta, either with or without an increase in the t1/2-alpha and/or the AUC or both, for instance as described in paragraph o) on page 57 of WO 08/020079.

[0128] Unless indicated otherwise, the terms "immunoglobulin" and "immunoglobulin sequence"--whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody--is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as V.sub.HH domains or V.sub.H/V.sub.L domains, respectively).

[0129] The term "domain" (of a polypeptide or protein) as used herein refers to a folded protein structure which has the ability to retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.

[0130] The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (such as e.g., a chain of a conventional 4-chain antibody or of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are characterized in that they retain the immunoglobulin fold characteristic of antibody molecules, which consists of a two-layer sandwich of about seven antiparallel beta-strands arranged in two beta-sheets, optionally stabilized by a conserved disulphide bond.

[0131] The term "immunoglobulin variable domain" as used herein means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain(s) that confer specificity to an antibody for the antigen by carrying the antigen-binding site, and in particular CDR1, CDR2 and/or CDR3.

[0132] The term "immunoglobulin single variable domain" ("ISV" or ISVD''), interchangeably used with "single variable domain", defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets ISVs apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VI) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation.

[0133] In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an ISV, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

[0134] In contrast, ISVs are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISV is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an ISV is formed by no more than three CDRs.

[0135] As such, the 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 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 essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

[0136] In one embodiment of the invention, the ISVs are heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, the ISVs can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody.

[0137] For example, the ISV may be a (single) domain antibody, an amino acid that is suitable for use as a (single) domain antibody, an immunoglobulin that is suitable for use as a (single) domain antibody, a "dAb" or sdAb, or an amino acid that is suitable for use as a dAb, or a Nanobody (as defined herein, and including but not limited to a VHH); a humanized VHH sequence, a camelized VH sequence, a VHH sequence that has been obtained by affinity maturation, other single variable domains, an immunoglobulin single heavy chain variable domain or any suitable fragment of any one thereof.

[0138] In particular, the ISV may be a Nanobody.RTM. (as defined herein) or a suitable fragment thereof. [Note: Nanobody.RTM. and Nanobodies.RTM. are registered trademarks of Ablynx N.V.] For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in WO 08/020079 (page 16).

[0139] "VHH domains", also known as VHHs, V.sub.HH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (variable) domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al. Nature 363: 446-448, 1993). The term "VHH domain" has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "V.sub.H domains" or "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "V.sub.L domains" or "VL domains"). For a further description of VHHs and Nanobodies, reference is for instance made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (=EP 1433793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the international application 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 in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the ISVs, Nanobodies, including humanization and/or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the ISVs, Nanobodies and their preparations can be found e.g. in WO 08/101985 and WO 08/142164. For a further general description of Nanobodies, reference is made to the prior art cited herein, such as e.g., described in WO 08/020079 (page 16).

[0140] "Domain antibodies", also known as "Dab" (s), "Domain Antibodies", and "dAbs" (the terms "Domain Antibodies" and "dAbs" being used as trademarks by the GlaxoSmithKline group of companies) have been described in e.g., EP 0368684, Ward et al. (Nature 341: 544-546, 1989), Holt et al. (Tends in Biotechnology 21: 484-490, 2003) and WO 03/002609 as well as for example WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammalians, in particular human 4-chain antibodies. In order to bind an epitope as a single antigen binding domain, i.e., without being paired with a VL or VH domain, respectively, specific selection for such antigen binding properties is required, e.g. by using libraries of human single VH or VL domain sequences. Domain antibodies have, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not require humanization for e.g. therapeutic use in humans.

[0141] It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629).

[0142] Thus, in the meaning of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" comprises polypeptides which are derived from a non-human source, preferably a camelid, preferably a camelid heavy chain antibody. They may be humanized, as previously described. Moreover, the term comprises polypeptides derived from non-camelid sources, e.g. mouse or human, which have been "camelized", as e.g., described in Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13: 475-479, 1995; Prot. Eng. 9: 531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999).

[0143] The amino acid residues of a VHH domain are numbered according to the general numbering for V.sub.H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids, as shown e.g., in FIG. 2 of Riechmann and Muyldermans Immunol. Methods 231: 25-38, 1999). Alternative methods for numbering the amino acid residues of V.sub.H domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the present description, claims and figures, the numbering according to Kabat applied to VHH domains as described above will be followed, unless indicated otherwise.

[0144] It should be noted that--as is well known in the art for V.sub.H domains and for VHH domains--the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0145] Determination of CDR regions may also be done according to different methods. In the CDR determination according to Kabat, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113.

[0146] In the present application, however, CDR sequences were determined according to Kontermann and Dubel (Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113 (according to Kabat numbering).

[0147] ISVs such as Domain antibodies and Nanobodies (including VHH domains) can be subjected to humanization. In particular, humanized immunoglobulin single variable domains, such as Nanobodies (including VHH domains) may be immunoglobulin single variable domains that are as generally defined for in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V.sub.HH sequence with the corresponding framework sequence of one or more closely related human V.sub.H sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V.sub.HH sequence (in any manner known per se, as further described herein) and the resulting humanized V.sub.HH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody (including VHH domains) may be partially humanized or fully humanized.

[0148] ISVs such as Domain antibodies and Nanobodies (including VHH domains and humanized VHH domains), can also be subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations result in an improved affinity of the resulting immunoglobulin single variable domain for its respective antigen, as compared to the respective parent molecule. Affinity-matured immunoglobulin single variable domain molecules of the invention may be prepared by methods known in the art, for example, as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas, et al. (Proc. Nat. Acad. Sci, USA 91: 3809-3813, 1994), Shier et al. (Gene 169: 147455, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226: 889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996).

[0149] The process of designing/selecting and/or preparing a polypeptide, starting from an ISV such as a Domain antibody or a Na nobody, is also referred to herein as "formatting" said ISV; and an ISV that is made part of a polypeptide is said to be "formatted" or to be "in the format of" said polypeptide. Examples of ways in which an ISV can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted ISV form a further aspect of the invention.

[0150] For example, and without limitation, one or more ISVs may be used as a "binding unit", "binding domain" or "building block" (these terms are used interchangeable) for the preparation of a polypeptide, which may optionally contain one or more further ISVs that can serve as a binding unit (i.e., against the same or another epitope on Aggrecan and/or against one or more other antigens, proteins or targets than Aggrecan).

[0151] The present invention provides Aggrecan binders, 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 that bind Aggrecan.

[0152] Aggrecan is also known as aggrecan 1, ACAN, AGC1, AGCAN, CSPGCP, MSK16, SEDK, cartilage-specific proteoglycan core protein (CSPCP) or chondroitin sulfate proteoglycan 1 (CSPG1). Aggrecan is in humans encoded by the ACAN gene, which is located at chromosome Chr 15: q26.1.

[0153] Aggrecan is a large, multimodular molecule (2317 amino acids). Its core protein is composed of three globular domains (G1, G2 and G3) and a large extended region (CS) between 62 and G3 onto which a multitude of N-linked oligosaccharides and chondroitin sulfate chains and keratan sulfate chains are attached. Aggrecan is the major proteoglycan in the articular cartilage. It plays an important role in the proper functioning of articular cartilage by providing a hydrated gel structure through its interaction with hyaluronan and link proteins, which endows the cartilage with load-bearing properties. The G1 domain interacts with hyaluronan acid and link proteins, forming stable ternary complexes in the extracellular matrix (ECM). The G2 domain is homologous to the tandem repeats of G1 and link proteins, and is involved in product processing. G3 makes up the carboxyl terminus of the core protein, and enhances glycosaminoglycan modification and product secretion. Also, the G3 domain links the proteoglycan aggregates to the ECM proteins (fibulins and tenascins). Degradation of Aggrecan appears to initiate at the C-terminus. The population of Aggrecan molecules without the 63 domain increases with aging. Aggrecan interacts with laminin, fibronectin, tenascin, and collagen, but it is also an enzymatic substrate of various A Disintegrin And Metalloprotease with Thrombo-spondin Motifs (ADAMTSs) such as ADAMTS4, ADAMTS5 and ADAMTS11 and matrix metallo-proteinases (MMPs) such as MMP8, MMP13, MMP19 and MMP20.

[0154] In one aspect, the invention relates to Aggrecan binders such as ISVs and polypeptides that specifically bind Aggrecan.

[0155] The Aggrecan binders of the invention are eventually intended for use as medicaments in humans. Accordingly, in one aspect the invention relates to Aggrecan binders, such as ISVs and polypeptides that specifically bind human Aggrecan (SEQ ID NO: 125).

[0156] The inventors identified Aggrecan binders with highly improved interspecies cross-reactivity and exquisite selectivity properties.

[0157] Accordingly, in an aspect the invention relates to an Aggrecan binder, such as an ISV or polypeptide, wherein said Aggecan binder specifically binds to human Aggrecan (P16112; SEQ ID NO: 125), dog Aggrecan (028343; SEQ ID NO: 126), bovine Aggrecan (P13608; SEQ ID NO: 127), rat Aggrecan (P07897; SEQ ID NO: 128); pig Aggrecan (core; Q29011, SEQ ID NO: 129); mouse Aggrecan (061282; SEQ ID NO: 130), rabbit Aggrecan (G1U677-1; SEQ ID NO: 131); cynomolgus Aggrecan (XP_005560513.1; SEQ ID NO: 132) and/or rhesus Aggrecan (XP_002804990.1; SEQ ID NO: 133) (cf. Table B).

[0158] The present inventors surprisingly observed that the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention have favorable characteristics over the prior art molecules; they are stable in joints, they retain in the cartilage for prolonged times and they are specific for cartilaginous tissue, e.g. do not bind substantially to Neurocan (014594, SEQ ID NO: 134) and/or Brevican (Q96GW7, SEQ ID NO: 135) (cf. Table B).

[0159] Accordingly, in one aspect the invention relates to an Aggrecan binder, such as an ISV or polypeptide, wherein said Aggrecan binder does not bind substantially to Neurocan (014594, SEQ ID NO: 134) and/or Brevican (Q96GW7, SEQ ID NO: 135), preferably wherein said Aggrecan binds to Neurocan and/or Brevican with a K.sub.D value greater than 10.sup.-5 mol/liter, such as 10.sup.-4 mol/liter.

[0160] In one aspect the invention relates to an Aggrecan binder, such as an ISV, wherein said Aggrecan binder has more than 10 fold, more than 100 fold, preferably more than 1000 fold selectivity over Neurocan and/or Brevican for binding to Aggrecan.

[0161] Preferred Aggrecan binders of the invention include immunoglobulins (such as heavy chain antibodies, conventional 4-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 4-chain antibody, the individual chains thereof, as well as all parts, domains or fragments 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).

[0162] It was observed that the Aggrecan binders of the invention had a pl over 8, with only one exception (cf. Table 2.2). Without being bound by theory, the present inventors hypothesized that the high positive charge of the Aggrecan may influence retention and cartilage penetration of the whole moiety, i.e. even when coupled to another building block such as in a multispecific polypeptide. Accordingly, the present invention relates to an Aggrecan binder, such as an ISV, polypeptide or construct of the invention, preferably an ISV of the invention, having a pl of more 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 more, such as 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8 or even 9.8.

[0163] Binding of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, to Aggrecan can be measured in various binding assays, commonly known in the art. Typical assays include (without being limiting) 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 microresonator (WGM), Resonant waveguide grating (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 binding of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, to Aggrecan is SPR, such as e.g. the SPR as described in the examples, wherein binding of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, to Aggrecan was determined. Some preferred KD values for binding of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, to Aggrecan will become clear from the further description and examples herein. Another particularly preferred assay is ELISA as detailed in the Examples (cf. Examples 1.2 and 2.4).

[0164] Binding of the Aggrecan binders of the invention to Aggrecan can also be measured in binding assays that preferably preserve the conformation of the Aggrecan target. Typical assays include (without being limiting) assays in which Aggrecan is exposed on a cell surface (such as e.g. CHO cells).

[0165] In an embodiment of the invention, the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, have an on rate constant (Kon) for binding to said Aggrecan selected from the group consisting of at least about 10.sup.2 M.sup.-1 s.sup.-1, at least about 10.sup.3 M.sup.1 s.sup.-1, at least about 10.sup.4 M.sup.-1 s.sup.-1, at least about 10.sup.5 M.sup.-1 s.sup.-1, at least about 10.sup.6 M.sup.-1 s.sup.-1, 10.sup.7 M.sup.-1 s.sup.-1, at least about 10.sup.8 M.sup.-1 s.sup.-1, at least about 10.sup.9 M.sup.-1 s.sup.-1, and at least about 10.sup.10 M.sup.-1 s.sup.-1, preferably as measured by surface plasmon resonance.

[0166] In an embodiment of the invention, the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, have an off rate constant {Koff} for binding to said Aggrecan selected from the group consisting of at most about 10.sup.-3 s.sup.-1, at most about 10.sup.-4 s.sup.-1, at most about 10.sup.-5 s.sup.-1, at most about 10.sup.-6 s.sup.-1, at most about 10.sup.-7 s.sup.-1, at most about 10.sup.-8 s.sup.-1, at most about 10.sup.-9 s.sup.-1, and at most about 10.sup.-10 s.sup.-1, preferably as measured by surface plasmon resonance.

[0167] In an embodiment of the invention, the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, bind to said Aggrecan with an average KD value of between 100 nM and 10 pM, such as at an average KD value of 90 nM or less, even more preferably at an average KD value of 80 nM or less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, 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 less such as less than 10 pM. Preferably, the KD is determined by SPR, for instance as determined by Proteon.

[0168] Some preferred EC50 values for binding of the immunoglobulins and/or polypeptides of the invention to Aggrecan will become clear from the further description and examples herein.

[0169] In an ELISA binding assay, the Aggrecan binders of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or G1-IGD-G2 domain, may have EC50 values in binding human Aggrecan of 10.sup.-8 M or lower, more preferably of 10.sup.-9 M or lower, or even of 10.sup.-10 M or lower. For example, in such ELISA binding assay, the immunoglobulins and/or polypeptides of the present invention may have EC50 values in binding human Aggrecan between 10.sup.-10 M and 10.sup.-8 M, such as between 10.sup.-9 M and 10.sup.-8 M or between 10.sup.-10 M and 10.sup.-9 M.

[0170] In such ELISA binding assay, the Aggrecan binders of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or G1-IGD-G2 domain, may have EC50 values in binding cynomolgus (cyno) Aggrecan of 10.sup.-7 M or lower, preferably of 10.sup.-8 M or lower, more preferably of 10.sup.-9 M or lower, or even of 10.sup.-10 M or lower. For example, in such ELISA binding assay, the polypeptides of the present invention may have EC50 values in binding cyno Aggrecan between 10.sup.-10 M and 10.sup.-7 M, such as between 10.sup.-10 M and 10.sup.-8 M, between 10.sup.-10 M and 10.sup.-9 M.

[0171] In such ELISA binding assay, the Aggrecan binders of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or G1-IGD-G2 domain, may have EC50 values in binding rat Aggrecan of 10.sup.-6 M or lower, preferably of 10.sup.-7 M or lower, preferably of 10.sup.-8 M or lower, more preferably of 10.sup.-9 M or lower, or even of 10.sup.-10 M or lower. For example, in such ELISA binding assay, the polypeptides of the present invention may have EC50 values in binding rat Aggrecan between 10.sup.-10 M and 10.sup.-6 M, such as between 10.sup.-10 M and 10.sup.-7 M, between 10.sup.-10 M and 10.sup.-8 M, between 10.sup.-10 M and 10.sup.-9 M.

[0172] In such ELISA binding assay, the Aggrecan binders of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or G1-IGD-G2 domain, may have EC50 values in binding dog Aggrecan of 10.sup.-5 M or lower, preferably of 10.sup.-7 M or lower, preferably of 10.sup.-8 M or lower, more preferably of 10.sup.-9 M or lower, or even of 10.sup.-10 M or lower. For example, in such ELISA binding assay, the polypeptides of the present invention may have EC50 values in binding dog Aggrecan between 10.sup.-10 M and 10.sup.-6 M, such as between 10.sup.-10 M and 10.sup.-7 M, between 10.sup.-10 M and 10.sup.-8 M, between 10.sup.-10 M and 10.sup.-9 M.

[0173] In such ELISA binding assay, the Aggrecan binders of the invention, such as ISVs and/or polypeptides of the present invention may, preferably binding the G1 domain and/or G1-IGD-G2 domain, have EC50 values in binding bovine Aggrecan of 10.sup.-6 M or lower, preferably of 10.sup.-7 M or lower, preferably of 10.sup.-8 M or lower, more preferably of 10.sup.-9 M or lower, or even of 10.sup.-10 M or lower. For example, in such ELISA binding assay, the polypeptides of the present invention may have EC50 values in binding bovine Aggrecan between 10.sup.-10 M and 10.sup.-6M, such as between 10.sup.-10 M and 10.sup.-7M, between 10.sup.-10 M and 10.sup.-8 M, between 10.sup.-10 M and 10.sup.-9M.

[0174] 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 space and relative amounts of collagen and proteoglycan. "Articular cartilage" is the cartilage found on the articular surface of bones and is mostly hyaline cartilage. Menisci are made entirely of fibrocartilage. Aggrecan is the main proteoglycan in the extracellular matrix (ECM) and accounts for ca. 50% of total protein content (the other ca. 50% are collagen II and some minor proteins, such as, e.g. collagen IX).

[0175] The Aggrecan binders of the invention demonstrated a preference to bind to cartilaginous tissues in a joint such as cartilage and meniscus over non-cartilaginous tissue such as synovial membrane, tendon, and/or epimysium. Accordingly, the present invention relates to an Aggrecan binder, such as an ISV or polypeptide, wherein said Aggrecan binder preferably binds to cartilaginous tissue such as cartilage and/or meniscus, preferably by at least a factor 1.5, a factor 2, a factor 3, a factor 4, a factor 5 or even more compared to non-cartilaginous tissue.

[0176] It is appreciated that joints are the areas where two or more bones meet. Most joints are mobile, allowing the bones to move. Joints consist of the following: cartilage, synovial membrane, ligaments, tendons, bursas and synovial fluid. Some joints also have a meniscus.

[0177] As demonstrated in the examples, the Aggrecan binders of the invention have various cartilage retention characteristics, which enables customizing retention in joints according to the specific needs (cf. Example 2.2). Preferably, the Aggrecan binders have the ability to retain in cartilage for prolonged periods of time following a relatively short exposure of the Aggrecan binders to the cartilage, which can be expected upon intra-articular injection. The cartilage retention can be measured via an ex vivo cartilage retention assay as set out in the examples section. The degree of retention can be measured by visual inspection of Western blots or via densitometric quantification. The scale used for determining the degree of retention can be defined by the person skilled in the art, for instance a scale from 0 to 6 RU (Retention Units), wherein 0 is no retention and 6 is full retention in this assay. If necessary, the scale can be quantified by using the Aggrecan binders of the invention in which each Aggrecan binder is assigned a score, e.g. full retention and no retention are fixed. In the alternative, the scale can be set by various intermediate scores, which are assigned via the Aggrecan binders of the invention, e.g. an Aggrecan binder comprising two 114F08=6 RU and a dummy Aggrecan binder, e.g. ALB26-ALB26=0 RU; or an Aggrecan binder comprising two 114F08=6; Aggrecan binders comprising 608A05=5; Aggrecan binder 604G01=4; Aggrecan binder comprising two 601D02=3; Aggrecan binder comprising two 606A07=2; Aggrecan binder 112A01=1; and a dummy Aggrecan binder, e.g. ALB26-ALB26=0 (cf. Table 2.2). Accordingly, the present invention relates to an Aggrecan binder, such as an ISV and/or polypeptide according to the invention wherein said Aggrecan binder has a cartilage retention of at least 2, such as at least, 3, 4, 5 or 6 RU in a cartilage retention assay.

[0178] The Aggrecan binders of the invention should preferably be stable. As a first prerequisite, the biophysical properties of the Aggrecan binders were tested as detailed in Example 3, in which it was demonstrated that these Aggrecan binders demonstrated favourable stability characteristics as shown by the high melting temperatures and the absence of signs of aggregation and multimerisation. Next, the Aggrecan binders were tested for their activity in the joints for prolonged periods by incubation in synovial fluids at 37.degree. C. (cf. Example 6). No degradation of any of the constructs could be detected, indicating that the constructs were stable under circumstances mimicking the in vivo situation.

[0179] In an aspect the invention relates to Aggrecan binders, such as ISVs wherein said Aggrecan binder has a stability of at least 3 days, 4 days, 5 days, 6 days, 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months in synovial fluid (SF) at 37.degree. C.

[0180] The present invention provides stretches of amino acid residues (SEQ ID NOs: 20-37 and 109, SEQ ID NOs: 38-55 and 110, and SEQ ID NOs: 56-74 and 111; Table A-2) that are particularly suited for binding to Aggrecan. In particular, the invention provides stretches of amino acid residues which bind to human Aggrecan and wherein the binding of said stretches to said Aggrecan retains the presence in cartilaginous tissue (as described above). These stretches of amino acid residues may be present in, and/or may be incorporated into, a construct or polypeptide of the invention, in particular in such a way that they form (part of) the antigen binding site of the polypeptide of the invention. These stretches of amino acid residues have been generated as CDR sequences of heavy chain antibodies or V.sub.HH sequences that were raised against Aggrecan. These stretches of amino acid residues are also referred to herein as "CDR sequence(s) of the invention" ("CDR1 sequence(s) of the invention", "CDR2 sequence(s) of the invention" and "CDR3 sequence(s) of the invention", respectively).

[0181] It should however be noted that the invention in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in a polypeptide of the invention, as long as these stretches of amino acid residues allow the polypeptide of the invention to bind to Aggrecan with a desired affinity and potency. Thus, generally, the invention in its broadest sense provides polypeptides (also referred to herein as "polypeptide(s) of the invention") that are capable of binding to Aggrecan with a certain specified affinity, avidity, efficacy and/or potency and that comprises one or more CDR sequences as described herein and, in particular a suitable combination of two or more such CDR sequences, that are suitably linked to each other via one or more further amino acid sequences, such that the entire polypeptide forms a binding domain and/or binding unit that is capable of binding to Aggrecan. It should however also be noted that the presence of only one such CDR sequence in a polypeptide of the invention may by itself already be sufficient to provide the polypeptide of the invention the capacity of binding to Aggrecan; reference is for example again made to the so-called "Expedite fragments" described in WO 03/050531.

[0182] In a specific, but non-limiting aspect, the Aggrecan binder of the invention such as the ISV and/or polypeptide of the invention, may essentially consist of or comprise at least one stretch of amino acid residues that is chosen from the group consisting of: [0183] i) CDR1 sequences: [0184] 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 [0185] b) amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24; [0186] and/or [0187] ii) CDR2 sequences: [0188] 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 [0189] d) amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42; [0190] and/or [0191] iii) CDR3 sequences: [0192] 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 [0193] f) amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, [0194] preferably, the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0195] In a further aspect, the Aggrecan binder of the invention, such as the polypeptide and/or ISV of the invention, may comprise at least one stretch of amino acid residues that is chosen from the group consisting of SEQ ID NOs: 20-74 and 109-111.

[0196] In particular, the Aggrecan binder of the invention, such as the polypeptide and/or ISV of the invention, may be an Aggrecan binder that comprises one antigen binding site, wherein said antigen binding site comprises at least one stretch of amino acid residues that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences as described above (or any suitable combination thereof). In a preferred aspect, however, the Aggrecan binder of the invention, such as the polypeptide and/or ISV of the invention, comprises more than one, such as two or more stretches of amino acid residues chosen from the group consisting of the CDR1 sequences of the invention, the CDR2 sequences of the invention and/or the CDR3 sequences of the invention. Preferably, the Aggrecan binder of the invention, such as the polypeptide and/or ISV of the invention, comprises three stretches of amino acid residues chosen from the group consisting of the CDR1 sequences of the invention, the CDR2 sequences of the invention and the CDR3 sequences of the invention, respectively. The combinations of CDR's that are mentioned herein as being preferred for the Aggrecan binder of the invention, such as the polypeptide and/or ISV of the invention, are listed in Table A-2, i.e. preferably the CDR combination shown on a single row in said table.

[0197] Representative polypeptides of the present invention having the CDRs described above are shown in Table A-1 (SEQ ID NO:s 1-19 and 114-118).

[0198] In a preferred embodiment, the present invention relates to an Aggrecan binder of the invention, such as an ISV and/or polypeptide of the invention, that comprises 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: [0199] CDR1 is chosen 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; [0200] CDR2 is chosen 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 [0201] CDR3 is chosen 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 binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0202] In a preferred embodiment, the present invention relates to an Aggrecan binder of the invention, such as an ISV and/or polypeptide of the invention, that comprises 3 complementarity determining regions (CDR1 to CDR3, respectively), wherein: [0203] CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; [0204] CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; [0205] CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; [0206] CDR1 is SEQ ID NO: 21, CDR2 is HQ ID NO: 39, and CDR3 is SEQ ID NO: 57; [0207] CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; [0208] CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59; [0209] CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; [0210] CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62; [0211] CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; [0212] CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64; [0213] COR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; [0214] CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66; [0215] CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; [0216] CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69; [0217] CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70; [0218] CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 71; [0219] CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 72; [0220] CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 73; [0221] CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 74; or [0222] CDR1 is SEQ ID NO: 109, CDR2 is SEQ ID NO: 110, and CDR3 is SEQ ID NO: 111; [0223] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0224] In a preferred embodiment, the present invention relates to an Aggrecan binder, such as an ISV, wherein said ISV has been chosen from the group consisting of SEQ ID NOs: 117, 5, 118, 13, 114-116, 1-4, 6-12 and 14-19.

[0225] It should be further noted that the invention is not limited as to the origin of the Aggrecan binder of the invention, such as the ISV and/or polypeptide of the invention, (or of the nucleic acid of the invention used to express it), nor as to the way that the Aggrecan binder of the invention, such as the ISV and/or polypeptide of the invention, or nucleic acid of the invention is (or has been) generated or obtained, Thus, the Aggrecan binder of the invention, such as the ISV and/or polypeptide of the invention, may be naturally occurring ISVs (from any suitable species) or synthetic or semi-synthetic ISVs and/or polypeptides.

[0226] Furthermore, it will also be clear to the skilled person that it is possible to "graft" one or more of the CDRs mentioned above onto other "scaffolds", including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be clear to the skilled person and are well known in the art, see for example 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 Mob. 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 the further references cited therein. For example, techniques known per se for grafting mouse or rat CDR's onto human frameworks and scaffolds can be used in an analogous manner 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 clear to the skilled person, and for example comprise binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as Affibodies.TM.), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al. Nat Biotech 23:1257, 2005), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al. Corn Chem High Throughput Screen 9:619-32, 2006).

[0227] In the Aggrecan binder of the invention, such as the ISV and/or polypeptide 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, in which said amino acid sequences are preferably framework sequences or are amino acid sequences that act as framework sequences, or together form a scaffold for presenting the CDRs.

[0228] According to a preferred embodiment, the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, comprise at least three CDR sequences linked to at least two framework sequences, in which preferably at least one of the three CDR sequences is a CDR3 sequence, with the other two CDR sequences being CDR1 or CDR2 sequences, and preferably being one CDR1 sequence and one CDR2 sequence. According to one specifically preferred, but non-limiting embodiment, the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which CDR1, CDR2 and CDR3 are as defined herein for the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, and FR1, FR2, FR3 and FR4 are framework sequences. In such an Aggrecan binder of the invention, such as an ISV and/or polypeptide 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, for example on the basis of the standard handbooks and the further disclosure and prior art mentioned herein.

[0229] Accordingly, an Aggrecan binder of the invention, such as an ISV and/or polypeptide of the invention, comprises 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0230] (i) CDR1 is chosen from the group consisting of: [0231] (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 [0232] (b) amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24 or with any of SEQ ID NOs: 20-23, 25-37 and 109; and/or [0233] (ii) CDR2 is chosen from the group consisting of: [0234] (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 [0235] (d) amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42 or with any of SEQ ID NOs: 38-41, 43-55 and 110; and/or [0236] (iii) CDR3 is chosen from the group consisting of: [0237] (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 [0238] (f) amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60 or with any of SEQ ID NOs: 56-59, 61-74 and 111 [0239] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0240] The Aggrecan binders of the invention could be mapped to the G1-region, the G1-IGD-G2 region or the G2 region of Aggrecan.

[0241] Accordingly, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that bind to the G2 domain of Aggrecan. As set out in the examples, these Aggrecan binders of the invention, such as ISVs and/or polypeptides have various preferred characteristics. Preferably, the Aggrecan binders of the invention, such as ISVs and/or polypeptides, have a pl of more than 8, and/or have a Koff of less than 2*10.sup.-2 s.sup.-1, and/or have an EC50 of less than 1*10.sup.-6M.

[0242] A comparison of the CDRs of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, revealed a number of permissible amino changes in the CDRs, while retaining binding to the G2 domain of Aggrecan. The sequence variability in the CDRs of all clones against the CDRs of 601D02, which was used as reference, is depicted in the Tables 1.5A, 1.58 and 1.5C.

[0243] In an embodiment, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides, in which: [0244] I) CDR1 is chosen from the group consisting of: [0245] a) SEQ ID NO:s 28, 22, 26, and 33; and [0246] b) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 28, wherein the amino acid difference(s) are defined as follows: [0247] at position 1 the G has been changed into R; [0248] at position 2 the P has been changed into S or R; [0249] at position 3 the T has been changed into I; [0250] at position 5 the S has been changed into N; [0251] at position 6 the R has been changed into N, M, or S; [0252] at position 7 the Y has been changed into R or is absent; [0253] at position 8 the A has been changed into F or is absent; and/or [0254] at position 10 the G has been changed into Y; [0255] and/or [0256] ii) CDR2 is chosen from the group consisting of: [0257] c) SEQ ID NO: 46, 40, 44, and 52; and [0258] d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 46, wherein the amino acid difference(s) are defined as follows: [0259] at position 1 the A has been changed into S, or Y; [0260] at position 4 the W has been changed into L; [0261] at position 5 the S has been changed into N; [0262] at position 6 the S is absent; [0263] at position 7 the G is absent; [0264] at position 8 the G has been changed into A; [0265] at position 9 the R has been changed into S, D, or T; and/or [0266] at position 11 the Y has been changed into N or R; [0267] and/or [0268] iii) CDR3 is chosen from the group consisting of: [0269] e) SEQ ID NO: 64, 58, 62, and 70; and [0270] f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 64, wherein the amino acid difference(s) are defined as follows: [0271] at position 1 the A has been changed into R, or F; [0272] at position 2 the R has been changed into 1, or L; [0273] at position 3 the I has been changed into H, or Q; [0274] at position 4 the P has been changed into G, or N; [0275] at position 5 the V has been changed into S; [0276] at position 6 the R has been changed into G, N, or F; [0277] at position 7 the T has been changed into R, W, or Y; [0278] at position 8 the Y has been changed into R, or S, or is absent; [0279] at position 9 the T has been changed into 5, or is absent; [0280] at position 10 the S has been changed into E, K or is absent; [0281] at position 11 the E has been changed into N, A, or is absent; [0282] at position 12 the W has been changed into D, or is absent; [0283] at position 13 the N has been changed into D, or is absent; [0284] at position 14 the Y is absent; and/or [0285] D and N are added after position 14 of SEQ ID NO: 64; [0286] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0287] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders, wherein: [0288] CDR1 is chosen from the group consisting of SEQ ID NOs: 28, 22, 26, and 33; [0289] CDR2 is chosen from the group consisting of SEQ ID NOs: 46, 40, 44, and 52; and [0290] CDR3 is chosen from the group consisting of SEQ ID NOs: 64, 58, 62, and 70; preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0291] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders, wherein: [0292] CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64; [0293] CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; [0294] COR1 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; preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0295] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders chosen from the group consisting of SEQ ID NOs: 9, 3, 7 and 15, and Aggrecan binders which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 9, 3, 7 and 15.

[0296] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that cross-block the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G2 domain of Aggrecan.

[0297] In an aspect, the present invention relates to a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to G2-domain of Aggrecan, and which competes for binding to the G2 domain of Aggrecan with Aggrecan binders of the invention, such as ISVs and/or polypeptides of the invention, preferably represented by any one of SEQ ID NOs: 9, 3, 7 and 15.

[0298] The present invention also relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that bind to the G1-IGD-G2 domain of Aggrecan. As set out in the examples, these Aggrecan binders of the invention, such as ISVs and/or polypeptides have various preferred characteristics. Preferably, the Aggrecan binders of the invention, such as ISVs and/or polypeptides have a pl of more than 8, and/or have a Koff of less than 2*10.sup.-2 s.sup.-1, and/or have an EC50 of less than 1*10.sup.-6M.

[0299] A comparison of the CDRs of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, revealed a number of permissible amino changes in the CDRs, while retaining binding to the G1-IGD-G2 domain of Aggrecan. The sequence variability in the CDRs of all clones against the CDRs of 604F02, which was used as reference, is depicted in the Tables 1.4A, 1.4B and 1.4C.

[0300] In an aspect the present invention also relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides, in which: [0301] i) COR1 is chosen from the group consisting of: [0302] a) SEQ ID NOs: 32, 30 and 23; and [0303] b) amino acid sequences that have 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 32, wherein the amino acid difference(s) are defined as follows: [0304] at position 2 the R has been changed into L; [0305] at position 6 the S has been changed into T; and/or [0306] at position 8 the T has been changed into A; [0307] and/or [0308] ii) CDR2 is chosen from the group consisting of: [0309] c) SEQ ID NOs: 50, 41, 48 and 51; and [0310] d) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 50, wherein the amino acid difference(s) are defined as follows: [0311] at position 7 the G has been changed into S or R; and/or [0312] at position 8 the R has been changed into T; [0313] and/or [0314] iii) CDR3 is chosen from the group consisting of: [0315] e) SEQ ID NOs: 68, 59, 66 and 69; and [0316] f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 68, wherein the amino acid difference(s) are defined as follows: [0317] at position 4 the R has been changed into V, or P; [0318] at position 6 the A has been changed into Y; [0319] at position 7 the S has been changed into T; [0320] at position 8 the S is absent; [0321] at position 9 the N has been changed into P; [0322] at position 10 the R has been changed into T or L; [0323] at position 11 the G has been changed into E; and/or [0324] at position 12 the L has been changed into T or V; [0325] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0326] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides, wherein: [0327] CDR1 is chosen from the group consisting of SEQ ID NOs: 32, 30 and 23; [0328] CDR2 is chosen from the group consisting of SEQ ID NOs: 50, 41, 48 and 51; and [0329] CDR3 is chosen from the group consisting of SEQ ID NOs: 68, 59, 66 and 69; [0330] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0331] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders, wherein: [0332] CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68; [0333] CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69; [0334] CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66; and [0335] CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59; [0336] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0337] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group consisting of Aggrecan binders with SEQ ID NOs: 118, 13, 4, 11 and 14, and Aggrecan binders which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 118, 13, 4, 11 and 14.

[0338] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that cross-block the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G1-IGD-G2 domain of Aggrecan.

[0339] In an aspect, the present invention relates to a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to the G1-IGD-G2 domain of Aggrecan, and which competes for binding to the G1-IGD-G2 domain of Aggrecan with the Aggrecan binder of the invention, such as the ISV and/or polypeptide of the invention, preferably represented by any one of SEQ ID NOs: 118, 13, 4, 11 and 14.

[0340] In a particularly preferred embodiment the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides of the invention, which bind to the G1 domain of Aggrecan. As set out in the examples, these Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, have various preferred characteristics. Preferably, the Aggrecan binders of the invention, such as ISVs and/or polypeptides have a pl of more than 8, and/or have a Koff of less than 2*10.sup.-2 s.sup.-1, and/or have an EC50 of less than 1*10.sup.-6M.

[0341] A comparison of the CDRs of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, revealed a number of permissible amino changes in the CDRs, while retaining binding to the G1 domain of Aggrecan. The sequence variability in the CDRs of all clones against the CDRs of 114F08, which was used as reference, is depicted in the Tables 1.3A, 1.3B and 1.3C.

[0342] In a preferred aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides of the invention that comprises 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0343] i) CDR1 is chosen from the group consisting of: [0344] a) SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, and 37; and [0345] b) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein the amino acid difference(s) are defined as follows: [0346] at position 2 the S has been changed into R, F, I, or T; [0347] at position 3 the T has been changed into I; [0348] at position 5 the I has been changed into S; [0349] at position 6 the I has been changed into S. T, or M; [0350] at position 7 the N has been changed into Y, or R; [0351] at position 8 the V has been changed into A, Y, T, or G; [0352] at position 9 the V has been changed into M; and/or at position 10 the R has been changed into G, K, or A; [0353] and/or [0354] ii) CDR2 is chosen from the group consisting of: [0355] c) SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, and 55; and [0356] d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein the amino acid difference(s) are defined as follows: [0357] at position 1 the T has been changed into A, or G; [0358] an S or N is inserted between position 3 and position 4 (position 2a, Table 1.38); [0359] at position 3 the S has been changed into R, W, N, or T; [0360] at position 4 the S has been changed into T or G; [0361] at position 5 the G has been changed into S; [0362] at position 6 the G has been changed into S, or R; [0363] at position 7 the N has been changed into S, T, or R; [0364] at position 8 the A has been changed into T; and/or [0365] at position 9 the N has been changed into D or Y; [0366] and/or [0367] iii) CDR3 is chosen from the group consisting of: [0368] e) SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73 and 74; and [0369] f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) are defined as follows: [0370] at position 1 the P has been changed into G, R, D, or E, or is absent; [0371] at position 2 the T has been changed into R, L, P, or V, or is absent; [0372] at position 3 the T has been changed into M, S, or R, or is absent; [0373] at position 4 the H has been changed into D, Y, G, or T; [0374] at position 5 the Y has been changed into F, V, T or G; [0375] at position 6 the G has been changed into L, D, S, Y, or W; [0376] an R, T, Y or V is inserted between position 6 and position 7 (position 6a, Table 1.3C); [0377] at position 7 the G has been changed into P, or S, [0378] at position 8 the V has been changed into G, T, H, R, L, or Y; [0379] at position 9 the Y has been changed into R, A, S, D or G; [0380] at position 10 the Y has been changed into N, E, G, W, or S; [0381] a W is inserted between position 10 and position 11 (position 10a, Table 1.3C); [0382] at position 11 the G has been changed into 5, K, or Y; [0383] at position 12 the P has been changed into E, or D, or is absent; and/or [0384] at position 13 the Y has been changed into L, or is absent; [0385] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0386] In a preferred aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, 37 and 109; CDR2 is chosen from the group consisting of SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, 55, and 110; and CDR3 is chosen from the group consisting of SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73, 74, and 111; preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0387] In a preferred aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders, wherein: [0388] CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60; [0389] CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56; [0390] CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57; [0391] CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61; [0392] CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63; [0393] CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65; [0394] CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; [0395] CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 71; [0396] CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 72; [0397] CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 73; [0398] CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55, and CDR3 is SEQ ID NO: 74; and [0399] CDR1 is SEQ ID NO: 109, CDR2 is SEQ ID NO: 110, and CDR3 is SEQ ID NO: 111; [0400] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0401] It has been demonstrated in the examples section that the exemplary clone 114108 has particularly preferred characteristics. Clone 114F08 represents a family or set of clones, further comprising clone 114A09 (SEQ ID NO: 114) and 114B04 (SEQ ID NO: 115), which have been grouped based on similarities in the CDRs (cf. Table A-2 and Tables 3.3A, 3,3B, and 3.3C), which translates into similarities in functional characteristics. Hence, in another particularly preferred aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that comprises 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0402] i) CDR' is chosen from the group consisting of: [0403] a) SEQ ID NO:s 24 and 109; and [0404] b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein the amino acid difference(s) are defined as follows: [0405] at position 7 the N has been changed into S; and/or [0406] at position 9 the V has been changed into M; [0407] and/or [0408] ii) CDR2 is chosen from the group consisting of: [0409] c) SEQ ID NO:s 42 and 110; and [0410] d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein the amino acid difference(s) are defined as follows: [0411] at position 1 the T has been changed into A; [0412] at position 3 the S has been changed into R; [0413] at position 4 the S has been changed into T; [0414] at position 8 the A has been changed into T; and/or [0415] at position 9 the N has been changed into D; [0416] and/or [0417] iii) CDR3 is chosen from the group consisting of: [0418] e) SEQ ID NO:s 60 and 111; and [0419] f) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) are defined as follows: [0420] at position 4 the H has been changed into R; and/or [0421] at position 8 the V has been changed into D; [0422] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0423] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides, chosen from the group of Aggrecan binders, wherein: [0424] CDR1 is chosen from the group consisting of SEQ ID NOs: 24 and 109; [0425] CDR2 is chosen from the group consisting of SEQ ID NOs: 42 and 110; and [0426] CDR3 is chosen from the group consisting of SEQ ID NOs: 60 and 111 [0427] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0428] It further has been demonstrated in the examples section that Aggrecan binders binding to the G1 region of Aggrecan and belonging to epitope bin 1 or epitope bin 4 are particularly effective in cartilage retention assays. In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that belong to epitope bin 1 or epitope bin 4.

[0429] A comparison of the CDRs of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, belonging to epitope bin 1 revealed a number of permissible amino changes in the CDRs, while retaining binding to the G1 domain of Aggrecan. The sequence variability in the CDRs of all clones against the CDRs of 608A05, which was used as reference, is depicted in the Tables 2.3D, 2.3E and 2.3F.

[0430] In a preferred aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that comprises 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0431] i) CDR1 is chosen from the group consisting of: [0432] a) SEQ ID NO:s 36, 20 and 29; and [0433] b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 36, wherein the amino acid difference(s) are defined as follows: [0434] at position 3 the T has been changed into S; [0435] at position 6 the T has been changed into S; [0436] at position 8 the T has been changed into A; and/or [0437] at position 9 the M has been changed into V; [0438] and/or [0439] ii) CDR2 is chosen from the group consisting of: [0440] c) SEQ ID NO:s 54, 38 and 37; and [0441] d) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 54, wherein the amino acid difference(s) are defined as follows: [0442] at position 1 the A has been changed into I; [0443] at position 4 the W has been changed into R; [0444] at position 7 the G has been changed into R; and/or [0445] at position 8 the T has been changed into S; [0446] and/or [0447] iii) CDR3 is chosen from the group consisting of: [0448] e) SEQ ID NO: 73, 56 and 65; and [0449] f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 73, wherein the amino acid difference(s) are defined as follows: [0450] at position 1 the R has been changed into G; [0451] at position 2 the P has been changed into R or L; [0452] at position 3 the R has been changed into L or S; [0453] at position 5 the Y has been changed into R; [0454] at position 6 the Y has been changed into S or A; [0455] at position 7 the Y has been changed into T, or is absent; [0456] at position 8 the S has been changed into P; [0457] at position 9 the L has been changed into H or R; [0458] at position 10 the Y has been changed into P or A; [0459] at position 11 the S has been changed into A or V; [0460] at position 12 the Y has been changed into O; [0461] at position 13 the D has been changed into F; [0462] at position 14 the Y has been changed into G, or is absent; and/or [0463] after position 14 an S is inserted; [0464] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0465] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders, wherein: [0466] CDR1 is chosen from the group consisting of SEQ ID NOs: 20, 29, and 36; [0467] CDR2 is chosen from the group consisting of SEQ ID NOs: 38, 47, and 54; and [0468] CDR3 is chosen from the group consisting of SEQ ID NOs: 56, 65, and 73; preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0469] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides belonging to epitope bin 1 that cross-block the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G1 domain of Aggrecan.

[0470] In an aspect, the present invention relates to a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to epitope bin 1 of the G1-domain of Aggrecan, and which competes for binding to the G1 domain of Aggrecan with the Aggrecan binders of the invention, such as ISVs and/or polypeptides that belong to epitope bin 1, preferably such as e.g. represented by any one of SEQ ID NO:s 1, 10 and 18.

[0471] A comparison of the CDRs of the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, belonging to epitope bin 4 revealed a number of permissible amino changes in the CDRs, while retaining binding to the G1 domain of Aggrecan. The sequence variability in the CDRs of all clones against the CDRs of 114F08, which was used as reference, is depicted in the Tables 2.3A, 2.3B and 2.3C.

[0472] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides that comprises 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: [0473] i) CDR1 is chosen from the group consisting of: [0474] a) SEQ ID NO: 24, 25 and 27; and [0475] b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein the amino acid difference(s) are defined as follows: [0476] at position 2 the S has been changed into I or F; [0477] at position 5 the I has been changed into S; [0478] at position 6 the I has been changed into S or M; [0479] at position 7 the N has been changed into R or Y; [0480] at position 8 the V has been changed into A or Y; [0481] at position 9 the V has been changed into M; and/or [0482] at position 10 the R has been changed into K; [0483] and/or [0484] ii) CDR2 is chosen from the group consisting of: [0485] c) SEQ ID NO: 42, 43 and 45; and [0486] d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acids) difference with the amino acid sequence of SEQ ID NO: 42, wherein the amino acid difference(s) are defined as follows: [0487] at position 1 the T has been changed into A or G; [0488] an N is inserted between position 2 and position 3 (position 2a Table 2.3B); [0489] at position 7 the N has been changed into R; [0490] at position 8 the A has been changed into T; and/or [0491] at position 9 the N has been changed into D; [0492] and/or [0493] iii) CDR3 is chosen from the group consisting of: [0494] e) SEQ ID NO: 60, 61 and 63; and [0495] f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acids) difference with the amino acid sequence of SEQ ID NO: 60, wherein the amino acid difference(s) are defined as follows: [0496] at position 1 the P is absent; [0497] at position 2 the T has been changed into R or is absent; [0498] at position 3 the T has been changed into M or is absent; [0499] at position 4 the H has been changed into D or Y; [0500] at position 5 the Y has been changed into F or V; [0501] at position 6 the G has been changed into L. or D; [0502] at position 8 the V has been changed into G or T; [0503] at position 9 the Y has been changed into R; [0504] at position 10 the Y has been changed into N or E; [0505] at position 11 the G has been changed into S or K; [0506] at position 12 the P has been changed into E or is absent; and/or [0507] at position 13 the Y has been changed into L or is absent; [0508] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0509] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group of Aggrecan binders, wherein: [0510] CDR1 is chosen from the group consisting of SEQ ID NOs: 24, 25, and 27; [0511] CDR2 is chosen from the group consisting of SEQ ID NOs: 42, 43, and 45; and [0512] CDR3 is chosen from the group consisting of SEQ ID NOs: 60, 61, and 63; [0513] preferably the Aggrecan binder, such as the ISV and/or polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

[0514] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides belonging to epitope bin 4 that cross-block the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation to the G1 domain of Aggrecan.

[0515] In an aspect, the present invention relates to a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to epitope bin 4 of the G1-domain of Aggrecan, and which competes for binding to the G1 domain of Aggrecan with the Aggrecan binders of the invention, such as ISVs and/or polypeptides that belong to epitope bin 4, such as e.g. represented by any one of SEQ ID NO:s 117, 114, 115, 116, 5, 6 and 8.

[0516] In an aspect, the present invention relates to Aggrecan binders of the invention, such as ISVs and/or polypeptides chosen from the group consisting of Aggrecan binders 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 which have more than 80%, such as 90% or 95%, or even more sequence identity with any one of SEQ ID NOs: 117, 118, 116, 114, 115, 5, 13, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19.

[0517] In a specific, but non-limiting aspect, the Aggrecan binder of the invention may be a stretch of amino acid residues that comprises an immunoglobulin fold or an Aggrecan binder that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold (i.e., by folding). Reference is inter alia made to the review by Halaby et al. (i. Protein Eng. 12: 563-71, 1999). Preferably, when properly folded so as to form an immunoglobulin fold, the stretches of amino acid residues may be capable of properly forming the antigen binding site for binding to Aggrecan. Accordingly, in a preferred aspect the Aggrecan binder of the invention is an immunoglobulin, such as e.g. an immunoglobulin single variable domain.

[0518] Accordingly, the framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by sequence optimization such as humanization or camelization). For example, the framework sequences may be framework sequences derived from an immunoglobulin single variable domain such as a light chain variable domain (e.g., a V.sub.L-sequence) and/or from a heavy chain variable domain (e.g., a V.sub.H-sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a V.sub.HH-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional V.sub.H sequences that have been camelized (as defined herein).

[0519] The framework sequences may preferably be such that the Aggrecan binder of the invention is an ISV such as a Domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); a single domain antibody (or an amino acid that is suitable for use as a single domain antibody); a "dAb" (or an amino acid that is suitable for use as a dAb); a Nanobody.RTM.; a V.sub.HH sequence; a humanized V.sub.HH sequence; a camelized V.sub.H sequence; or a V.sub.HH sequence that has been obtained by affinity maturation. Again, suitable framework sequences will be clear to the skilled person, for example on the basis of the standard handbooks and the further disclosure and prior art mentioned herein.

[0520] Another particularly preferred class of ISVs of the invention comprises ISVs with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V.sub.H domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V.sub.H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V.sub.HH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the description herein. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and/or are present at the V.sub.H--V.sub.L interface, and/or at the so-called Camelidae hallmark residues, well known to the person skilled in the art and which have been defined for example in WO 94/04678 and Davies and Riechmann (1994 and 1996). Preferably, the V.sub.H sequence that is used as a starting material or starting point for generating or designing the camelized ISVs is preferably a V.sub.H sequence from a mammal, more preferably the V.sub.H sequence of a human being, such as a V.sub.H3 sequence. However, it should be noted that such camelized ISVs of the invention can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V.sub.H domain as a starting material.

[0521] For example, again as further described herein, both "humanization" and "camelization" can be performed by providing a nucleotide sequence that encodes a naturally occurring V.sub.HH domain or V.sub.H domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence in such a way 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, so as to provide the desired ISVs of the invention. Alternatively, based on the amino acid sequence of a naturally occurring V.sub.HH domain or V.sub.H domain, respectively, the amino acid sequence of the desired humanized or camelized ISVs of the invention, respectively, can be designed and then synthesized de nova using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring V.sub.HH domain or V.sub.H domain, respectively, a nucleotide sequence encoding the desired humanized or camelized ISVs of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired ISVs of the invention.

[0522] In particular, the framework sequences present in the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, may contain one or more of Hallmark residues for instance as defined in WO 08/020079 (Tables A-3 to A-8), such that the Aggrecan binder of the invention is a Nanobody. 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 (in particular V.sub.HH sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more "Hallmark residues" in one or mare of the framework sequences (as e.g., further described in WO 08/020079, page 61, line 24 to page 98, line 3). As used herein "represented by" in the context of any SEQ ID NO is equivalent to "comprises or consists of" said SEQ ID NO and preferably equivalent to "consists of" said SEQ ID NO.

[0523] More in particular, the invention provides Aggrecan binders comprising at least one ISV that is an amino acid sequence with the (general) structure

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

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which COR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and which: [0524] 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), in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded. In this respect, 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 [0525] ii) combinations of framework sequences as depicted in Table A-2; [0526] and in which: [0527] 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 chosen from the Hallmark residues such as, e.g. mentioned in Table A-3 to Table A-8 of WO 08/020079.

[0528] Accordingly, the present invention relates to an ISV and/or polypeptide, wherein said ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and said 3 complementarity determining regions CDR1 to CDR3, e.g. the ISV that specifically binds Aggrecan consists of 4 framework regions (FR1 to FR4, respectively) and said 3 complementarity determining regions CDR1 to CDR3, the therapeutic ISV, e.g. the ISV that binds to a member 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 consists of 4 framework regions (FR1 to FR4, respectively) and said 3 complementarity determining regions CDR1 to CDR3; the ISV binding serum albumin essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively).

[0529] The Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, may also contain the specific mutations/amino acid residues described in the following co-pending US provisional applications, all entitled "Improved immunoglobulin variable domains": U.S. 61/994,552 filed May 16, 2014; U.S. 61/014,015 filed Jun. 18, 2014; U.S. 62/040,167 filed Aug. 21, 2014; and U.S. 62/047,560, filed Sep. 8, 2014 (all assigned to Ablynx N.V.).

[0530] In particular, the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, may suitably contain (i) a K or Q at position 112; or (ii) a K or Q at position 110 in combination with a V at position 11; or (iii) a T at position 89; or (iv) an L on position 89 with a K or Q at position 110; or (v) a V at position 11 and an L at position 89; or any suitable combination of (i) to (v).

[0531] As also described in said co-pending US provisional applications, when the Aggrecan binder of the invention, such as the ISV and/or polypeptide of the invention, contain the mutations according to one of (i) to (v) above (or a suitable combination thereof): [0532] the amino acid residue at position 11 is preferably chosen from L, V or K (and is most preferably V); and [0533] the amino acid residue at position 14 is preferably suitably chosen from A or P; and [0534] the amino acid residue at position 41 is preferably suitably chosen from A or P; and [0535] the amino acid residue at position 89 is preferably suitably chosen from T, V or L; and [0536] the amino acid residue at position 108 is preferably suitably chosen from Q or L; and [0537] the amino acid residue at position 110 is preferably suitably chosen from T, K or Q; and [0538] the amino acid residue at position 112 is preferably suitably chosen from S, K or Q.

[0539] As mentioned in said co-pending US provisional applications, said mutations are effective in preventing or reducing binding of so-called "pre-existing antibodies" to the ISVs, polypeptides and constructs of the invention. For this purpose, the Aggrecan binders of the invention, such as the ISVs and/or polypeptides of the invention, may also contain (optionally in combination with said mutations) a C-terminal extension (X)n (in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)), for which reference is again made to said US provisional applications as well as to WO 12/175741. In particular, an Aggrecan binder of the invention, such as an ISV and/or polypeptide of the invention, may contain such a C-terminal extension when it forms the C-terminal end of a protein, polypeptide or other compound or construct comprising the same (again, as further described in e.g. said US provisional applications as well as WO 12/175741).

[0540] An Aggrecan binder of the invention may be an immunoglobulin, such as an ISV, derived in any suitable manner and from any suitable source, and may for example be naturally occurring V.sub.HH sequences (i.e., from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to "humanized" (as defined herein) Nanobodies or VHH sequences, "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when an immunoglobulin comprises a V.sub.HH sequence, said immunoglobulin may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized immunoglobulins of the invention. Similarly, when an immunoglobulin comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), said immunoglobulin may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized immunoglobulins of the invention.

[0541] In an aspect, the present invention provides an Aggrecan binder of the invention, such as an ISV, wherein said Aggrecan binder is chosen from the group consisting of SEQ ID NO:s 117, 118, 116, 115, 114 and 1-19.

[0542] The ISVs may be used as a "building block" for the preparation of a polypeptide, which may optionally contain one or more further "building blocks", such as ISVs, against the same or another epitope on To Aggrecan and/or against one or more other antigens, proteins or targets than Aggrecan, e.g. building blocks having a therapeutic mode of action, e.g. therapeutic ISVs.

[0543] Generally, proteins or polypeptides or constructs that comprise or essentially consist of a single building block, single ISV or single Nanobody will be referred to herein as "monovalent" proteins or polypeptides or as "monovalent constructs", respectively. Polypeptides or constructs that comprise two or more building blocks or binding units (such as e.g., ISVs) will also be referred to herein as "multivalent" polypeptides or constructs, and the building blocks/ISVs present in such polypeptides or constructs will also be referred to herein as being in a "multivalent format". For example, a "bivalent" polypeptide may comprise two ISVs, optionally linked via a linker sequence, whereas a "trivalent" polypeptide may comprise three ISVs, optionally linked via two linker sequences; whereas a "tetravalent" polypeptide may comprise four ISVs, optionally linked via three linker sequences, etc.

[0544] In a multivalent polypeptide or construct, the two or more ISVs, such as Nanobodies may be the same or different, and may be directed against the same antigen or antigenic determinant (for example against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. Polypeptides or constructs that contain at least two building blocks (such as e.g., ISVs) in which at least one building block is directed against a first antigen (i.e., Aggrecan) and at least one building block is directed against a second antigen (i.e., different from Aggrecan, such as e.g. a therapeutic target) will also be referred to as "multispecific" polypeptides or multispecific constructs, respectively, and the building blocks (such as e.g., ISVs) present in such polypeptides or constructs will also be referred to herein as being in a "multispecific format". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide 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., different from Aggrecan, such as e.g. a therapeutic target), whereas a "trispecific" polypeptide of the invention is a polypeptide 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., different from Aggrecan, such as e.g. a therapeutic target) and at least one further ISV directed against a third antigen (i.e., different from both Aggrecan and the second antigen); etc.

[0545] "Multiparatopic" polypeptides and "multiparatopic" constructs, such as e.g., "biparatopic" polypeptides or constructs and "triparatopic" polypeptides or constructs, comprise or essentially consist of two or more building blocks that each have a different paratope.

[0546] Accordingly, the ISVs of the invention that bind Aggrecan can be in essentially isolated form (as defined herein), or they may form part of a construct or polypeptide, which may comprise or essentially consist of one or more ISVs that bind Aggrecan and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). The present invention relates to a polypeptide or construct that comprises or essentially consists of at least one ISV according to the invention, such as one or more ISVs of the invention (or suitable fragments thereof), binding Aggrecan.

[0547] The one or more ISVs of the invention can be used as a binding unit or building block in such a polypeptide or construct, so as to provide a monovalent, multivalent or multiparatopic polypeptide or construct of the invention, respectively, all as described herein. The present invention thus also relates to a polypeptide which is a monovalent construct comprising or essentially consisting of one monovalent polypeptide or ISV of the invention. The present invention thus also relates to a polypeptide or construct which is a multivalent polypeptide or multivalent construct, respectively, such as e.g., a bivalent or trivalent polypeptide or construct comprising or essentially consisting of two or more ISVs of the invention (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is e.g. also made to Conrath et al. (J. Biol. Chem. 276: 7346-7350, 2001), as well as to for example WO 96/34103, WO 99/23221 and WO 2010/115998.

[0548] The invention further relates to a multivalent polypeptide (also referred to herein as a "multivalent polypeptide(s) of the invention") that comprises or (essentially) consists of at least one ISV, such as one or two ISVs (or suitable fragments thereof) directed against Aggrecan, preferably human Aggrecan, and one additional ISV.

[0549] In an aspect, in its simplest form, the multivalent polypeptide or construct of the invention is a bivalent polypeptide or construct of the invention comprising a first ISV, such as a Nanobody, directed against Aggrecan, and an identical second ISV, such as a Nanobody, directed against Aggrecan, wherein said first and said second ISVs, such as Nanobodies, may optionally be 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, such as Nanobody, directed against Aggrecan, an identical second ISV, such as Nanobody, directed against Aggrecan and a third ISV, such as a Nanobody, directed against an antigen different from Aggrecan, such as e.g. a therapeutic target, in which said first, second and third ISVs, such as Nanobodies, may optionally be linked via one or more, and in particular two, linker sequences.

[0550] 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, such as a Nanobody, directed against Aggrecan, and a second ISV, such as a Nanobody, directed against a second antigen, such as e.g. a therapeutic target, in which said first and second ISVs, such as Nanobodies, may optionally be linked via a linker sequence (as defined herein); whereas a multivalent polypeptide or construct of the invention may also be a trispecific polypeptide or construct of the invention, comprising a first ISV, such as a Nanobody, directed against Aggrecan, a second ISV, such as a Nanobody, directed against a second antigen, such as e.g. a therapeutic target, and a third ISV, such as a Na nobody, directed against a third antigen, such as e.g. also therapeutic target but different from said second antigen, in which said first, second and third ISVs, such as Nanobodies, may optionally be linked via one or more, and in particular two, linker sequences.

[0551] 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 in its simplest form 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 a Nanobody, directed against another antigen, such as e.g. a therapeutic target, in which said first, second and third ISVs, such as Nanobodies, may optionally be linked via one or more, and in particular two, linker sequences.

[0552] 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 of the invention (as defined herein), comprising two ISVs, such as Nanobodies, against Aggrecan, wherein said ISVs against Aggrecan may be the same or different and a third ISV, such as a Nanobody, directed against another antigen, such as e.g. a therapeutic target, in which said first, second and third ISVs, such as Nanobodies, may optionally be linked via one or more, and in particular two, linker sequences.

[0553] Particularly preferred trivalent, bispecific polypeptides or constructs in accordance with the invention are those shown in the Examples described herein and in Tables E-1 and E-2.

[0554] In another aspect, the polypeptide of the invention is a bispecific polypeptide or construct. A bispecific polypeptide or construct of the invention 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 e.g. a therapeutic target, in which said first and second ISVs, such as Nanobodies, may optionally be linked via a linker sequence.

[0555] In a preferred aspect, the multivalent polypeptide or construct of the invention comprises or essentially consists of two or more ISVs directed against Aggrecan. In an aspect, the invention relates to a polypeptide or construct that comprises or essentially consists of at least two ISVs according to the invention, such as 2, 3 or 4 ISVs (or suitable fragments thereof), binding Aggrecan. The two or more ISVs may optionally be linked via one or more peptidic linkers.

[0556] The two or more ISVs present in the multivalent polypeptide or construct of the invention may consist of a light chain variable domain sequence (e.g., a V.sub.L sequence) or of a heavy chain variable domain sequence (e.g., a V.sub.H-sequence); they may consist of a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or of a heavy chain variable domain sequence that is derived from heavy chain antibody. In a preferred aspect, they consist of a Domain antibody (or an amino acid that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid that is suitable for use as a single domain antibody), of a "dAb" (or an amino acid that is suitable for use as a dAb), of a Nanobody.RTM. (including but not limited to V.sub.HH), of a humanized V.sub.HH sequence, of a camelized V.sub.H sequence; or of a V.sub.HH sequence that has been obtained by affinity maturation. The two or more ISVs may consist of a partially or fully humanized Nanobody or a partially or fully humanized VHH.

[0557] In an aspect of the invention, the first ISV and the second ISV present in the multiparatopic (preferably biparatopic or triparatopic) polypeptide or construct of the invention do not (cross)-compete with each other for binding to Aggrecan and, as such, belong to different families. Accordingly, 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 an aspect, the first ISV of this multiparatopic (preferably biparatopic) polypeptide or construct of the invention does not cross-block the binding to Aggrecan of the second ISV of this multiparatopic (preferably biparatopic) polypeptide or construct of the invention and/or the first ISV is not cross-blocked from binding to Aggrecan by the second ISV. In another aspect, the first ISV of a multiparatopic (preferably biparatopic) polypeptide or construct of the invention cross-blocks the binding to Aggrecan of the second ISV of this multiparatopic (preferably biparatopic) polypeptide or construct of the invention and/or the first ISV is cross-blocked from binding to Aggrecan by the second ISV.

[0558] In a preferred aspect, the polypeptide or construct of the invention comprises or essentially consists of two or more ISVs, of which at least one ISV is directed against Aggrecan. In a particularly preferred aspect, the polypeptide or construct of the invention comprises or essentially consists of three or more ISVs, of which at least two ISVs are directed against Aggrecan. It will be appreciated that said at least two ISVs directed against Aggrecan can be the same or different, can be directed against the same epitope or different epitopes of Aggrecan, can belong to the same epitope bin or to different epitope bins, and/or can bind to the same or different domains of Aggrecan.

[0559] In a preferred aspect, the polypeptide or construct of the invention comprises or essentially consists of at least two ISVs, wherein said at least two ISVs can be the same or different, which are independently chosen from the group consisting of SEQ ID NOs: 117, 118, 116, 115 and 1-19, more preferably said at least two ISVs are chosen from the group consisting of SEQ ID NOs: 117, 5, 6, 8, 114-116 and/or said at least two ISVs are chosen from the group consisting of SEQ ID NOs: 118 and 13.

[0560] In a further aspect, the invention relates to a multiparatopic (preferably biparatopic) polypeptide or construct comprising two or more immunoglobulin single variable domains directed against Aggrecan that bind the same epitope(s) as is bound by any one of SEQ ID NOs: 117, 118, 114, 115, 116 and 1-19.

[0561] It is anticipated that the final format of a molecule for clinical use comprises one or two building blocks, such as ISVs, binding Aggrecan and one or more building blocks, such as ISVs, with a therapeutic mode of action, and possibly further moieties. In the examples section it is demonstrated that such formats retain both Aggrecan binding and retention properties as well as the therapeutic effect, e.g. enzymatic and/or inhibitory functions. The one or more building blocks, such as ISVs, with a therapeutic mode of action can be any building block having a therapeutic effect ("therapeutic building block" or "therapeutic ISV") in diseases in which Aggrecan is involved, such as arthritic disease, osteoarthritis, spondyloepimetaphyseal dysplasia, lumbar disk degeneration disease, Degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, aggrecanopathies and/or in which Aggrecan is used for directing, anchoring and/or retaining other, e.g. therapeutic, building blocks at the desired site, such as e.g. in a joint. The present invention thus pertains to a polypeptide or construct according to the invention, wherein the one or more further building block(s), e.g. further ISV(s), retain activity.

[0562] The present invention relates to a polypeptide or construct that comprises or essentially consists of at least one ISV according to the invention, such as one or more ISVs of the invention (or suitable fragments thereof), binding Aggrecan, and at least one further ISV, in particular a therapeutic ISV, wherein said at least one further ISV preferably binds to a therapeutic target, such as binds to a member 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.

[0563] In an aspect the present invention relates to a polypeptide or construct of the invention essentially consisting of or comprising at least one ISV binding Aggrecan and at least one further ISV which has a therapeutic effect, e.g. a therapeutic building block. The therapeutic effect can be any desired effect which ameliorates, treats or prevents a disease as will be further detailed below. Preferably the further ISV, e.g. a therapeutic ISV, inhibits or decreases a protease activity, e.g. inhibits or decreases an activity of a therapeutic target, i.e. of a member 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. Inhibiting or decreasing an activity may be achieved by binding to the active site or by modifying the structure of a protease or proteinase, thereby preventing and/or decreasing the hydrolysis of the target protein of the protease or proteinase.

[0564] In an aspect the present invention relates to a polypeptide or construct of the invention chosen from the polypeptides and constructs of Table E-1 and Table E-2.

[0565] In an aspect the present invention relates to an ISV, polypeptide or construct of the invention having a stability of at least 7 days, such as at least 14 days, 21 days, 1 month, 2 months or even 3 months in synovial fluid (SF) at 37.degree. C.

[0566] In an aspect the present invention relates to an ISV, polypeptide or construct of the invention having cartilage retention of at least 2, such as at least, 3, 4, 5 or 6 RU in a cartilage retention assay.

[0567] In an aspect the present invention relates to an ISV, polypeptide or construct of the invention penetrating into the cartilage by at least 5 .mu.m, such as at least 10 .mu.M, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m or even more.

[0568] The stability of a polypeptide, construct or ISV of the invention can be measured by routine assays known to the person skilled in the art. Typical assays include (without being limiting) assays in which the activity of said polypeptide, construct or ISV is determined, followed by incubating in Synovial Fluid for a desired period of time, after which the activity is determined again, for instance as detailed in the examples section (cf. Example 6).

[0569] The desired activity of the therapeutic building block in the multivalent polypeptide or construct of the invention can be measured by routine assays known to the person skilled in the art. Typical assays include assays in which GAG release is assayed as detailed in the examples section (cf. Example 8).

[0570] The relative affinities may depend on the location of the ISVDs in the polypeptide. It will be appreciated that the order of the ISVDs in a polypeptide of the invention (orientation) may be chosen according to the needs of the person skilled in the art. The order of the individual ISVDs as well as whether the polypeptide comprises a linker is a matter of design choice. Some orientations, with or without linkers, may provide preferred binding characteristics in comparison to other orientations. For instance, the order of a first ISV (e.g. ISV 1) and a second ISV (e.g. ISV 2) in the polypeptide of the invention may be (from N-terminus to C-terminus): (i) ISV 1 (e.g. Nanobody 1)--[linker]ISV 2 (e.g. Nanobody 2)--[C-terminal extension]; or (ii) ISV 2 (e.g. Nanobody 2)-[linker]-ISV 1 (e.g. Nanobody 1)-[C-terminal extension]; (wherein the moieties between the square brackets, i.e. linker and C-terminal extension, are optional). All orientations are encompassed by the invention. Polypeptides that contain an orientation of ISVs that provides desired binding characteristics may be easily identified by routine screening, for instance as exemplified in the examples section. A preferred order is from N-terminus to C-terminus: therapeutic ISV-[linker]-ISV binding Aggrecan-[C-terminal extension], wherein the moieties between the square brackets are optional. Another preferred order is from N-terminus to C-terminus: therapeutic ISV-[linker]-ISV binding Aggrecan-[linker]-ISV binding Aggrecan-[C-terminal extension], wherein the moieties between the square brackets are optional.

[0571] The Aggrecan binders of the invention, such as the 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 binders, such as polypeptides and/or ISVs (with or without 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 further groups, residues, moieties or binding units may or may not provide further functionality to the Aggrecan binder such as the polypeptide and/or ISV and may or may not modify the properties of the Aggrecan binder such as the polypeptide and/or ISV.

[0572] For example, such further groups, residues, moieties or binding units may be one or more additional 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 chosen from the group consisting of Domain antibodies, amino acids that are suitable for use as a domain antibody, single domain antibodies, amino acids that are suitable for use as a single domain antibody, dAbs, amino acids that are suitable for use as a dAb, Nanobodies (such as e.g. VHH, humanized VHH or camelized VH sequences).

[0573] As described above, additional binding units, such as ISVs having different antigen specificity can be linked to form multispecific polypeptides. By combining ISVs of two or more specificities, bispecific, trispecific etc. polypeptides or constructs can be formed. For example, a polypeptide according to the invention may comprise one, two or more ISVs directed against Aggrecan and at least one ISV domain against another target. Such constructs and modifications thereof, which the skilled person can readily envisage, are all encompassed by the term "compound of the invention, construct of the invention and/or polypeptide of the invention" as used herein.

[0574] In the compounds, constructs and/or polypeptides described above, the one, two, three or more ISVs and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting polypeptide is a fusion (protein) or fusion (polypeptide).

[0575] The one or more further groups, residues, moieties or binding units may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the polypeptide of the invention, and may or may not add further functionality to the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the polypeptide of the invention.

[0576] Examples of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson (Nature Biotechnology 23: 1126-1136, 2005).

[0577] For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the compound, construct and/or polypeptide of the invention, compared to polypeptide of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).

[0578] In a specific aspect of the invention, a construct or polypeptide of the invention may have a moiety conferring an increased half-life, compared to the corresponding construct or polypeptide of the invention without said moiety. Some preferred, but non-limiting examples of such constructs and polypeptides of the invention will become clear to the skilled person based on the further disclosure herein, and for example comprise ISVs or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); Aggrecan binders of the invention, such as ISVs and/or polypeptides of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention which comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) which increases the half-life of the amino acid sequence of the invention. Examples of constructs of the invention, such as polypeptides of the invention, which comprise such half-life extending moieties or ISVs will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the 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 one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, ISVs that are suitable for use as a domain antibody, single domain antibodies, ISVs that are suitable for use as a single domain antibody, dAbs, ISVs that are suitable for use as a dAb, or Nanobodies that can bind 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 mentioned herein); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more ISVs of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins, such as, for instance, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489, WO2008/068280, WO2009/127691 and PCT/EP2011/051559.

[0579] In an aspect the present invention provides a construct of the invention, such as a polypeptide, wherein said polypeptide further comprises a serum protein binding moiety or a serum protein.

[0580] Preferably, said serum protein binding moiety binds serum albumin, such as human serum albumin.

[0581] Generally, the constructs or polypeptides of the invention with increased half-life preferably have 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, greater than the half-life of the corresponding constructs or polypeptides of the invention per se, i.e. without the moiety conferring the increased half-life. For example, the constructs or polypeptides of the invention with increased half-life may have a half-life e.g., in humans that is increased with more than 1 hours, 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 constructs or polypeptides of the invention per se, i.e. without the moiety conferring the increased half-life.

[0582] In a preferred, but non-limiting aspect of the invention, the constructs of the invention, such as polypeptides of the invention, have a serum half-life e.g. in humans that is increased with more than 1 hours, 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 constructs or polypeptides of the invention per se, i.e. without the moiety conferring the increased half-life.

[0583] In another preferred, but non-limiting aspect of the invention, such constructs of the invention, such as polypeptides of the invention, exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

[0584] In a particularly preferred but non-limiting aspect of the invention, the invention provides a construct of the invention, such as a polypeptide of the invention, comprising besides the one or more building blocks binding Aggrecan and possibly the one or more therapeutic building blocks, at least one building block binding serum albumin, such as an ISV binding serum albumin, such as human serum albumin as described herein, wherein said ISV binding serum albumin comprises or essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG and CDR3 is GGSLSR. Preferably, said ISV binding human serum albumin is chosen from the group consisting of Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11 (S11210-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG, Alb92 or Alb223 (cf. Table C).

[0585] In an embodiment, the present invention relates to construct of the invention, such as a polypeptide comprising a serum protein binding moiety, wherein said serum protein binding moiety is a non-antibody based polypeptide.

[0586] In an aspect, the present invention relates to a compound or construct as described herein comprising one or more other groups, residues, moieties or binding units, preferably chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

[0587] In an embodiment, the present invention relates to construct of the invention, such as a polypeptide comprising a moiety conferring half-life extension, wherein said moiety is a PEG. Hence, the present invention relates to a construct or polypeptide of the invention comprising PEG.

[0588] The further amino acid residues may or may not change, alter or otherwise influence other (biological) properties of the polypeptide of the invention and may or may not add further functionality to the polypeptide of the invention. For example, such amino acid residues: [0589] a) can comprise an N-terminal Met residue, for example as result of expression in a heterologous host cell or host organism. [0590] b) may form a signal sequence or leader sequence that directs secretion of the polypeptide from a host cell upon synthesis (for example to provide a pre-, pro- or prepro-form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention). Suitable secretory leader peptides will be clear to the skilled person, and may be as further described herein. Usually, such a leader sequence will be linked to the N-terminus of the polypeptide, although the invention in its broadest sense is not limited thereto; [0591] c) may form a "tag", for example an amino acid sequence or residue that allows or facilitates the purification of the polypeptide, for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g. by chemical or enzymatical cleavage) to provide the polypeptide (for this purpose, the tag may optionally be linked to the amino acid sequence or polypeptide sequence via a cleavable linker sequence or contain a cleavable motif). Some preferred, but non-limiting examples of such residues are multiple histidine residues, glutathione residues and a myc-tag such as AAAEQKLISEEDLNGAA; [0592] d) may be one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups will be clear to the skilled person and include, but are not limited to, the amino acid residues and functional groups mentioned herein for the derivatives of the polypeptides of the invention.

[0593] In the constructs of the invention, such as the polypeptides of the invention, the two or more building blocks, such as e.g. ISVs, and the optionally one or more other groups, drugs, agents, residues, moieties or binding units may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof. Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing constructs, proteins or polypeptides that are intended for pharmaceutical use.

[0594] For instance, the polypeptide of the invention may, for example, be a trivalent, trispecific polypeptide, comprising one building block, such as an ISV, binding Aggrecan, one therapeutic building block, such as an ISV, and one building block, such as an ISV, binding (human) serum albumin, in which said first, second and third building blocks, such as ISVs, may optionally be linked via one or more, and in particular two, linker sequences. Also, the present invention provides a construct or polypeptide of the invention comprising a first ISV binding Aggrecan and/or a second ISV and/or possibly a third ISV and/or possibly an ISV binding serum albumin, wherein said first ISV and/or said second ISV and/or possibly said third ISV and/or possibly said ISV binding serum albumin are linked via a linker.

[0595] Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, it should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent V.sub.H and V.sub.L domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each ISV, such as a Nanobody, by itself forms a complete antigen-binding site).

[0596] For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly.sub.x ser.sub.y).sub.z, such as (for example (gly.sub.4 ser).sub.3 or (gly.sub.3 ser.sub.2).sub.3, as described in WO 99/42077 and the GS30, GS15, G59 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 regions of naturally occurring heavy chain antibodies or similar sequences (such as for instance described in WO 94/04678). Preferred linkers are depicted in Table D (SEQ ID NO:s 154-170).

[0597] Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, polyethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.

[0598] It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final the construct of the invention, such as the polypeptide of the invention, including but not limited to the affinity, specificity or avidity for a chemokine, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific construct of the invention, such as the polypeptide of the invention, optionally after some limited routine experiments.

[0599] For example, in multivalent polypeptides of the invention that comprise building blocks, ISVs or Nanobodies directed against Aggrecan and another target, the length and flexibility of the linker are preferably such that it allows each building block, such as an ISV, of the invention present in the polypeptide to bind to its cognate target, e.g. the antigenic determinant on each of the targets. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific construct of the invention, such as a polypeptide of the invention, optionally after some limited routine experiments.

[0600] It is also within the scope of the invention that the linker(s) used, confer one or more other favourable properties or functionality to the constructs of the invention, such as the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g., as described herein for the derivatives of the ISVs of the invention). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

[0601] Finally, when two or more linkers are used in the constructs such as polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific construct or polypeptide of the invention, optionally after some limited routine experiments.

[0602] Usually, for the ease of expression and production, a construct of the invention, such as a polypeptide of the invention, will be a linear polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a construct of the invention, such as a polypeptide of the invention, comprises three of more building blocks, ISVs or Nanobodies, it is possible to link them by use of a linker with three or more "arms", which each "arm" being linked to a building block, ISV or Nanobody, so as to provide a "star-shaped" construct. It is also possible, although usually less preferred, to use circular constructs.

[0603] Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein said ISVs are directly linked to each other or are linked via a linker.

[0604] Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein a first ISV and/or a second ISV and/or possibly an ISV binding serum albumin are linked via a linker.

[0605] Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein said linker is chosen from the group consisting of linkers of 5GS, 765, 9GS, 10GS, 1565, 1865, 20GS, 25GS, 3065, 3565, poly-A, 8G5, 40GS, G1 hinge, 9GS-G1 hinge, llama upper long hinge region, and 63 hinge.

[0606] Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein said polypeptide is chosen from the group consisting of polypeptides of Table E-1 and Table E-2.

[0607] Also encompassed in the present invention are compounds, constructs and/or polypeptides that comprise an ISV or polypeptide of the invention and further comprise tags or other functional moieties, e.g., toxins, labels, radiochemicals, etc.

[0608] The other groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more ISVs or polypeptides of the invention so as to provide a "derivative" of the polypeptide of the invention.

[0609] Accordingly, the invention in its broadest sense also comprises compounds, constructs and/or polypeptides that are derivatives of the polypeptides of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g., enzymatic) modification, of the polypeptides of the invention and/or of one or more of the amino acid residues that form a polypeptide of the invention.

[0610] Examples of such modifications, as well as examples of amino acid residues within the polypeptide sequences that can be modified in such a manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person (see also Zangi et al., Nat Biotechnol 31(10):898-907, 2013).

[0611] For example, such a modification may involve the introduction (e.g., by covalent linking or in any other suitable manner) of one or more (functional) groups, residues or moieties into or onto the polypeptide of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the polypeptide of the invention. Examples of such functional groups will be clear to the skilled person.

[0612] For example, such modification may comprise the introduction (e.g., by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or the absorption of the polypeptide of the invention, that reduce the immunogenicity and/or the toxicity of the polypeptide of the invention, that eliminate or attenuate any undesirable side effects of the polypeptide of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the polypeptide of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv's and single domain antibodies), for which reference is for example made to Remington (Pharmaceutical Sciences, 16.sup.th ed., Mack Publishing Co., Easton, Pa., 1980). Such functional groups may for example be linked directly (for example covalently) to a polypeptide of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

[0613] One specific example is a derivative polypeptide of the invention wherein the polypeptide of the invention has been chemically modified to increase the half-life thereof (for example, by means of pegylation). This is one of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins and comprises attachment of a suitable pharmacologically acceptable polymer, such as polyethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments, such as e.g. (single) domain antibodies and ScFv's; reference is made to for example Chapman (Nat. Biotechnol. 54: 531-545, 2002), Veronese and Harris (Adv. Drug Deliv. Rev. 54: 453-456, 2003), Harris and Chess (Nat. Rev. Drug. Discov. 2: 214-221, 2003) and WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA.

[0614] Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Yang et al. (Protein Engineering 16: 761-770, 2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a polypeptide of the invention, a polypeptide of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of a polypeptide of the invention, all using techniques of protein engineering known per se to the skilled person.

[0615] Preferably, for the polypeptides of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000.

[0616] Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the polypeptide of the invention.

[0617] Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled polypeptide of the invention. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals, such as, .sup.152Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes (such as .sup.3H, .sup.125I, .sup.32P, .sup.35S, .sup.14C, .sup.51Cr, .sup.36Cl, .sup.57Co, .sup.58Co, .sup.59Fe, and .sup.75Se), metals, metal chelates or metallic cations (for example metallic cations such as .sup.99mTC, .sup.123I, .sup.111In, .sup.131I, .sup.97Ru, .sup.67Cu, .sup.67Ga, and .sup.68Ga or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, such as (.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Cr, and .sup.56Fe)), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy.

[0618] Such labelled polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.

[0619] As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0620] Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the polypeptide of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a polypeptide of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated polypeptide of the invention may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the polypeptide of the invention to a carrier, including carriers suitable for pharmaceutical purposes. See, for instance, the liposomal formulations described by Cao and Suresh (Journal of Drug Targeting 8: 257, 2000). Such binding pairs may also be used to link a therapeutically active agent to the polypeptide of the invention.

[0621] Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g, to study function-activity relationships). Reference is for example made to Lundblad and Bradshaw (Biotechnol. Appl. Biochem. 26: 143-151, 1997).

[0622] Preferably, the compounds, constructs, polypeptides and/or derivatives are such that they bind to Aggrecan, with an affinity (suitably measured and/or expressed as a K.sub.0-value (actual or apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value, as further described herein) that is as defined herein (i.e. as defined for the polypeptides of the invention).

[0623] Such compounds, constructs and/or polypeptides of the invention and derivatives thereof may also be in essentially isolated form.

[0624] In an aspect, the present invention relates to a construct of the invention, that comprises or essentially consists 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.

[0625] In an aspect, the present invention relates to a construct of the invention, in which one or more other groups, residues, moieties or binding units are chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

[0626] The invention further relates to methods for preparing the compounds, constructs, polypeptides, nucleic acids, host cells, and compositions described herein.

[0627] The multivalent polypeptides of the invention can generally be prepared by a method which comprises at least the step of suitably linking the ISV and/or monovalent polypeptide of the invention to one or more further ISVs, optionally via the one or more suitable linkers, so as to provide the multivalent polypeptide of the invention. Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.

[0628] A method for preparing multivalent polypeptides of the invention may comprise at least the steps of linking two or more ISVs of the invention and for example one or more linkers together in a suitable manner. The ISVs of the invention (and linkers) can be coupled by any method known in the art and as further described herein. Preferred techniques include the linking of the nucleic acid sequences that encode the ISVs of the invention (and linkers) to prepare a genetic construct that expresses the multivalent polypeptide. Techniques for linking amino acids or nucleic acids will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the examples below.

[0629] Accordingly, the present invention also relates to the use of an ISV of the invention in preparing a multivalent polypeptide of the invention. The method for preparing a multivalent polypeptide will comprise the linking of an ISV of the invention to at least one further ISV of the invention, optionally via one or more linkers. The ISV of the invention is then used as a binding domain or building block in providing and/or preparing the multivalent polypeptide comprising 2 (e.g., in a bivalent polypeptide), 3 (e.g., in a trivalent polypeptide), 4 (e.g., in a tetravalent) or more (e.g., in a multivalent polypeptide) building blocks. In this respect, the ISV of the invention may be used as a binding domain or binding unit in providing and/or preparing a multivalent, such as bivalent, trivalent or tetravalent polypeptide of the invention comprising 2, 3, 4 or more building blocks.

[0630] Accordingly, 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 the preparation of the multivalent polypeptide will comprise the linking of the ISV of the invention to at least one further ISV of the invention, optionally via one or more linkers.

[0631] The polypeptides and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the polypeptides and nucleic acids include the methods and techniques described herein.

[0632] The method for producing a polypeptide of the invention may comprise the following steps: [0633] the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said polypeptide of the invention (also referred to herein as a "nucleic acid of the invention"), optionally followed by: [0634] isolating and/or purifying the polypeptide of the invention thus obtained. In particular, such a method may comprise the steps of: [0635] cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one polypeptide of the invention; optionally followed by: [0636] isolating and/or purifying the polypeptide of the invention thus obtained.

[0637] Accordingly, the present invention also relates to a nucleic acid or nucleotide sequence that encodes a polypeptide, ISV or construct of the invention (also referred to as "nucleic acid of the invention").

[0638] A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA. According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein. The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, e.g. expression vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form. Accordingly, the present invention also relates to an expression vector comprising a nucleic acid or nucleotide sequence of the invention.

[0639] The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least two nucleic acids encoding ISVs of the invention and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner. Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g, to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as to the Examples below.

[0640] In a preferred but non-limiting embodiment, a genetic construct of the invention comprises [0641] a) at least one nucleic acid of the invention; [0642] b) operably connected to one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also [0643] c) one or more further elements of genetic constructs known per se; in which the terms "regulatory element", "promoter", "terminator" and "operably connected" have their usual meaning in the art.

[0644] The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.

[0645] The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e., for expression and/or production of the polypeptide 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 as well as all other host cells or (non-human) hosts known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the skilled person. Reference is also made to the general background art cited hereinabove, 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 the further references cited herein. Furthermore, the polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above. The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention. Accordingly, the present invention relates to a host or host cell comprising a nucleic acid according to the invention, or an expression vector according to the invention. Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g., under suitable conditions), a polypeptide of the invention (and in case of a host organism; in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention, which may for instance be obtained by cell division or by sexual or asexual reproduction.

[0646] To produce/obtain expression of the polypeptides of the 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 invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

[0647] The polypeptide of the invention may then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g., using a specific, cleavable amino acid sequence fused with the polypeptide of the invention) and/or preparative immunological techniques (i.e. using antibodies against the polypeptide to be isolated).

[0648] In an aspect the invention relates to method for producing a construct, polypeptide or ISV according to the invention comprising at least the steps of: (a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid sequence according to the invention; optionally followed by (b) isolating and/or purifying the construct, polypeptide or ISV according to the invention.

[0649] In an aspect the invention relates to a composition comprising a construct, polypeptide, ISV or nucleic acid according to the invention.

[0650] Generally, for pharmaceutical use, the constructs, polypeptides and/or ISVDs of the invention may be formulated as a pharmaceutical preparation or composition comprising at least one construct, polypeptide and/or ISVD of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (such as intra-articular administration), for administration by inhalation, by a skin patch, by an implant, by a suppository, etc., wherein the intra-articular administration is preferred. Such suitable administration forms--which may be solid, semi-solid or liquid, depending on the manner of administration--as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein. Such a pharmaceutical preparation or composition will generally be referred to herein as a "pharmaceutical composition".

[0651] Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least 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 further active substances. In a particular aspect, the invention relates to a pharmaceutical composition that comprises 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 further active substances.

[0652] Generally, the constructs, polypeptides, and/or ISVs of the invention can be formulated and administered in any suitable manner known per se. Reference is for example 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 the standard handbooks, such as Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21.sup.st Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages 252-255).

[0653] In a particular aspect, the invention relates to a pharmaceutical composition that comprises a construct, polypeptide, ISV or nucleic acid according to the invention, and which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.

[0654] The constructs, polypeptides, and/or ISVs of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including SaFv's and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (e.g., intra-articular, transdermal or intradermal) administration.

[0655] Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include those mentioned on page 143 of WO 08/020079. Usually, aqueous solutions or suspensions will be preferred.

[0656] The constructs, polypeptides, and/or ISVs of the invention can also be administered using methods of delivery known from gene therapy, see, e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference for its gene therapy delivery methods. Using a gene therapy method of delivery, primary cells transfected with the gene encoding a construct, polypeptide, and/or ISV of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, joints or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression.

[0657] The constructs, polypeptides, and/or ISVs of the invention may also be administered intravenously, intra-articularly or intraperitoneally by infusion or injection. Particular examples are as further described on pages 144 and 145 of WO 08/020079 or in PCT/EP2010/062975 (entire document).

[0658] Useful dosages of the constructs, polypeptides, and/or ISVs of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; see for example U.S. Pat. No. 4,938,949.

[0659] The amount of the 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 and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the constructs, polypeptides, and/or ISVs of the invention varies depending on the target cell, tumor, joint, tissue, graft, or organ.

[0660] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. Preferably, the dose is administered once per week or even less frequent, such as once per two weeks, once per three weeks, once per month or even once per two months.

[0661] An administration regimen could include long-term treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See for instance Remington's Pharmaceutical Sciences (Martin, E. W., ed. 4.sup.th), Mack Publishing Co., Easton, Pa. The dosage can also be adjusted by the individual physician in the event of any complication.

[0662] The art is in need of more effective therapies for disorders affecting cartilage in joints, such as osteoarthritis. Even when administered intra-articularly, the residence time of most drugs for treating affected cartilage is insufficient. The present inventors hypothesized that the efficacy of a therapeutic drug could be increased significantly by coupling the therapeutic drug to a moiety which would "anchor" the drug in the joint and consequently increase retention of the drug, but which should not disrupt the efficacy of said therapeutic drug (also indicated as "cartilage anchoring protein" or "CAP"). This anchoring concept not only increases the efficacy of drug, but also the operational specificity for a diseased joint by decreasing toxicity and side-effects, thus widening the number of possible useful drugs. The present inventors further hypothesized that Aggrecan binders might potentially function as such an anchor, although Aggrecan is heavily glycosylated and degraded in various disorders affecting cartilage in joints. Moreover, in view of the costs and extensive testing in various animal models required before a drug can enter the clinic, such Aggrecan binders should preferentially have a broad cross-reactivity, e.g. the Aggrecan binders should bind to Aggrecan of various species. Using various ingenious immunization, screening and characterization methods, the present inventors were able to identify various Aggrecan binders with superior selectivity, stability and specificity features, which enabled prolonged retention and activity in the joint.

[0663] In an aspect the present 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.

[0664] In an aspect the present invention relates to a method for reducing and/or inhibiting the efflux of a composition, a polypeptide or a construct from a joint, wherein said method comprises administering 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 to a person in need thereof.

[0665] In the present invention the term "reducing and/or inhibiting the efflux" means reducing and/or inhibiting the outward flow of the composition, polypeptide or construct from within a joint to the outside. Preferably, the efflux is reduced and/or inhibited by at least 10% such as at least 20%, 30%, 40% or 50% or even more such as at least 60%, 70%, 80%, 90% or even 100%, compared to the efflux of the aforementioned composition, polypeptide or construct in a joint under the same conditions but without the presence of the Aggrecan binder of the invention, e.g. ISV(s) binding Aggrecan.

[0666] It is anticipated that the Aggrecan binders of the invention can be used in various diseases affecting cartilage, such as arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costochondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis (commonly indicated herein as "Aggrecan associated diseases").

[0667] In an aspect the present invention relates to a composition, an ISV, a polypeptide, and/or a construct according to the invention for use in preventing or treating an Aggrecan associated disease, such as e.g. arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis.

[0668] In an aspect the present invention relates to a method for preventing or treating arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of at least a composition, ISV, polypeptide, or construct according to the invention to a person in need thereof.

[0669] In an aspect the present 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 arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis.

[0670] It is expected that by binding to Aggrecan, the Aggrecan binders of the invention may reduce or inhibit an activity of a member of the serine protease family, cathepsins, matrix metallo-proteinases (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.

[0671] Accordingly, in an aspect the invention relates to a method for reducing or inhibiting an activity of a member of the serine protease family, cathepsins, matrix metallo-proteinases (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, wherein said method comprises administering a pharmaceutically active amount of at least an ISV, polypeptide, construct or composition according to the invention to a person in need thereof.

[0672] In the context of the present invention, the term "prevention and/or treatment" not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

[0673] The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases, disorders and conditions mentioned herein.

[0674] Generally, the treatment regimen will comprise the administration of one or more ISVs, polypeptides, compounds and/or constructs of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to be administered can be determined by the clinician, again based on the factors cited above.

[0675] Generally, depending on the specific disease, disorder or condition to be treated, the potency of the specific ISV, polypeptide, compound and/or construct of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the clinician will be able to determine a suitable daily dose.

[0676] Usually, in the above method, an ISV, polypeptide, compound and/or construct of the invention will be used. It is however within the scope of the invention to use two or more ISVs, polypeptides and/or constructs of the invention in combination.

[0677] The ISVs, polypeptides and/or constructs of the invention may be used in combination with one or more further pharmaceutically active compounds or principles, i.e., as a combined treatment regimen, which may or may not lead to a synergistic effect.

[0678] Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgment.

[0679] In particular, the ISVs, polypeptides and/or constructs of the invention may 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, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.

[0680] When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

[0681] 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 used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also 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 action. This may for example be useful for avoiding, limiting or reducing any unwanted side-effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

[0682] The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease, disorder or condition involved, as will be clear to the clinician. The clinician will also be able, where appropriate and on a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.

[0683] Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

[0684] In another aspect, the invention relates to the use of an ISV, polypeptide, compound and/or construct of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment of at least an Aggrecan associated disease; and/or for use in one or more of the methods of treatment mentioned herein.

[0685] The 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 or disorder that can be prevented and/or treated by modulating Aggrecan, e.g. inhibiting Aggrecan degradation.

[0686] The 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 an ISV, polypeptide, compound and/or construct of the invention to a patient.

[0687] The invention further relates to an ISV, polypeptide, compound and/or construct of the invention or a pharmaceutical composition comprising the same for use in the prevention and/or treatment of at least one Aggrecan associated disease.

[0688] The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. In veterinary applications, the subject to be treated includes any animal raised for commercial purposes or kept as a pet. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases, disorders and conditions mentioned herein.

[0689] Again, in such a pharmaceutical composition, the one or more ISVs, polypeptides, compounds and/or constructs of the invention, or nucleotide encoding the same, and/or a pharmaceutical composition comprising the same, may also be suitably combined with one or more other active principles, such as those mentioned herein.

[0690] The invention also relates to a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for use, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multi-cellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a disease, disorder or condition of the invention).

[0691] It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless explicitly stated otherwise.

[0692] Sequences are disclosed in the main body of the description and in a separate sequence listing according to WIPO standard ST.25. A SEQ ID specified with a specific number should be the same in the main body of the description and in the separate sequence listing. By way of example SEQ ID NO.: 1 should define the same sequence in both, the main body of the description and in the separate sequence listing. Should there be a discrepancy between a sequence definition in the main body of the description and the separate sequence listing (if e.g. SEQ ID NO.: 1 in the main body of the description erroneously corresponds to SEQ ID NO.: 2 in the separate sequence listing) then a reference to a specific sequence in the application, in particular of specific embodiments, is to be understood as a reference to the sequence in the main body of the application and not to the separate sequence listing. In other words a discrepancy between a sequence definition/designation in the main body of the description and the separate sequence listing is to be resolved by correcting the separate sequence listing to the sequences and their designation disclosed in the main body of the application which includes the description, examples, figures and claims.

[0693] The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures.

[0694] The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove.

EXAMPLES

Example 1 Immunization of Llamas with Aggrecan, Cloning of the Heavy Chain-Only Antibody Fragment Repertoires and Preparation of Phage

[0695] The present inventors realized that the purpose of animal models of OA is to controllably reproduce the scale and progression of joint damage, so that opportunities to detect and modulate symptoms and disease progression can be identified and new therapies developed. An ideal animal model is of relatively low cost and displays reproducible disease progression with a magnitude of effect large enough to detect differences within a short period of time. If the model progresses too rapidly to end-stage degeneration, intermediate time points, which are representative of OA pathophysiology, may not be obtainable and in IT the absence of this information, subtle effects of potential interventions may be missed. Recognizing that OA is an end-stage phenotype, the result of an interaction of mechanical and biochemical processes, animal models allow these factors to be studied in a controlled environment (cf. Teeple et al. 2013 AAPS J. 15: 438-446).

[0696] The final goal of animal models is to reproduce human diseases (cf. Cohen-Solal et al. 2013 Bonekey Rep. 2: 422). Given the heterogeneity of profiles in human OA, many models are needed. They are either spontaneous or induced. Most of them focus on one factor that favors the development of OA such as aging, mechanical stress (surgery), chemical defect (enzyme) or in genetic factors. All of them differ in terms of severity, localization of lesions and pathogenesis. However, no animal model addresses all aspects of developing OA.

[0697] Thus, in order to be useful in different animal models as well as ultimately in the human patient, the CAP-binder preferably has a broad cross-reactivity, e.g. 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, pig Aggrecan, mouse Aggrecan, rabbit Aggrecan, cynomolgus Aggrecan and/or rhesus Aggrecan.

[0698] Moreover, the present inventors realized that degradation of Aggrecan appears to initiate within the C-terminal region. The population of Aggrecan molecules without the G3 domain increases also with aging. A major feature of cartilage degeneration associated with arthritis is the loss of Aggrecan due to proteolytic cleavage within the interglobular region between the G1 and G2 domains. Hence, 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, the IGD, or the G2 domain. Most preferably, the Aggrecan binder would bind to the G1 domain, which remains present in chondrocytes and the ECM.

1.1 Immunizations

[0699] Five llamas were immunized with recombinant (rec) human Aggrecan (G1-IGD-G2 domains, R&D Systems it 1220-PG) (see Example 1.2). Serum samples were taken after antigen administrations and titers were determined by ELISA against human recombinant Aggrecan G1-IGD-G2. All llamas gave specific serum titers.

1.2 Primary Screening

[0700] RNA was extracted from PBLs (primary blood lymphocytes) and used as template for RT-PCR to amplify ISV encoding gene fragments. These fragments were cloned into phagemid vector pAX212 enabling production of phage particles displaying ISVs fused with His6- and FLAG3-tags. Phages were prepared and stored according to standard protocols (cf. Phage Display of Peptides and Proteins: A Laboratory Manual 1.sup.st Edition, Brian K. Kay, Jill Winter, John McCafferty, Academic Press, 1996).

[0701] Phage Display selections were performed with five immune libraries and two synthetic ISV libraries. The libraries were subjected to two to three rounds of enrichment against different combinations of recombinant human and (biotin-)rat Aggrecan G1-IGD-G2 domain, full length extracted bovine Aggrecan or intact bovine cartilage. Individual clones from the selection outputs were screened for binding in ELISA (using periplasmic extracts from E. coli cells expressing the ISVs) against the human G1-IGD-G2 domain. Sequencing of 542 ELISA-positive clones identified 144 unique ISV sequences. ISVs were assessed for species cross-reactivity and mapped by ELISA for binding to the individual human G1, IGD and G2 domains. Only a few ISVs showed similar binding levels to recombinant human, rat, dog and bovine Aggrecan G1-IGD-G2. The limited species cross-reactivity was particularly evident for G1 domain binders, for which binding to especially bovine and dog Aggrecan was poor. To identify more species cross-reactive G1 domain-binding ISVs, Phage Display selections against bovine G1-IGD-G2, dog G1-IGD-G2 and human G1 domains were performed. Of 1245 clones screened in ELISA for binding to human, cynomolgus, rat, dog and bovine G1-IGD-G2, only 15 novel species cross-reactive ISVs were identified of which nine could be mapped to the G1-domain.

[0702] A total of 19 unique clones were selected as `Lead panel` for further characterization. An overview of the domain-mapping and species cross-reactivity data for this lead panel is provided in Table 1.2.

TABLE-US-00001 TABLE 1.2 Overview of periplasmic extract-based screening data for the lead panel. Periplasmic extract ELISA. OD 450 nm Hu G1- Cy G1- Rat G1- Dog G1- Boy G1- Mapping Clone IGD-G2 IGD-G2 IGD-G2 IGD-G2 IGD-G2 G1 C0101PMP601E08 2.28 1.32 2.49 0.57 1.68 G1 C0101PMP102G11 1.69 0.60 0.16 1.02 0.32 G1 C0101PMP114F08 2.38 2.32 2.05 1.90 1.18 G1 C0101PMP112A01 2.50 2.50 2.03 1.57 2.41 G1 C0101PMP115B08 1.65 1.18 1.85 1.80 0.84 G1 C0101PMP117G09 2.21 2.21 2.29 1.68 0.76 G1 C0101PMP604B05 2.48 2.04 1.98 1.27 1.63 G1 C0101PMP606A05 0.25 1.24 0.93 0.51 0.19 G1 C0101PMP606A07 0.71 2.41 2.31 1.47 0.10 G1 C0101PMP608A05 2.33 2.48 2.39 0.86 2.27 G1 C0101PMP609C09 2.10 1.83 0.97 1.52 1.08 G2 C0101PMP112A03 2.51 2.36 1.69 1.47 0.73 G2 C0101PMP117D05 2.25 2.12 2.35 1.53 1.92 G2 C0101PMP604G09 2.41 1.57 1.40 1.16 1.21 G1-IGD-G2 C0101PMP113A01 2.56 2.57 2.53 2.51 2.54 G1-IGD-G2 C0101PMP601D02 2.58 nd 2.59 2.58 nd G1-IGD-G2 C0101PMP601E09 2.59 nd 2.61 2.57 nd G1-IGD-G2 C0101PMP604F02 2.41 1.37 0.78 1.04 0.82 G1-IGD-G2 C0101PMP604G01 2.27 1.25 0.60 1.55 0.68 control cAbLys3 0.05 0.06 0.06 0.06 0.06 control cAbLys3 0.05 0.05 0.06 0.06 0.05 Nd: not determined.

1.3 G1 Binders

[0703] The sequence variability in the CDRs of the G1-binders has been determined against clone 114F08. The amino acid sequences of the CDRs of clone 114F08 were used as reference, against which the CDRs of all other clones (G1-binders) were compared, and are depicted in the 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-US-00002 TABLE 1.3A G1 CDR1* absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G S T F I I N V V R sequence mutation R I S S Y A M G mutation F M R G K mutation I T Y A mutation T T *up to 2 CDR1 mutations in one clone

TABLE-US-00003 TABLE 1.3B G1 CDR2* absolute 1 2 2a 3 4 5 6 7 8 9 numbering wildtype T I -- S S G G N A N sequence mutations A S R T S S S T D G N W G R T Y T R N *up to 5 CDR2 mutations in one clone

TABLE-US-00004 TABLE 1.3C G1 CDR3* absolute 1 2 3 4 5 6 6a 7 8 9 10 10a 11 12 13 numbering wildtype P T T H Y G -- G V Y Y -- G P Y sequence mutations -- -- -- D F L R P G R N W S -- -- G R M Y V D T S T A E K E L D L S G T S Y H S G Y D R P R T G Y V R D W E V W L G S Y *up to 5 CDR3 mutations in one clone

1.4 G1-IGD-G2 Binders

[0704] The sequence variability in the CDRs of the G1-IGD-G2 (GIG) binders has been determined against clone 604F02. The amino acid sequences of the CDRs of clone 604F02 were used as reference, against which the CDRs of all other clones (GIG binders) were compared, and are depicted in the 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-US-00005 TABLE 1.4A GIG CDR1* absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G R T F S S Y T M G sequence mutation L T A *up to 2 CDR1 mutations in one clone

TABLE-US-00006 TABLE 1.4B GIG CDR2* absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype A I S W S G G R T Y sequence mutations S T R *up to 2 CDR2 mutations in one clone

TABLE-US-00007 TABLE 1.4C GIG CDR3* absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 numbering wildtype Y R R R R A S S N R G L W D Y sequence mutations V Y T -- P T E T P L V *up to 5 CDR3 mutations in one clone

1.5 G2 Binders

[0705] The sequence variability in the CDRs of the G2-binders has been determined against clone 601D02. The amino acid sequences of the CDRs of clone 601002 were used as reference, against which the CDRs of all other clones (G2 binders) were compared, and are depicted in the 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-US-00008 TABLE 1.5A G2 CDR1* absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G P T F S R Y A M G sequence mutation R S I N N R F Y mutation R M -- -- S *up to 5 CDR1 mutations in one clone

TABLE-US-00009 TABLE 1.5B G2 CDR2* absolute 1 2 3 4 5 6 7 8 9 10 11 numbering wildtype A I T W S S G G R T Y sequence mutations S L N A S N Y D R T *up to 5 CDR2 mutations in one clone

TABLE-US-00010 TABLE 1.5C G2 CDR3* absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 numbering wildtype A R I P V R T Y T S E W N Y -- -- sequence mutations R I H G S G R R S E N D D -- D N F L Q N N W S -- K A -- -- F Y -- -- -- *up to 5 CDR3 mutations in one clone

1.6 Sequence Optimization of ISVs

[0706] Various ISVs were subjected to a sequence optimisation process. Sequence optimisation is a process in which a parental ISV sequence is mutated. This process covers the humanisation (i) of the ISV and knocks-out post-translational modifications (ii) as well as epitopes for potential pre-existing antibodies (iii). [0707] (i) for humanisation purposes the parental ISV sequence is mutated to yield a ISV sequence which is more identical to the human IGHV3-IGHJ germline consensus sequence. Specific amino acids in the framework regions (with the exception of the so-called hallmark residues) that differ between the ISV and the human IGHV3-IGHJ germline consensus are altered to the human counterpart in such a way that the protein structure, activity and stability are kept intact. A handful of hallmark residues are known to be critical for the stability, activity and affinity of the ISV and are therefore not mutated. [0708] (ii) the amino acids present in the CDRs and for which there is experimental evidence that they are sensitive to post-translational modifications (PTM) are altered in such a way that the PTM site is inactivated while the protein structure, activity and stability are kept intact. [0709] (iii) the sequence of the ISV is optimised, without affecting protein structure, activity and stability, to minimise binding of any naturally occurring pre-existing antibodies and reduce the potential to evoke a treatment-emergent immunogenicity response.

[0710] For the generation of sequence optimised formatted ISVs, the ISV building were produced in Pichia pastoris as tagless proteins and purified via Protein A affinity chromatography, followed by desalting, all according to standard protocols.

[0711] Various sequence optimised formatted ISVs are shown in Tables A-1 and A-2.

Example 2 Characterization of the Lead Panel (Purified ISVs) Aggrecan

[0712] After the primary screening, initial assessment of binding via ELISA, determination of off-rate and species cross-reactivity, the ISVs of the Lead panel were subjected to further characterization.

2.1 Formatting Aggrecan Lead Panels with ALB26 (n=19)

[0713] It is anticipated that the final format of a molecule for clinical use comprises one or two Aggrecan binding ISVs ("anchors") and also one, two or more ISVs or other moieties with a therapeutic mode of action. Hence, the 19 selected clones were fused in monovalent or bivalent format to ALB26 (CAP-ALB26 or ALB26-CAP-CAP) and expressed in P. pastoris. ALB26 is a variant of ALB11 (Albumin binding ISV) with w two mutations in CDR1, which completely abolish binding to Albumin from different species. The fusion to ALB26 was performed in order to mimic the size of a final polypeptide format comprising an Aggrecan binder. Without being bound by any theory, the inventors hypothesized that the pl may influence cartilage penetration and retention. As negative control, or `dummy`, bivalent ALB26 (C01010030) was used.

2.2 Ex Vivo Bovine Cartilage Retention

[0714] Since there is no established assay for assessing cartilage retention, the inventors developed reliable and reproducible ex vivo cartilage retention assay using bovine cartilage.

[0715] Bovine bones were typically collected from the local slaughter house. Cartilage was cut off the bones in .about.1 mm thick strips and further cut into circular discs with a diameter of 3 mm with biopsy cutters. The cartilage discs were preferentially taken from fresh cartilage.

[0716] The ability of the ISVs to be retained in the cartilage for a prolonged period of time, following a relatively short exposure of the Nanobody to the cartilage (which can be expected upon intra-articular injection), was determined. The assay consisted of incubating ex vivo cartilage, typically 3 mm bovine discs (.about.10 mg wet weight) with 10 .mu.g/ml. Nanobody (100 .mu.l) ON, followed by washing for up to 5 days (PBS/0.1% BSA/0.1% NaN.sub.3/100 mM NaCl). Hereafter, bound (retained) Nanobody was released from the cartilage in SDS-containing SDS-PAGE sample buffer (LDS sample buffer Invitrogen) and analysed by Western Blot (WB). The assay was typically performed with 4 cartilage discs per Nanobody sample; 2 discs were analysed right after the Nanobody incubation (t.sub.0) to determine the initial amount of bound Nanobody; 2 discs were analysed after washing (t.sub.1-5 days). The degree of retention was defined as the ratio of the amount of Nanobody detected at t.sub.3-5 days and t.sub.0. To increase the throughput of the assay, the determination of this ratio was performed by visual inspection of the Western Blots giving a score from 0-6, where 0 is no retention and 6 is furl retention.

[0717] A summary of the results is shown in Table 2.2.

TABLE-US-00011 TABLE 2.2 Epitope binning and cartilage retention of the ALB26-formatted Aggrecan Lead Panel. Epitope Cartilage Target bin C01010# Construct pl retention * G1 4 118 ALB26-114F08- 9.09 6.00 114F08 G1 1 131 ALB26-601E08- 9.00 6.00 601E08 G1-IGD-G2 8 106 ALB26-604F02- 9.61 6.00 604F02 G1-IGD-G2 8 94 604F02-ALB26 9.47 5.33 G1 4 54 114F08-ALB26 9.02 5.00 G1 4 93 117G09-ALB26 9.13 5.00 G1 1 97 608A05-ALB26 9.09 5.00 G1 1 109 ALB26-608A05- 8.95 5.00 608A05 G2 7 115 ALB26-117D05- 8.73 5.00 117D05 G1-IGD-G2 8 47 601E09-ALB26 9.13 4.83 G2 6 108 ALB26-604G09- 9.13 4.00 604G09 G1-IGD-G2 8 95 604G01-ALB26 6.96 4.00 G1-IGD-G2 8 116 ALB26-113A01- 8.73 4.00 113A01 G1-IGD-G2 8 88 113A01-ALB26 8.53 3.50 G2 6 45 601D02-ALB26 9.15 3.40 G2 7 99 117D05-ALB26 9.10 3.33 G2 6 96 604G09-ALB26 8.99 3.00 G2 6 130 ALB26-601D02- 9.24 3.00 601D02 G1 1 46 601E08-ALB26 8.96 2.60 G1 5 60 606A07-ALB26 9.09 2.25 G1 5 113 ALB26-606A07- 8.62 2.00 606A07 G1 4 119 ALB26-115B08- 9.49 2.00 115B08 G2 6 117 ALB26-112A03- 9.12 2.00 112A03 G2 6 62 112A03-ALB26 9.21 1.66 G1 4 104 115B08-ALB26 8.66 1.50 G1 1 40 102G11-ALB26 9.20 1.33 G1 2 53 112A01-ALB26 9.17 1.00 G1 2 111 ALB26-112A01- 8.64 1.00 112A01 G1 3 56 604B05-ALB26 9.89 0.66 G1 3 59 606A05-ALB26 9.19 0.33 G1 2 98 609C09-ALB26 9.72 0.33 G1 2 110 ALB26-609C09- 8.13 0.00 609C09 G1 3 112 ALB26-604B05- 9.06 0.00 604B05 G1 3 114 ALB26-606A05- 9.03 0.00 606A05 Dummy 30 ALB26-ALB26 8.75 0.00 * The table lists average scores from a number (n) of independent ex vivo bovine cartilage retention assays on a scale from 0-6, in which 0 is no retention and 6 is full retention.

[0718] It was found that 9 constructs were retained very well (scores 5-6) in the cartilage. This `top-9` included both monovalent and bivalent constructs for the Aggrecan binding moiety binding to all of the recombinant G1, G2 or G1-IGD-G2 domains. 14 constructs showed moderate retention (scores between <5 and 2) and 5 constructs showed low albeit detectable retention (scores between <2 and 1) in this assay. It is notable that all Aggrecan constructs, except one, had pl values ranging from 8 to above 9.

2.3 Epitope Binning

[0719] For epitope-binning the purified ALB26-fused Nanobodies constructs were screened against the same set of Nanobodies fused with a FLAG-tag in a competition ELISA.

[0720] In short, the assay set up was as follows. Monoclonal phage ELISA were incubated at half-saturating dilution of phage with or without 1 .mu.M purified Nanobody (or 5 .mu.g/mL mAb). The ratio between the absorbance at 450 nm in the presence and absence of purified Nanobody (or mAb) was used to determine if the Nanobodies recognised overlapping or non-overlapping epitopes.

[0721] 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. There appears to be, however, no direct correlation between binding to bovine Aggrecan G1-IGD-G2 as measured by ELISA and bovine cartilage retention. Without being bound to any theory, the inventors hypothesized that these epitopes may not be easily accessible in the native cartilage tissue.

[0722] The sequence variability of the CDRs of clones belonging to a bin is depicted below and above (i.e. bin 8 with 604F02 as reference compound; Tables 1.4A-C).

[0723] The sequence variability of the G1-binders of epitope bin 4 against 114F08 is depicted in the Tables 2.3A, 2.3B and 2.3C below. The amino acid sequences of the CDRs of clone 114F08 were used as reference, against which the CDRs of all other clones (epitope bin 4 binders) were compared (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

TABLE-US-00012 TABLE 2.3A (114F08) G1 bin 4 CDR1* absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G S T F I I N V V R sequence mutations I S S R Y M K F M Y A *Up to 2 CDR1 mutations in one clone

TABLE-US-00013 TABLE 2.3B (114F08) G1 bin 4 CDR2* absolute 1 2 2a 3 4 5 6 7 8 9 numbering wildtype T I -- S S G G N A N sequence mutations A N R T D G *Up to 2 CDR2 mutations in one clone

TABLE-US-00014 TABLE 2.3C (114F08) G1 bin 4 CDR3* absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 numbering wildtype P T T H Y G G V Y Y G P V sequence mutations -- -- -- D F L G R N S -- -- R M Y V D T E K E L *Up to 5 CDR3 mutations in one clone

[0724] The sequence variability of the G1-binders of epitope bin 1 against 608A05 is depicted in the Tables 2.30, 2.3E and 2.3F below. The amino acid sequences of the CDRs of clone 608A05 were used as reference, against which the CDRs of all other clones (epitope bin 1 binders) were compared (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

TABLE-US-00015 TABLE 2.3D (608A05) G1 bin 1 CDR1* absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G R T F S T Y T M G sequence mutation S S A V *up to 2 CDR1 mutations in one clone

TABLE-US-00016 TABLE 2.3E (608A05) G1 bin 1 CDR2* absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype A I S W S G G T T Y sequence I R R S mutations *up to 2 CDR2 mutations in one clone

TABLE-US-00017 TABLE 2.3F (608A05) G1 bin1 CDR3* absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 numbering wilcitype R P R Y Y Y Y S L Y S Y D Y -- sequence mutations G L L R S T P H P Y D F G S R S A -- R A A *up to 5 CDR3 mutations in one clone

2.4 Binding Characteristics--ELISA and SPR

[0725] Based on the ex viva bovine cartilage retention and the epitope binning data, some exemplary constructs from different epitope bins were selected for further characterization. Binders to the G2-domain were excluded from further characterization at this stage for the reasons set out before.

[0726] The selected constructs were characterized in ELISA on the recombinant G1-IGD-G2 region from human, cynomolgus, rat, dog and bovine Aggrecan to determine their species cross-reactivity and on recombinant human Neurocan and Brevican to determine selectivity. The determined EC.sub.50 values are listed in Table 2.4A.

[0727] SPR (ProteOn) experiments were carried out for the "monovalent" Aggrecan-ALB26 formats in order to determine off-rates. The interaction of the Nanobodies with the Aggrecan surface was found to be heterogeneous. The heterogeneity could be due to re-binding events, a heterogeneous population of immobilized Aggrecan and/or heterogeneous glycosylation patterns. As a consequence, the calculated off-rates are only indicative. Overall it appears that the dissociation kinetics were fast for the Aggrecan comprising Nanobodies (Table 2.4B).

TABLE-US-00018 TABLE 2.4A Characterization of the ALB26-formatted Aggrecan Lead panel by ELISA. EC50 (M) Epitope C01010 Neuro- Brevi- Target bin # Construct Hu Cy Rat Dog Bov can can G1 4 54 114F08-ALB26 6.0E-09 4.4E-09 7.6E-09 3.0E-09 5.6E-09 No bind No bind G1 4 118 ALB26-114F08-114F08 1.1E-10 7.6E-11 1.9E-10 2.4E-10 3.7E-10 No bind No bind G1 1 97 608A05-ALB26 2.4E-10 2.1E-10 3.3E-10 2.5E-08 2.8E-10 No bind No bind G1 1 109 ALB26-608A05-608A05 1.0E-10 9.1E-11 9.5E-11 3.3E-10 7.7E-11 No bind No bind G1 1 46 601E08-ALB26 5.1E-09 6.8E-09 3.2E-10 6.1E-10 1.2E-09 No bind No bind G1 5 60 606A07-ALB26 1.2E-08 5.4E-09 8.4E-09 6.9E-09 No fit No fit No bind G1 5 113 AL326-606A07-606A07 6.7E-10 3.0E-10 1.2E-10 3.0E-09 No fit 8.7E-10 No bind G1-IGD-G2 8 94 604F02-ALB26 1.2E-09 2.2E-09 5.9E-09 2.6E-09 1.6E-09 No bind No bind G1-IGD-G2 8 106 ALB26-604F02-604F02 6.6E-11 6.8E-11 1.0E-10 9.7E-11 No fit No bind No bind Dummy 30 ALB26-ALB26 No bind No bind No bind No bind No bind No bind No bind

TABLE-US-00019 TABLE 2.4B Characterization of the `monovalent` ALB26-formatted Aggrecan Lead Panel (n = 5) by SPR (off-rate). Off-rates are only indicative due to heterogeneous binding patterns. C01010 G1-IGD-G2 (kd 1/s) Target # Construct human Cyno Rat Dog Bovine G1 54 114F08-ALB26 1.3E-02 6.9E-03 6.5E-01 1.1E-02 4.7E-01 G1 97 608A05-ALB26 2.5E-03 1.8E-03 1.5E-03 8.3E-02 2.7E-03 G1 46 601E08-ALB26 3.4E-03 3.1E-03 2.5E-04 7.1E-03 1.3E-03 G1 60 606A07-ALB26 2.1E-02 2.0E-02 2.1E-02 3.8E-02 2.7E-02 G1-IGD-G2 94 604F02-ALB26 1.7E-01 1.5E-01 2.6E-01 1.2E-01 2.6E-01

Example 3 Biophysical Characterization of Monovalent Lead Constructs--Aggrecan

[0728] Since all selected constructs demonstrated various favourable characteristics, whether or not in combination, the ISVs 114F08 and 604F02 and their corresponding ALB26-formats (C010100054, -118 and -094) were used as exemplary constructs representing the Lead panel for further characterization,

3.1 Expression of Monovalent 114F08 and 604F02 in E. coli and P. pastoris

[0729] For biophysical characterization, the monovalent Nanobodies 114F08 and 604F02 were expressed with FLAG.sub.3-His.sub.6-tags in E. coli and/or P. pastoris and purified according to standard protocols (e.g. Maussang et al. 2013 J Biol Chem 288(41): 29562-72).

3.2 pl, Tm and analytical SEC of 114F08 and 604F02

[0730] For the Thermal shift assay (TSA), 5 .mu.L purified monovalent Nanobody (800 .mu.g/ml) was incubated with 5 .mu.L of the fluorescent probe Sypro Orange (Invitrogen, 56551) (final concentration 10.times.) in 10 .mu.L buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, 115 mM NaCl, buffered at different pH ranging from 3.5 to 9). The samples were heated in a LightCycler 48011 machine (Roche), from 37 to 99.degree. C. at the rate of 4.4.degree. C./s, after which they were cooled down to 37.degree. C. at a rate of 0.03.degree. C./s. Upon heat-induced unfolding, hydrophobic patches of the proteins are exposed to which the 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).

[0731] The Analytical size exclusion chromatography (Analytical SEC) experiments were performed on an Ultimate 3000 machine (Dionex) in combination with a Biosep-SEC-3 (Agilent) column using 10 mM phosphate, 300 mM Arg-HCl, pH 6.0 as mobile phase. 8 .mu.g of Nanobody sample (0.5 mg/mL in d-PBS) were injected.

[0732] The isoelectric points of the two Aggrecan ISVs are relatively basic. The sequences are shown in Table A-1). The melting temperature was determined to be 61.0.degree. C. for 114F08 and 70.0.degree. C. for 604F02. None of the clones showed signs of aggregation or multimerisation as determined by analytical SEC.

[0733] Accordingly, next to the positive functional properties, the ISVs demonstrate favourable biophysical properties.

3.3 114F08 Family Members

[0734] The sequence variability in the CDRs of the family members of 114F08 is depicted in the Tables 3.3A, 3.3B and 3.3C below. The amino acid sequences of the CDRs of clone 114F08 were used as reference, against which the CDRs of all other clones (114F08 family members) were compared (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

TABLE-US-00020 TABLE 3.3A 114F08 CDR1* Kabat 26 27 28 29 30 31 32 33 34 35 numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G S T F I I N V V R sequence mutations S M *Up to 2 CDR1 mutations in one clone

TABLE-US-00021 TABLE 3.3B 114F08 CDR2* Kabat 50 51 52 53 54 55 56 57 58 numbering absolute 1 2 3 4 5 6 7 8 9 numbering wildtype T I S S G G N A N sequence mutations A R T T D *Up to 5 CDR2 mutations in one done

TABLE-US-00022 TABLE 3.3C 114F08 CDR3* Kabat 95 96 97 98 99 100 100a 100b 100c 100d 100e 100f 100g numbering absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 numbering wildtype P T T H Y G G V Y Y G P Y sequence mutations . . . R . . . D . . . . . *Up to 2 CDR3 mutations in one clone

Example 4 Ex Vivo Binding to Cartilage from Various Species

[0735] The exemplary CAP comprising polypeptides (also designated herein as "CAP comprising constructs" or "constructs") were shown to bind recombinant/extracted human proteins and bovine cartilage in the bovine ex vivo cartilage retention assay. In order to demonstrate that these exemplary CAP comprising constructs also bind to cartilage from other species, experiments as set out above with bovine cartilage were repeated in essence with human cartilage and rat cartilage.

4.1 Binding to Ex Vivo Human Cartilage

[0736] In order to confirm that the exemplary CAP comprising constructs also bind to human cartilage, selected constructs were tested in the ex vivo cartilage binding assay using frozen human cartilage chips. Binding was determined after a 30 min wash by means of Western Blot.

[0737] The results are summarized in Table 4.1.

[0738] It was found that all constructs bound better to the human cartilage than the Dummy construct.

4.2 Binding to Ex Vivo Rat Cartilage

[0739] To facilitate testing of constructs in a rat in vivo model, binding to rat cartilage was assessed. Therefore, an assay was set up using femurs from rat with intact cartilage. Exemplary constructs C010100054, -118, and -094 were incubated with the rat cartilage overnight, followed by a 30 min wash, release of bound constructs followed by Western Blot analysis.

[0740] The results are shown in Table 4.2.

[0741] It was found that all the tested constructs bound well to Rat cartilage.

Example 5 Tissue Specificity

[0742] It was demonstrated above that the constructs of the invention bind specifically to Aggrecan both in vitro and ex vivo. In addition, these constructs should also bind preferably to the cartilage of a joint, while not or less to other tissues in a joint.

[0743] Binding of exemplary CAP comprising constructs to synovial membrane, tendon, epimysium and meniscus was assessed using the same set up as for the ex vivo cartilage binding assay. Construct release and Western Blot analysis were performed following a brief wash of the tissues (30 min) after ON incubation with the constructs.

[0744] The results are summarized in Table 5.

[0745] The results show that CAP binders show preferential binding to the cartilaginous tissues, including meniscus, over the other tissues found in the joint.

TABLE-US-00023 TABLE 5 Tissue specificity. Binding of the ALB26-formatted Lead Panel (n = 10) to articular cartilage, synovial membrane, tendon, epimysium and meniscus. C010100 Synovial Target # Construct Cartilage Membrane Tendon Epimysium Meniscus G1 054 114F08-AB26 +++++ +/- + +/- nd G1 118 ALB26-114F08-114F08 +++++ + + + nd G1-IGD-G2 094 604F02-ALB26 +++++ + + +/- nd G1 046 601E08-ALB26 ++++ +/- + nd +++ Dummy 030 ALB26-ALB26 +/- +/- +/- - -

Example 6 Nanobody Stability in Bovine Synovial Fluid

[0746] For various reasons, including patient convenience and safety, it is preferred that the constructs remain stable for longer periods in the synovium.

[0747] Accordingly, the stability of the exemplary ALB26-fused CAP constructs in Synovial Fluid (SF) was assessed by incubation of the constructs in non-arthritic bovine SF for up to 7 days at 37.degree. C.

[0748] The results are summarized in Table 6.

TABLE-US-00024 TABLE 6 Stability of ALB26-formatted Lead Panel in bovine SF. Stability in Bovine Target C010100# Construct SF, 37.degree. C. G1 054 114F08-ALB26 >7 days G1 118 ALB26-114F08-114F08 >7 days G1-IGD-G2 094 604F02-ALB26 >7 days Dummy 030 ALB26-ALB26 >7 days

[0749] No degradation of any of the constructs could be detected.

Example 7 Retention in IL-1.alpha.-Stimulated Explant Cartilage

[0750] Up to this point, all experiments addressing cartilage binding and retention of the CAP comprising Nanobodies were performed in healthy (non-arthritic) ex vivo cartilage. Arthritic cartilage is characterized by degraded Collagen and Aggrecan. It is therefore of relevance to also assess binding and retention of the Aggrecan-binders in cartilage where degradation of these proteins has taken place. To this end, the exemplary ALB26-fused CAP constructs were tested in a cartilage explant assay in which cartilage was stimulated to induce degradation.

[0751] In short, the exemplary CAP comprising constructs were incubated overnight (ON) with bovine cartilage explants that were cultured with, or without, IL-1.alpha. and Oncostatin M, followed by 5 days of culture with daily change of medium (wash). IL-1.alpha. and Oncostatin M primarily induce the degradation of Aggrecan within the 6 days of the experiment. The cartilage explants were analysed for construct binding and retention by WB. Two independent experiments were performed (Exp A and Exp B).

[0752] The results of the Western Blots are depicted in Table 7.1.

[0753] The results of the CAP comprising construct retention in stimulated cartilage explants are summarized in Table 7.2.

TABLE-US-00025 TABLE 7.2 Summary of CAP binding and retention in stimulated bovine cartilage explant assay. Binding stimulated vs non- Retention Target C010100# Construct stimulated day 5 G1 054 114F08-ALB26 Reduced Partial G1 118 ALB26-114F08- Equal Full 114F08 G1-IGD-G2 094 604F02-ALB26 Reduced Partial G1 045 601D02-ALB26 Reduced Partial Dummy 030 ALB26-ALB26 No binding No binding

[0754] The results show that the constructs C01010054 ("054" or "54") and C01010045 ("045" or "45") have reduced retention in stimulated cartilage after 5 days of wash as compared to non-stimulated cartilage, while constructs C01010118 ("118") and C01010094 ("094" or "94") showed little sensitivity to stimulation.

[0755] It further appears that binding to the G2 Aggrecan domain (as exemplified by C01010045) is reduced more than binding to the other domains, which would be consistent with the hypothesis that Aggrecan degradation proceeds from the C-terminus.

Example 8 ADAMTS5-CAP GAG-Release Assay

[0756] In order to address the possible impact of CAP, the cartilage anchoring moiety, on the potency of a protease inhibiting Nanobody in cartilage tissue, the exemplary CAP constructs were fused to an ADAMTS5 (ATS5) blocking ISV and tested in a GAG (GlycosAminoGlycan)-release cartilage explant assay.

[0757] Before testing the constructs in the GAG-release cartilage explant assay, the in vitro cartilage binding and ADAMTS5 inhibiting properties were confirmed. For the latter, an enzymatic peptide assay was performed that showed that the enzyme-blocking function of the ADAMTS5 ISV was not impaired in any of the CAP-fusion constructs in vitro.

[0758] In the GAG-release assay, bovine cartilage explants were cultured for 5 days in the presence of IL-1.alpha. and Oncostatin M (for induction of ADAMTS5) and a dose range of constructs followed by quantification of the released GAG content in the culture supernatant.

[0759] The tested constructs and the results of the GAG-release assay are summarized in Table 8.

TABLE-US-00026 TABLE 8 Summary of ADAMTS5-CAP GAG-release assay. IC50 (nM) Peptide GAG- Target ID Construct assay release ADAMTS-5-G1 C010100270 ATS5-114F08 0.11 4.17 ADAMTS-5-G1 C010100276 ATS5-114F08- 0.06 19.15 114F08 ADAMTS-5-G1- C010100271 ATS5-604F02 0.19 2.15 IGD-G2 ADAMTS-5-G1 C011400510 ATS5 (Tag-less) 0.12 0.87

[0760] The results show that adding the anchoring arm (CAP-ISV construct) to the ADAMTS5 inhibitor still allowed for efficient inhibition of GAG-release.

Example 9 In Vivo Bio-Imaging of CAP-Constructs

[0761] In parallel to the in vitro and ex vivo characterization of the exemplary Aggrecan CAP constructs, in vivo bio-distribution was determined for several of the ALB26-fusion constructs, in order to confirm the retention properties.

9.1 Biodistribution Studies of ALB26-CAP Constructs

[0762] The Na nobodies were labeled with .sup.125I (via Lysine coupling of .sup.125I-SIB). The constructs were injected into the knee joints of healthy rats. Autoradiography images of the joints were produced for different time points up to 4 weeks post injection. These images allowed assessing the retention and the tissue (cartilage) specificity of the constructs in an in vivo-setting.

[0763] Representative images are shown in FIG. 1.

[0764] From the results it can be concluded that all constructs showed specific binding to the cartilage. A clear staining--even 4 weeks post injection--was observed for both `monovalent` and `bivalent` Aggrecan binders.

9.2 MARG of ALB26-CAP Constructs

[0765] The biodistribution study described above (Example 9.1) demonstrated specific retention in the cartilage of the ALB26-CAP constructs. However, the resolution of the images did not allow investigation of the depth of penetration into the cartilage. In order to increase the resolution of the imaging and thus to be able to evaluate penetration into the cartilage, MARG (Micro-Auto-Radio-Graphy) was used.

[0766] The exemplary constructs that went into the study are listed in Table 9.2A. For this study, the Nanobodies were labelled with .sup.3H (via lysine coupling of .sup.3H-NSP (N-Succinimidyl propionate)) and injected into the healthy and osteoarthritic (surgically induced via transection of the anterior cruciate ligament) rat joints; 8 rats per group. 7 to 14 days after injection the rats were sacrificed and the injected healthy and OA-induced joints were processed for MARG.

[0767] Representative MARG images are shown in FIG. 2.

TABLE-US-00027 TABLE 9.2A Exemplary Nanobody constructs tested Target C010100# Construct Aggrecan #54 114F08-ALB26 Aggrecan #626 ALB26-114F08-114F08 SO Aggrecan #94 604F02-ALB26 Dummy #30 ALB26-ALB26

[0768] All of the Aggrecan binders generally showed penetration into the healthy cartilage. Construct 626 occasionally also showed some more intense staining on the surface. Various degrees of cartilage staining and penetration were seen in the operated knee: no staining was observed with monovalent construct 054; staining was absent or mild with monovalent construct 094 while the bivalent construct 626 resulted in a somewhat more consistent staining albeit with varying depths of penetration (see Table 9.2B)

TABLE-US-00028 TABLE 9.2B Summary MARG staining results. Healthy Knee joint Operated Knee joint Silver grain Penetration Silver grain Penetration Construct* evaluation Depth evaluation Depth 030 0% of samples na 0% of samples na stained stained 054 100% samples C 0% of samples na with minimal stained staining 094 83% samples C 60% samples C with mostly with mostly mild staining mild staining 626 100% samples B-C 100% samples A-B-C with mostly with minimal mild staining to mild staining *Overall results of 8 animals are presented, based on a silver grain evaluation. Scoring of distribution: A = surface of cartilage with virtually no deeper staining, B = Surface of cartilage with some deeper staining, C = Staining in deeper layers of cartilage with no accumulation at surface

Example 10 In Vivo Rat MMT DMOAD Demonstrated a Statistical Significant Effect

[0769] In order to further demonstrate the in vivo efficacy of the CAP binders of the invention, a surgically induced Medial Meniscal Tear (MMT) model in rats was used. In short, CAP binders of the invention were coupled to an anti-MMP13 ISV (designated as "0754" or "C010100754") or an anti-ADAMTS5 ISV (designated as "0954" or "C010100954"). Rats were operated in one knee to induce OA-like symptoms. Treatment started 3 days post-surgery by IA injection. Histopathology was performed at day 42 post surgery. Interim and terminal serum samples were taken for exploratory biomarker analysis. The medial and total substantial cartilage degeneration width was determined, as well as the percentage reduction of cartilage degeneration. 20 animals were used per group.

[0770] The inhibition of cartilage degradation by Nanobodies in the medial tibia is shown in FIG. 3.

[0771] The results demonstrate that the cartilage width was substantially reduced by the ADAMTS5-CAP construct and the MMP13-CAP construct after 42 days compared to the vehicle. These results suggest that the CAP-moiety (a) has no negative impact on the activity of either the anti-MMP13 ISV (0754) or the anti-ADAMTS5 ISV (0954); and (b) enables the retention of these constructs for prolonged extension of time in the joints.

Example 11 Retention of CAP Binders in Healthy and Osteoarthritic Rats is Similar In Vivo

[0772] It was demonstrated in a cartilage retention study in healthy rats that the polypeptides of the invention were measurable in cartilage up to 112 days after intra-articular (I.A.) injection (data not shown). Since the cartilage composition can have an influence on cartilage binding and absorption in systemic circulation, the pharmacokinetics of the polypeptides of the invention were compared in diseased osteoarthritis and healthy rats in vivo by following the serum level of the polypeptides in time.

[0773] In particular, the surgically induced Medial Meniscal Tear (MMT) model in rats was used as described in Example 10, but with some modifications. In short, the polypeptides of the invention were coupled to an anti-MMP13 ISV and an anti-ADAMTS5 ISV, resulting in an MMP13-ADAMTS5-CAP-CAP construct (designated as "0949" or "C010100949" Nanobodies). Rats were operated in one knee to induce OA-like symptoms (OA-group). Each treatment group (healthy and OA) comprised of 15 animals, and received a single I.A. injection of 400 .mu.g/30 .mu.l Nanobody at day 7 (healthy) or 7 days post-surgery (MMT). Serum samples were collected from anesthetized rats at day 0, at day 7 (at 0 h=pre-dose sample) at day 8 (at different times post treatment up to 24 h), day 9 (48 h post-treatment), d10 (3 days post-treatment), d14 (7 days post-treatment), d21 (14 days post-treatment) and d42 (35 days post-treatment). Collected serum samples were used for the determination of the polypeptide concentrations in an electrochemoluminescence (ECL) based total PK assay format, followed by a non-compartmental analysis.

[0774] The retention of the polypeptides in the serum of healthy and OA rats is shown in FIG. 4.

[0775] The results demonstrate that no obvious differences can be seen in the serum concentrations of the polypeptides in healthy rats and OA rats. These results suggest that cartilage degradation has no influence on the pharmacokinetics of the polypeptides of the invention.

TABLE-US-00029 TABLE A-1 Amino acid sequences of monovalent Aggrecan binders (''ID'' refers to the SEQ ID NO as used herein) Name In Amino acid sequence 102G11 1 EVQLVESGGGLVQAGGSLRLSCAASGRSESSYAMGWERQAPGKEREFVSIISWSGGSTVYADS- VKGRFTI SRDNAKNTVYLQMNSLKPEDTAIYYCAAGRLYRATPRPADFGSWGQGTQVTVSS 112A01 2 EVQLVESGGGLVQTGGSLRLSCVASGRAFSNYIMGWERQAPGKERDEVAAINWNGVTTHYTDS- VKGRFTI SRDNAKSTSYLQMDSLKPDDTAVYFCAARGTVYSRTYGVSEEGYMYWGQGTQVTVSS 112A03 3 EVQLVESGGGLVQPGGSLRLSCAASGSIFSNREMYWYRQAPGKQRELVASITLSGSTNYADSV- KGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCNTFLQNSFYWGQGTQVTVSS 113A01 4 EVOLVESGGGLVQPGGSLRLSCSASOFTFSGSWMFWVRQAPGKDYEWVASINSSGGRTYYDDS- VKGRFTI SRDSAKNTLYLEMNNLKPEDTALYFCARSPRVGSWGQGTQVTVSS 114F08 5 EVQLVESGGGLVQAGGSLRLSCAASGSTFIINVVRWYRRTPGKQRELVATISSGGNANYVDSV- RGRFSIS RDGAKNAVDLQMNGLKBEDTAVYYCNVPTTHYGGVYYGPYWGQGTQVTVSS 115B08 6 KVQLVESGGGINQPGGSLRLSCAASGFTESMYAMKWVRQAPGKGLEWVSGINSSGGRTNYAGS- VKGRFTI SRDNAKNTLYLQMNSLKPEDTAVYYCATDFLGGRNSRGQGTQVTVSS 117D05 7 KVQLVESGGGLVQAGGSLRLSCAASRRTFNMMGWFRQAPGKEREFVAYITWNGGDTRYAESVK- GRFTVSR DDVKNTMALQMNRLDPLDTAVYYCGVRINGSNWSTKADDYDNWGQGTQVTVSS 117G09 8 EVQLVESGGGSALPGGSLRLSCAASGITESSRYMRWYRQAPGRQRELVAAISSGGRTDYVDSV- RGRFTLS INNAKNTVYLQMNDLKPEDTAVYYCYRPRMYVDGTYEKELWGQGTLVTVES 601D02 9 DVQLVESGGGLVQPGGSLRLSCAASGPTESRYAMGWFRQAPGKEREFVAAITWSSGGRTYYAD- SVKGRFT ISRDNSKNTVYLQMNSLRFEDTAVYYCAAARIPVRTYTSEWNYWGQGTLVTVES 601E08 10 DVOLVESGGGLVQPGGSLRLSCTASGRTESSYAVGWERQAPGKEREFVAAISRSGRSTYYADS- VKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCAAGLSYYSPHAYYDYWGQGTLVTVSS 601E09 11 DVQLVESGGGLVQPGGSLRLSCAASGLTESTYAMGWERQAPGKEREFVAAISWSGSRTYYADS- VKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCAAYRRPRYSPTGTWDYWGQGTLVTVSS 604B05 12 DVQLVESGGGLVQPGGSLRLSCVASGRTFSIYTMAWFRQARGKEREFVAAISWSSGRTYYADS- VKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTAYTGPRSGYDYWGQGTLVTVSS 604E02 13 DVQLVESGGGLVQPGGSLRLSCAASGRTESSYTMGWERQAPGKEREFVAAISWSGGRTYYADS- VKGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCAAYRRRRASSNRGLWDYWGQGTLVTVSS 604G01 14 DVQLVESGGGLVQPGGSLRLSCAASGRTESSYTMGWERQAPGKEREFVAAISWSGRTTYYADS- VKGRFTI SRDNSKNTVYLQMNSLRFEDTAVYYCAAYRRVRYTNLEVWDYWGQGTLVTVSS 604G09 15 DVQLVESGGGLVQPGGSLRLSCVASGRTESSYAMGWFRQAPGKEREFVAATTWSSATTYYADS- VKGRETT SRDNSKNTVYLQMNSLRPEDTAVYYCAAARIPVGRRSENWDYWGQGTLVTVSS 606A05 16 DVQLVESGGGLVQPGGSLRLSCVASGRTFSIYTMGWERQAPGKEREFVAAISWSGGRTYYADS- VYGRFTI SRDNSKNTVYLQMNSLRPEDTAVYYCTAYTGRSYGSYDYWGQGTLVTVSS 606A07 17 DVQLVESGGGLVQPGGSLRLSCVASGRTFSIYGMGWERQAPGKEREFVAAINGGSRTYYADSV- KGRFTIS RDNSKNTVYLQMNSLRFEDTAVYYCAADRSGYGTSLDWWYDYWGQGTLVTVSS 608A05 18 DVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTMGWFRQAPGKEREFVAAISWSGGTTYYADS- VKGRFTI SRDNSKNIVYLQMNSLRPEDTAVYYCAARPRYYYYSLYSYDYWGQGTLVTVSS 609C09 19 DVQLVESGGGLVQPGGSLRLSCAASGTIFSINVMGWYRQAPGKEREFVAAITTGGRTNYADSV- KGRFTIS RDNSKNTVYLQMNSLRPBDTAVYYCNAEVTTGWVGYSWYDYWGQGTMVTVSS 114A09 114 EVQLVESGGGINQAGGSLRLSCAASGSTFIISVMRWYRQAPGKQRELVAAIRTGGNTDYAGPV- RGRESIS RDGAKNAVDLQMNGLKPEDTAVYYCNVPTTRYGGDYYGPYWGQGTQVTVSS 114B04 115 EVQLVESGGGLVQAGGSLRLSCAASGSTFIISVMRWYRQAPGKQRELVAAIRTGGNTDYAGPV- RGRESIS RDGAKDAVDLQMNGLKPEDTAVYYCNVPTTRYGGDYYGPYWGQGTQVTVSS 00269 116 EVQLVESGGGLVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVR- GRFTIS SO114F08 RDNSKNTVYLQMNSLRPEDTAVYYCNVPTTHYGGVYYGPYWGQGTLVTVSS 00745 117 EVQLVESGGGVVQPGGSLRLSCAASGSTFTINVVRWYRRAPGKQRELVATISSGGNANYVDSVR- GRFTIS PEA114F08 RDNSKNTVYLOMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 00747 118 EVQLVESGGGVVQPGGSLRLSCAASGRTFSSYTMGWERQAPGKEREFVAAISWSGGRTYYADSV- KGRFTI PEA604F02 SRDNSKNTVYLQMNSTAPEDTALYYCAAYRRRRASSNRGLWDYWGQGTLVTVSSA

TABLE-US-00030 TABLE A-2 Sequences for CDRs and frameworks, plus preferred combinations as provided in formula I, namely FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (the following terms: ''ID'' refers to the given SEQ ID NO) Nano- ID body ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 1 102G11 75 EVQLVESGGGLVQ 20 GRSFSSYAMG 85 WFRQAPGKEREFVS 38 IISWSGGSTV 94 YADSVKGRFTISRDNAKNTV 56 GRLYRATPRPADFGS 105 WGQGTQVTV AGGSLRLSCAAS YLQMNSLKPEDTAIYYCAA SS 2 112A01 76 EVQLVESGGGLVQ 21 GRAFSNYIMG 86 WFRQAPGKERDFVA 39 AINWNGVTTH 95 YTDSVKGRFTISRDNAKSTS 57 RGTVYSRTYGVSEEG 105 WGQGTQVTV TGGSLRLSCVAS YLQMDSLKPDDTAVYFCAA YMY SS 3 112A03 77 EVQLVESGGGLVQ 22 GSIFSNRFMY 87 WYRQAPGKQRELVA 40 SITLSGSTN 96 YADSVKGRFTISRDNAKNTV 58 FLQNSFY 105 WGQGTQVTV PGGSLRLSCAAS YLQMNSLKPEDTAVYYCNT SS 4 113A01 78 EVQLVESGGGLVQ 23 GFTFSGSWMF 88 WVRQAPGKDYEWVA 41 SINSSGGRTY 97 YDDSVKGRFTISRDSAKNTL 59 SPRVGS 105 WGQGTQVTV PGGSLRLSCSAS YLEMNNLKPEDTALYFCAR SS 5 114F08 75 EVQLVESGGGLVG 24 GSTFIINVVR 89 WYRRTPGKQRELVA 42 TISSGGNAN 98 YVDSVRGRFSISRDGAKNAV 60 PTTHYGGVYYGPY 105 WGQGTQVTV AGGSLRLSCAAS DLQMNGLKPEDTAVYYCNV SS 6 115B08 79 KVQLVESGGGLVQ 25 GFTFSMYAMK 90 WVRQAPOKGLEWVS 43 GINSSGGRTN 99 YAGSVKGRFTISRDNAKNTL 61 DFLGGRNS 106 RGQGTQVTV PGGSLRLSCAAS YLQMNSLKPEDTAVYYCAT SS 7 117D05 80 KVQLVESGGGLVQ 26 RRTFNMMG 91 WFRGAPGKEREFVA 44 YITWNGGDTR 100 YAESVKGRFTVSRDDVKNTM 62 RIHGSNWSTKADDYD 105 WGQGTQVTV AGGSLRLSCAAS ALQMNRLDPLDTAVYYCGV N SS 8 117G09 81 EVQLVESGGGSAL 27 GITFSSRYMR 92 WYRQAPGRQRELVA 45 AISSGGRTD 101 YVDSVRGRFTLSINNAKNTV 63 PRMYVDGTYEKEL 107 WGQGTLVTV PGGSLRLSCAAS YLQMNDLKPEDTAVYYCYR SS 9 601D02 82 DVQLVESGGGLVQ 28 GPTFSRYAMG 91 WFRQAPGKEREFVA 46 AITWSSGGRTY 102 YADSVKGRFTISRDNSKNTV 64 ARIPVRTYTSEWNY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRPEDTAVYYCAA SS 10 601E08 83 DVQLVESGGGLVQ 29 GRTFSSYAVG 91 WFRQAPGKEREFVA 47 AISRSGRSTY 102 YADSVKGRFTISRDNSKNTV 65 GLSYYSPHAYYDY 107 WGQGTLVTV PGGSLRLSCTAS YLQMNSLRPEDTAVYYCAA SS 11 601E09 82 DVQLVESGGGLVQ 30 GLTSTYANIG 91 WFRQAPGKEREFVA 48 AISWSGSRTY 102 YADSVKGRFTISRDNSKNTV 66 YRRPRYSPTGTWDY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRPEDTAVYYCAA SS 12 604B05 84 DVQLVESGGGLVQ 31 GRTFSIYTMA 91 WFRQAPGKEREFVA 49 AISWSSGRTY 183 YADSVKGRFTISRDNSKNTV 67 YTGPRSGYDY 107 WGQGTLVTV PGGSLRLSCVAS YLQMNSLRPEDTAVYYCTA SS 13 604F02 82 DVQLVESGGGLVQ 32 GRTFSSYTMG 91 WFRQAPGKERFFVA 50 AISWSGGRTY 102 YADSVKGRFTISRDNSKNTV 68 YRRRRASSNRGLWDY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRPEDTAVYYCAA SS 14 604G01 82 DVQLVDSGGGLVQ 32 GRTFSSYTMG 91 WFRQAFGKEREEVA 51 AISWSGRTTY 102 YADSVKGRFTISRDNSKNTV 69 YRRVRYTNLEVWDY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRPEDTAVYYCAA SS 15 604G09 84 DVQLVESGGGLVQ 33 GRTFSSYAMG 91 WFRQAPGKEREEVA 52 AITWSSATTY 102 YADSVKGRFTISRDNSKNTV 70 ARIPVGRRSENWDY 107 WGQGTLVTV PCCSDRLSCVAS YLQMNSLRFEDTAvYYCAA SS 16 606A05 84 DVQLVESGGGLVQ 34 GRTFSIYTMG 91 WFRQAPGKEREFVA 50 AISWSGGRTY 103 YADSVKGRFTISRDNSKNTV 71 YTGRSYGSYDY 147 WGQGTLVTV PGGSLRLSCVAS YLQMNSLRPEDTAVYYCTA SS 17 606A07 84 DVQLVESGGGLVQ 35 GRTFSIYGMG 91 WFRQAPGKEREFVA 53 AINGGSRTY 102 YADSVKGRFTISRDNSKNTV 72 DRSGYGTSLDWWYDY 107 WGQGTLVTV PGGSLRLSCVAS YLQMNSLRFEDTAVYYCAA SS 18 608A05 82 DVQLVESGGGLVQ 36 GRTFSTYTMG 91 WFRQAPGKEREFVA 54 AISWSGGTTY 102 YADSVKGRFTISRDNSKNTV 73 RPRYYYYSLYSYDY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRPEDTAVYYCAA SS 19 609C09 82 DVQLVESGGGLVQ 37 GTIFSINVMG 93 WYRQAPGKEREEVA 55 AITTGGRTN 104 YADSVKGRFTISRDNSKNTV 74 EVTTGWVGYSWYDY 108 WGQGTMVTV PGGSLRLSCAAS YLQMNSLRPEDTAVYYCNA SS 114 114A09 75 EVQLVESGGGLVQ 109 GSTFIISVMR 87 WYRQAPGKQRELVA 110 AIRTGGNTD 112 YAGPVRGRFSISRDGAKNAV 111 PTTRYGGDYYGPY 105 WGQGTQVTV AGGSLRLSCAAS DLQMNGLKPEDTAVYYCNV SS 115 114B04 75 EVQLVESGGGLVQ 109 GSTFIISVMR 87 WYRQAPGKQRELVA 110 AIRTGGNTD 113 YAGFVRGRFSISRDGAKDAV 111 PTTRYGGDYYGPY 105 WGQGTQVTV AGGSLRLSCAAS DLOMNGLKPEDTAVYYCNV SS 116 0269 77 EVQLVESGGGLVQ 24 GSTFIINVVR 121 WYRRAPGKQRELVA 42 TISSGGNAN 122 YVDSVRGRFTISRDNSKNTV 60 PTTHYGGVYYGPY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRPEDTAVYYCNV SS 117 0745 119 EVQLVESOGGVVQ 24 GSTFIINVVR 121 WYRRAPGKQRELVA 42 TISSGGNAN 123 YVDSVRGRFTISMNSKNTV 60 PTTHYGGVYYGPY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRFEDTALYYCNV SS 118 0747 120 DVQLVESGGGVVQ 32 GRTFSSYTMG 91 WFRQAPGKEREFVA 50 AISWSGGRTY 124 YADSVKGRFTISRDNSKNTV 68 YRRRRASSNRGLWDY 107 WGQGTLVTV PGGSLRLSCAAS YLQMNSLRPEDTALYyCAA SS

TABLE-US-00031 TABLE B Aggrecan sequences and others from various species (''ID'' refers to the SEQ ID NO as used herein) Name ID Amino acid sequence human 125 MTTLLWVFVTLRVITAAVTVETSDHIDNSLSVSIPUSPLRVLLGTSLTIPCYFIDPMHPVTTAP- STAPLA Aggrecan PRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEVQSLASNDSGVYRCE- VMHGIE DSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVR YPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARL ATTGHVYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYVHANQTGYPDPSSRYDAICYT GEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTF APEIGATAFAEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGPTRYS LTFEEAQQACPGTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGV RPSTETYDVYCFVDRLEGEVFFATRLEQFTFQEALEFCESHNATATTGQLYAAWSRGLDKCYAGWLADGS LRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGISAVPSPGEEEGGITTSPSGVEEWI VTQVVPGVAAVPVEEETTAVPSGETTATLEFTTEPENQTEWEPAYTENGTSPLPGILPTWPPTCAETEES TEGPSATEVPSASEEPSPSEVPFPSEEPSPSEEPFPSVRPFPSVELFPSEEPFPSKEPSPSEEPSASEEP YTPSPPEPSWTELPSSGEESGAPDVSGDFTGSGDVSMILDFSGQLSGDRASGLPSGDLDSSGLTSTVGSG LTVESGLPSGDEERIEWPSTPTVGELPSGAEILEGSASGVGDLSGLPSGEVLETSASGVGDLSGLPSGEV LETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISG LPSGEVLETTAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPG VEDISCLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVL ETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGL PSGEVLETAAPGVEDISGLPSGEVLETTAPGVERISGLPSGEVLETTAPGVDEISGLPSGEVLETTAPGV EEISGLPSGEVLETSTSAVGDLSGLPSGGEVLEISVSGVEDISGLPSGEVVETSASGIEDVSELPSGEGL ETSASGVEDLSRLFSGEEVLEISASGFGDLSGVPSGGEGLETSASEVGTDLSGLPSGREGIETSASGAED LSGLPSGKEDLVGSASGDLDLGKLIDSGTLGSWAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSG FPTVSLVDSTINEVVTASTASELEGRGTIGISGAGEISGLPSSELDISGRASGLPSGTELSGQASGSPDV SGEIPGLFGVSGQPSGFPDTSGETSGVTELSGLSSGQPGVSGEASGVLYGTSQPFGITDLSGETSGVPDL SGQRSGLPGFSGATSGVPDLVSGTTSGSGESSGITFVDTSLVEVAPTTFKEEEGLGSVELSGLPSGEADL SGKSGMVDVSGQFSGTVDSSGFTSQTPEFSGLPSGIAEVSGESSRAEIGSSLPSGAYYGSGTPSSFPTVS LVDRTLVESVTQAPTAQEAGEGPSGILELSGAHSGAPDMSGEHSGPLDLSGLQSGLIEPSGEPPGTPYFS GDFASTTNVSGESSVAMGTSGEASGLPEVTLITSEFVEGVTEPTISQELGQRPPVTHTPQLFESSGKVST AGDISGATPVLPGSGVEVSSVPESSSETSAYPEAGFGASAAPEASREDSGSPDLSETTSAPHEANLERSS GLGVSGSTLTFQEGEASAAPEVSGESTTTSDVGTEAPGLPSATPTASGDRTEISGDLSGHTSQLGVVIST SIPESEWTQQTQRPAETHLEIESSSLLYSGEETHTVETATSPTDASIPASPEWKRESESTAAAPARSCAE EPCGAGTCKETEGHVICLCPPGYTGEHCNIDQEVCEEGWNKYQGHCYRHFPDRETWVDAERRCREQQSHL SSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDMFFAAGEDCVVMIWHEKG EWNDVPCNYHLPFTCKKGTVACGEPPVVEHARTFGQKKDRYEINSLVRYQCTEGFVQRHMPTIRCQRSGH WEEPRITCTDATTYKRRLQKRSSRHPRRSRPSTAH dog 126 MTTLLWVFVTLRVITAASSEETSDHDNSLSVSIPEPSPMRVLLGSSLTIPCYFIDPMHPVTTAPST- APLA Aggrecan PRINNSRITKEKEVVLLVATEGQVRINSAYQDKVSLPNYPAIPSDATLEIQNLRSNDSGIYECE- VMHGIE DSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVR YPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVLYATSPEKETEQEAANECRRLGARL ATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDESSRYDATCYT GEDFVDIPENFFGVGGEEDITIQTVTWPDVELPLPRNITEGEARGNVILTVKPIFDLSPTAPEPEEPFTF VPEPEKPFTFATDVGVTAFPEAENRTGEATRPWGVPEESTPGPAFTAFTSEDIWVQVTAVPGAAEVPGQP RLPGGVVEHYRPGSARYSLTFEEAQQACLETGAVIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPR TPCVGDKDSSPGVRTYGVRPPSETYDVYCYVDKLEGEVFFITRLEQFTFQEALAFCESHNATIASTGQLY AAWRQGLDKCYAGWLSDGSLRYPIVTPRPSCGGDKPGVRTVYLYPNQTGLPDPLSRHHVECFRGVSGVPS PGEEEGGTPTPSVVEDWIPTQVGPVVPSVPMGEETTAILDFTIEPENQTEWEPAYSPAGTSPLPGIPPTW PPTSTATEESTEGPSGTEVPSVSEEPSPSEEPFPWEELSTLSPPGPSGTELPGSGEASGVPEVSGDFTGS GEVSGHPDSSGQLSGESASGLESEDLDSSGLTSAVGSGLASGDEDRITLSSIPKVEGEGLETSASGVEDL SGLPSGREGLETSTSGVGDLSGLPSGEGLEVSASGVEDLSGLPSGEGPETSTSGVGDLSRLPSGEGPEVS ASGVGDLSGLPSGREGLETSTSGVEDLSGLPSGEGPEASTSGVGDLSRLPSGEGPEVSASGVEDLSGLPS GEGLEASASGVGDLSGLPSGEGPEASASGVGDLSRLPSGEGPEVSASGVEDLSGLSSGESPEASASGVGD LSGLPSGREGLETSASGVGDLSGLPSGEGQEASASGVEDLSRLESGEGPEASASGVGELSGLPSGREGLE TSASGVGDLSGLPSGEGPEAEASGVEDLSILPSGEGPEASASGVGDLSGLPSGREGLETSTSGVGDLSGL PSGREGLETSTSGVGDLSGLPSGEGPEASASGIGDISGLESGREGLETSSSGVEDHPETSASGVEDLSGL PSGVEGHPETSASGVEDLSDLSEGGEGLETSASGAEDLSGEPSGKEDLIGSASGALDEGRIPSGTLGSGQ APEASSLPSGESGEYSGVDEGSGPISGLPDFSGLPSGEPTISLVDTTLVEVITTTSASELEGRGTIGISG AGETSGLPVSELDISGAVSGLPSGAELSGQASGSPDMSGETSGFFGVSGQPSGFPDISGGTSGLFEVSGQ PSGFSGETSGVTELSGLYSGQPDVSGEASGVPSGSGQPFGMTDLSGETSGVEDISGQPSGLPEFSGTTSG IPDLVSSTMSGSGESSGITFVDTSLVEVTPTTFKEKKRLGSVELSGLPSGEVDLSGASGTMDISGQSSGA TDSSGLTSHLPKESGLPSGAAEVSGESSGAEVGSSLPSGTYEGSGNEHEAFPTVELVDETLVESVTQAPT AQEAGEGPSGILELSGAHSGAPDVSGDESGSLDLSGMQSGLVEPSGEPSSTPYFSGDFSGTMDVTGEPST AMSASGEASGLLEVTLITSEFVEGVTEPTVSQELAQRPPVTHTPQLFESSGERSASGEISGATPAPPGSG LEASSVPESSSETSDEPERAVGVSAAPEASGGASGAPDVSEATSTFPEADVEGASGLGVSGGTSAFPEAP REGSATPEVINEPTTSYDVGREALGWPSATPTASGDRIEVSGDLSGHTSGLDVVISTSVPESEWIQQTQR PAEAHLEIEASSPLESGEETQTAETATSPTDDASIPTSPSGTDESAPAIPDIDECLSSPCLNGATCVDAI DSFTCLCLPSYRGDLCEIDQELCEEGWTKEQGHCYRYFPDRESWVDAESECEAQQSELSSIVTPEEQEEV NNNAQDYQWIGLNDRTIEGDFRWSDGHSLQFENWRPNQPDNEEVSGEDCVVMIWHEKGEWNDVPCNYYLP FTCKKGTVACGDPPVVEHARTEGQKKDRYEINSLVRYQCTEGEVQRHVPTIRCQPSGHWEKPRITCTDES TYKRRLQKRSSRAPRRSRPSTAH bovine 127 MTTLLLVFVTLRVITAAISVEVSEPDNSLSVSIPEPSPLRVLLGSSLTIPCYFIDPMHPVTTA- PSTAPLA Aggrecan PRIKWSRISKEKEVVLLVATEGRVRVNSAYQDKVTLPNYPAIPSDATLEIQNMRSNDSGILRCE- VMHGIE DSQATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVR YPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARL ATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYT GEDFVDTPESPFGVGGEEDITIQTVTWPDVELPLPRNITEGEARGSVILTAKPDFEVSPTAPEPEEPFTF VPEVRATAFPEVENRTEEATRPWAFPRESTPGLGAPTAFTSEDLVVQVTLAPGAAEVPGQPRLPGGVVFH YRPGSSRYSLTFEEAKQACLRTGAIIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPTVSPRTPCVGDKDS SPGVRTYGVRPPSETYDVYCYVDRLEGEVFFATRLEQFTFWEAQEFCESQNATLATTGQLYAAWSRGLDK CYAGWLADGSLRYPTVTPRPACGGDKPGVRTVYLYPNQTGLLDPLSRHHAFCFRGVSAAPSPEEEEGSAP TAGPDVEEWMVTQVGPGVAAVPIGEETTAIPGFTVEPENKTEWELAYTPAGTLPLPGIPPTWPPTGEATE EHTEGPSATEVPSASEKPFPSEEPFPPEEPFPSEKPFPPEELFPSEKPFPSEKPFPSEEPFPSEKPFPPE ELFPSEKPIPSEEPFPSEEPFPSEKPFPPEEPFPSEKPIPSEEPFPSEKPFPSEEPPPSEEPSTLSAPVP SRTELFSSGEVSGVPEISGDFTGSGEISGHLDFSGQPSGESASGLPSEDLDSSGLTSTVGSGLPVESGLP SGEEERITWTSAPKVDRLPSGGEGPEVSGVEDISCLPSGGEVHLETSASGVEDISGLPSGGEVHLEISAS GVEDLSRIPSGEGETISASGVEDISGLPSGEEGHLEISASGVEDLSGIPSGEGPEVSASGVEDLIGLPSG EGPEVSASGVEDLSRLPSGEGPEVSASGVEDLSGLPSGEGPEVSVSGVEDLSRLPSGEGFEVSASGVEDL SRLPSGEGPEISVSGVEDISILPSGEGPEVSASGVEDLSVLPSGEGHLEISTSGVEDLSVLPSGEGHLET SSGVEDISRLPSGEGPEVSASGVEDLSVLPSGEDHLEISASGVEDLGVLPSGEDHLEISASGVEDISRLP SGEGPEVSASGVEDLSVLPSGEGHLEISASGVEDLSRLPSGGEDHLETSASGVGDLSGLPSGREGLEISA SGAGDLSGLTSGKEDLTGSASGALDLGRIPSVTLGSGQAPEASGLPSGFSGEYSGVDLESGPSSGLPDFS GLPSGFPTVSLVDTTLVEVVTATTAGELEGRGTIDISGAGETSGLPFSELDISGGASGLSSGAELSGQAS GSPDISGETSGLFGVSGQPSGFPDISGETSGLLEVSGQPSGFYGEISGVTELSGLASGQPEISGEASGIL SGLGPPFGITDLSGEAPGIPDLSGQPSGLPEFSGTASGTPDLVSSAVSGSGESSGITFVDTSLVEVTPTT FKEEEGLGSVELSGLPSGELGVSGTSGLADVSGLSSGAIDSSGFTSQPPEFSGLPSGVTEVSGEASGAES GSSLPSGAYDSSGLPSGFPTVSFVDRTINESVTQAPTAQEAGEGPSGILELSGAPSGAPDMSGDHLGSLD QSGLQSGLVEPSGEPASTPYFSGDFSGTTDVSGESSAATSTSGEASGLPEVTLITSELVEGVTEPTVSQE LGQRPPVTYTPQLFESSGEASASGDVPRFPGSGVEVSSVPESSGETSAYPEAEVGASAAPEASGGASGSP NLSETTSTFHEADLEGTSGLGVSGSPSAFFEGPTEGLATPEVSGESTTAFDVSVEASGSPSATPLASGDR TDTSGDLSGHTSGLDIVISTTIPESEWTQQTQRPAEARLEIESSSPVHSGEESQTADTATSPTDASIPAS AGGTDDSEATTTDIDECLSSPCLNGATCVDAIDSFTCLCLPSYQGDVCEIQKLCEEGWTKFQGHCYRHFP DRATWVDAESQCRKQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDKTIEGDFRWSDGHSLQFENWRPNQP DNFFATGEDCVVMIWHEKGEWNDVPCNYQLPFTCKKGTVACGEPPVVEHARIFGQKKDRYEINALVRYQC TEGFIQGHVPTIRCQPSGHWEEPRITCTDPATYKRRLQKRSSRPLRRSHPSTAH rat 128 MTTLLLVFVTLRVIAAVISEEVETHDNSLSVSIPQPSPLKALLGTSLTIPCYFIDPMHPVTTAPST- APLT Aggrecan PRIKWSRVSKEKEVVLLVATEGQVRVNSIYQDKVSLPNYPAIPSDATLEIQNLRSNDSGIYRCE- VMHGIE DSEATLEVIVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVR YPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRTVGARL ATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDATCYT GEDFVDIPENFFGVGGEEDITIQTVTWPDLELPLPRNITEGEARGNVILTAKPIFDMSPTVSEPGEALTL APEVGTTVFPEAGERTEKTTRPWGFPEEATRGPDSATAFASEDLVVRVTISPGAVEVPGQPRLPGGVVFH YRPGSTRYSLTFEEAQQACIRTGAAIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPRTPCVGDKDS SPGVRTYGVRPSSETYDVYCYVDKLEGEVFFATQMEQFTFQEAQAFCAAQNATLASTGQLYAAWSQGLDK CYAGWLADGTLRYPIVNPRPACGGDKPGVRTVYLYPNQTGLPDPLSKBHAFCFRGVSVVPSPGGTPTSPS DIEDWIVTRVEPGVDAVPLEPETTEVPYFTTEPEKQTEWEPAYTPVGTSPLPGIPPTWLPTVPAAEEHTE SPSASQEPSASQVPSTSEEPYTPSLAVPSGTELPSSGDTSGAPDLSGDFTGSTDTSGRLDSSGEPSGGSE SGLPSGDLDSSGLGPTVSSGLPVESGSASGDGEIPWSSTPTVDRLPSGGESLEGSASASGTGDLSGLPSG GEITETSASGTEEISGLPSGGDDLETSTSGIDGASVLPTGRGGLETSASGVEDLSGLPSGEEGSETSTSG IEDISVLPTGESPETSASGVGDLSGLPSGGESLETSASGVEDVTQLPTERGGLETSASGIEDITVLPTGR ENLETSASGVEDVSGLPSGKEGLETSASGIEDISVFPTEAEGLETSASGGYVSGIPSGEDGTETSTSGVE GVSGLPSGGEGLETSASGVEDLGLPTRDSLETSASGVDVTGYPSGREDTETSVPGVGDDLSGLPSGQEGL ETSASGAEDLGGLPSGKEDLVGSASGALDFGKLPSGTLGSGQTPEASGLPSGFSGEYSGVDIGSGPSSGL PDFSGLPSGFPTVSLVDSTLVEVITATTASELEGRGTISVSGSGEESGPPLSELDSSADTSGLPSGTELS GQTSGSLDVSGETSGFFDVSGQPFGSSGTGEGTSGTPEVSGQAVRSPDTTEISELSGLSSGQPDVSGEGS GILFGSGQSSGITSVSGETSGISDLSGQPSGFPVLSGTTPGTPDLASGANSGSGDSSGITFVDTSLIEVT PTTFREEEGLGSVELSGLPSGETDLSGTSGMVDVSGQSSGAIDSSGLISPTPEFSGLPSGVAEVSGEVSG VETGSSLSSGAFDGSGLVSGFPTVSLVDRTLVESITLAPTAQEAGEGPSSILEFSGAHSGTPDISGDLSG SLDQSTWQPGWTEASTEPPSSPYFSGDFSSTTDASGESITAPTGSGETSGLPEVTLITSELVEGVTEPTV SQELGEGPSMTYTPRLFEASGEASASGDLGGPVTIFPGSGVEASVPEGSSDPSAYPEAGVGVSAAPEASS QLSEFPDLHGITSASRETDLEMTTPGTEVSSNPWTFQEGTREGSAAPEVSGESSTTSDIDAGTSGVPFAT PMTSGDRTEISGEWSDHTSEVNVTVSTTVPESRWAQSTQHPTETLQEIGSPNPSYSGEETQTAETAKSLT DTPTLASPEGSGETESTAADQEQCEEGWTKFQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEF VNKNAQDYQWIGLNDRTIEGDFRWSDGHSLQFEKWRPNQPDNFFATGEDCVVMIWHERGEWNDVPCNYQL PFTCKKGTVACGEPPAVEHARTLGQKKDRYEISSLVRYQCTEGFVQRHVPTIRCQPSADWEEPRITCTDP NTYKERLQKRTMRPTRRSRPSMAH Pig 129 AISVEVSEPDNSLSVSIPQPSPLRVLLGGSLTIPCYFIDPMHPVXTAPXTAPLAPRIKWSRVSKEK- EVVL Aggrecan LVATEGQVRVNSAYQDRVTLPNYPAIPSDATLETQNLRSNDSGIYRCEVMHGIEDSEATLEVVV- KGIVPH (core) YRAISXRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYG- DKDE FPGVITYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGQLYLAWRGGMDM CSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGG EEDITIQTVTWPDVELPLPRNITEGEARGTVILTVKPVFEFSPTAPEPEEPFTFAPGTGATAFPEAENRT GEATRPWAFPEESTPGLGAPTAFTSEDINVQVTSAATEEGTEGPSATEAPSTSEEPFPSEKPFPSEEPFP SEEPFPSEKPSASEEPFPSEQPSTLSAPVPSRTELPGSGEVSGAPEV mouse 130 MTTLLLVEVTLRVIAAVISEEVPDHDNSLSVSIPQPSPLKVLLGSSLTIPCYPIDPMHPVTTAP- STAPLT Aggrecan PRIKWSRVSKEKEVVLLVATEGQVRVNSIYQDKVSLPNYPAIPSDATLEIQNLRSNDSGIYRCE- VMHGIE DSEATLEVIVKGIVFHYRAISTRYTLDFDRAQRACLQNSAITATPEQLQAAYEDGFHQCDAGWLADQTVR YPIHTPREGCYGDKEEFPGVRTYGIRDTNETYDVYCFAEEMEGEVEYATSPEKFTFQEAANECRRLGARL ATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYT GEDFVDIPENFFGVGGEDDITIQTVTWPDLELPLPRNVTEGEALGSVILTAKPIFDLSPTISEPGEALTL APEVGSTAFPEAEERTGEATRPWGEPAEVTRGPDSATAFASEDLVVRVTISPGAAEVPGQPRLPGGVVFH YRPGSTRYSLTFEEAQQACMHTGAVIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPRTPCVGDKDS SPGVRTYGVRPSSETYDVYCYVDKLEGEVEFATRLEQFTEQEARAFCAAQNATLASTGQLYAAWSQGLDK CYAGWLADGTLRYPIITPRPACGGDKPGVRTVYLYPNQTGLPDPLSKHHAFCFRGVSVAPSPGEEGGSTP TSPSDIEDWIVTQVGPGVDAVPLEPKTTEVPYFTTEPRKQTEWEPAYTPVGTSPQRGIPPTWLPTLPAAE EHTESPSASEEPSASAVPSTSEEPYTSSFAVPSMTELPGSGEASGAPDLSGDFTGSGDASGRLDSSGQPS GGIESGLPSGDLDSSGLSPTVSSGLPVESGSASGDGEVPWSHTPTVGRLPSGGESPEGSASASGTGDLSG LPSGGEITETSISGAEETSGLPSGGDGLETSTSGVDDVSGIPTGREGLETSASGVEDLSGLPSGEEGSET STSGIEDISVLPTGGESLETSASGVGDLSGLPSGGESLETSASGAEDVTQLPTERGGLETSASGVEDITV LPTGRESLETSASGVEDVSGLPSGREGLETSASGIEDISVEPTEAEGLDTSASGGYVSGIPSGGDGTETS ASGVEDVSGLPSGGEGLETSASGVEDLGPSTRDSLETSASGVDVTGFPSGRGDPETSVSGVGDDFSGLPS GKEGLETSASGAEDLSGLPSGKEDLVGSASGALDEGKLPPGTLGSGQTPEVNGEPSGFSGEYSGADIGSG PSSGLPDFSGLPSGFPTVSLVDSTLVEVITATTSSELEGRGTIGTSGSGEVSGLPLGELDSSADISGLPS GTELSGQASGSPDSSGETSGFEDVSGQPEGSSGVSEETSGIPEISGQPSGTPDTTATSGVTELNELSSGQ PDVSGDGSGILFGSGQSSGITSVSGETSGISDLSGQPSGFPVFSGTATRTPDLASGTISGSGESSGITFV DTSFVEVTPTTFREEEGLGSVELSGFPSGETELSGTSGTVDVSEQSSGAIDSSGLTSPTPEFSGLPSGVA EVSGEFSGVETGSSLPSGAEDGSGINSGFPTVSLVDRTLVESITQAPTAQEAGEGPSGILEFSGAHSGTP DISGELSGSLDLSTLQSGQMETSTETPSSPYFSGDFSSTTDVSGESIAATTGSGESSGLPEVTLNTSELV EGVTEPTVSQELGHGPSMTYTPRLFEASGDASASGDLGGAVINFPGSGIEASVPEASSDLSAYPEAGVGV SAAPEASSKLSEFPDLHGITSAFHETDLEMTTPSTEVNSNPWTFQEGTREGSAAPEVSGESSTTSDIDTG TSGVPSATPMASGDRTEISGEWSDHTSEVNVAISSTITESEWAQPTRYPTETLQEIESPNPSYSGEETQT AETTMSLTDAPTLSSSEGSGETESTVADQEQCEEGWTKEQGHCYRHEHDRETWVDAERRCREQQSHLSSI VTPEEQEFVNKNAQDYQWIGLNDRTIEGDFRWSDGESLQFEKWRPNQPDNEFATGEDCVVMIWHERGEWN DVPCNYQLPFTCKKGTVACGDPPVVEHARTLGQKKERYEISSLVRYQCTEGFVQRHVPTIRCQPSGHWEE PRITCTDPNTYKERLQKRSMRPTRRSRPSMAH rabbit 131 MTTLLLVLVALRVIAAAISGDVSDLDNALSVSIPQPSPVRALLGTSLTIPCYFIDPVHPVTTA- PSTAPLT Aggrecan PRIKWSRISKDKEVVLLVANEGAVRINSAYQDKVSLPNYPAIPSDATLEIQSLRSNDSGIYRCE- VMHGLE DEEATLEVVVKGVVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVR YPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAASECRRLGARL ATTGQLYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYVHANQTGYPDPSSRYDAICYT GEDFMDIPENFFGVGGEEDITVQTVTWPDVELPVPRNITEGEARGSVVLTAKPVLDVSPTAPQPEETFAP GVGATAFPGVENGTEEATRPRGFADEATLGPSSATAFTSADLVVQVTAAPGVAEVPGQPRLPGGVVFHYR PGPTRYSLTFEEAQQACLIRTGAAMASAEQLQAAYEAGYEQCDAGWLQWTVRYPIVSPRTPCVGDKDSSP GVRTYGVRPPSETYDVYCYVDRLEGEVFFATRLEQFTFQEALESCESHNATIASTGQLYAAWSRGLDRCY AGWLADGSLRYPTVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFROTSEAPSPGPEEGGTATP ASGLEDWIVTQVGPGVAATPRAEERTAVPSFATEPGNQTGWEAASSPVGTSLLPGIPPTWPPTGTAAEGT TEGLSTAAMPSASEGPYTPSSLVARETELPGLGVTSVPPDISGDLTSSGEASGLFGPTGQPLGGSASGLP SGELDSGSLTPTVGSGLPIGSGLASGDEDRTQWSSSTEVGGVTSGAEIPETSASGVGTDLSGLPSGAEIP ETFASGVGTDLSGLPSGAEIPETFASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASGV GTDLSGLPSGAETLETSASGVGTDLSGLPSGAETRETFASGVGTDLSGLPSGAETLETSASGVGTDLSGL PSGAEIPETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEIL ETSASGVGTDLSGLFSGAEILETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGGEIPETFASGV GDLSGLPPGREDLETLTSGVGDLSGLSSGKDGLVGSASGALDFGGTLGSGQIPETSGLPSGYSGEYSEVD LGSGPSSGLPDFSGLPSGEPTVSLVDTPLVEVVTATTARELEGRGTIGISGAGETSGLPSSELDVSGGTS GADISGEADVGGEASGLIVRGQPSGFPDTSGEAFGVTEVSGLSSGQPDLSGEASGVLFGSGPPPGITDLS GEPSGQPSGLPEFSGTTHRIPDLVSGATSGSGESSGIAFVDTSVVEVTPTTLREEEGLGSVEFSGFPSGE TGLSGTPETIDVSGQSSGTIDSSGFTSLAPEVSGSPSGVAEVSGEASCTEITSGLPSGVFDSSGLPSGFP TVSLVDRTLVESVTQAPTAINAEGPSDILELSGVHSGLPDVSGAHSGFLDPSGLQSGLVEPSGEPPRTPY FSGDFPSTPDVSGEASAATSSSGDISGLPEVTLVTSEFMEGVTRPTVSQELGQGPPMTHVPKLFESSGEA LASGDTSGAAPAPPGSGLEASSVPESHGETSAYAEPGTKAAAAPDASGEASGSPDSGEITSVFREAAGEG ASGLEVSSSSLASQQGPREGSASPEVSGESTTSYEIGTETSGLPLATPAASEDRAEVSGDLSGRTPVPVD VVTNVPEAEWIQHSQRPAEMWPETKSSSPSYSGEDTAGTAASPASADTPGEPGPTTAAPRSCAEEPCGPG TCQETEGRVTCLCPPGHTGEYCDTDIDECLSSPCVNGATCVDASDSFTCLCLPSYGGEILCETWEVCEEG WTKFQGHCYRHFPDRETWVDAEGRCREQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDG HPLQFENWRPNQPDNFFATGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGDPEWVEHARTFGQKK DRYEINSLVRYQCAEGFTQRHVPTIRCQPSGHWEEPRITCTHPTTYKRRVQKRSSRTLQRSQASSAP cynomolgus 132 MTTLLWVFVTLRVIAAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTAPSTAPLA Aggrecan PRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEIQSLRSNDSGVYRCE- VMHGIE DSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVR YPIHTPREGCYGDKDEFPGVRTYGIRDINETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARL ATTGQLYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYT GEDFVDIPENFFGVGGEEDTTVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFDVSPSPLEPEEPFTF APEIGATAFPEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVPHYRPGSTRYS LTFEEAQQACLRTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGV RPSTETYDVYCYVDRLEGEVFFATRLEQFTFQEALEFCESHNATLATTGQLYAAWSRGLDKCYAGWLADG SLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGVSAVPSPGEEEGGTPTSPSGVEDW TATQVVPGVAAVPVEEETTAVPLGETTAILEFTTEPENQTEWEPAYTPMGTSPLPGILPTWPPTGTATEE STEGPSATEVITASKEPSPPEVPFPSEEPSPSEEPFPSVRPFPSVEPSPSEEPFPSVEPSPSEEPSASEE PYTPSPPVPSWTELPGSGEESGAPDVSGDFIGSGDVSGHLDFSGQLSGDRISGLPSGDLDSSGLTSTVGS GLPVDSGLASGDEERTEWSSTPTVGELPSGAEILEGSASEVGDLSGLPSGENLETSASGVGDLSGLPSGE VLETSASGVGDLSGLPSGEVLETSTSGVGDLSGLPSGEVLETSTSGVGDLSGLPSAGEVLETTASGVEDT

SGLPSGEVLETTASGVEDISGFPSGEVLETTASGVEDISGLPSGEVLETTASGVEDISGLPSGEVLETTA SGVGDLGGLPSGEVLETSTSGVGDLSGLPSGEVVETSTSGVEDLSGLPSGGEVLETSTSGVEDISGLPSG EVLETTASGIEDVSELPSGEGLETSASGVEDLSRLPSGEVLETSASGVGDISGLPSGGEVLETSASGVGD LSGLPSGGEGLETSASGVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDLVGPASGDLDLGKLPSGTL RSGQAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTI GISGAGEISGLPSSELDISGEASGLPSGTELSGQASGSPDVSRETPGLFDVSGQPSGFPDISGGTSGISE VSGQPSGFPDTSGETSGVTELSGLPSGQPGVSGEASGVPYGSSQPFGITDISGETSGVPDLSGQPSGLPG FSGATSGVPDLVSGATSGSGESSGITFVDTSLVEVTPTTFKEREGLGSVELSGLPSGEADLSGRSGMVDV SGQFSGTVDSSGFTSQTPEFSGLPTGIAEVSGESSGAETGSSLPSGAYYGSGLPSGFPTVSLVDRTINES VTQAPTAQEAGEGPPGILELSGTHSGAPDMSGDHSGFLDVSGLQFGLVEPSGEPPSTPYFSGDFASTTDV SGESSAAMGTSGEASGLPGVTLITSEFMEGVTEPTVSQELGQRPPVTHTPQLFESSGEASAAGDISGATP VLPGSGVEVSSVPESSSETSAYPEAGVGASAAPETSGEDSGSPDLSETTSAFHEADLERSSGLGVSGSTL TFQEGEPSASPEVSGESTTTGDVGTEAPGLPSATPTASGDRTEISGDLSGHTSGLGVVISTSIPESEWTQ QTQRPAEAHLETESSSLLYSGEETHTAETATSPTDASIPASPEWTGESESTVADIDECLSSPCLNGATCV DAIDSFTCLCLPSYGGDLCEIDQEVCEEGWTKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQ EFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNY HLPFTCKKGTVACGEPPMVQHARTFGQKKDRYEINSLVRYQCTEGFVQRHVPTIRCQPSGHWEEPRITCT DATAYKRRLQKRSSRHPRRSRPSTAH rhesus 133 MTTLLWVFVTLRVIAAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTA- PSTAPLA Aggrecan PRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEIQSLRSNDSGVYRCE- VMHGIE XM_ DSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTV- R 002804944.1 YPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARL ATTGQLYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYT GEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFDVSPSPLEPEEPFTF APEIGATAFPEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGSTRYS LTFEEAQQACLRTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGG RPSTETYDVYCYVDRLEGEVFFATRLEQFTFQEALEFCESHNATLATTGQLYAAWSRGLDKCYAGWLADG SLRYPIVTPRPACCGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGVSAVPSPGEEEGGTPTSPSGVEDW IATQVVPGVAAVPVEEETTAVPLGETTAILEFTTEPENQTEWEPAYTPMGTSPLPGILPTWPPTGTATEE STEGPSATEVLTASKEPSPPEVPFPSEEPSPSEEPFPSVRPFPSVEPSPSEEPFPSVEPSPSEEPSASEE PYTPSPPVPSWTELPGSGEESGAPDVSGDFIGSGDVSGHLDFSGQLSGDRISGLPSGDLDSSGLTSTVGS GLPVDSGLASGDEERIEWSSTPTVGELPSGAEILEGSASEVGDLSGLPSGDVLETSASGVGDLSGLPSGE VLETSVSGVGDLSGLPSGEVLETSTSGVGDLSGLPSGEVLETSTSGVGDLSGLPSAGEVLETTASGVEDI SGLPSGEVLETTASGVEDISGFPSGEVLETTASGVEDISGLPSGEVLETTASGVEDISCIPSGEVLETTA SGVGDLGGLPSGEVLETSTSGVGDLSGLPSGEVVETSTSGVEDLSGLPSGGEVLETSTSGVEDISGLPSG EVLETTASGTEDVSELPSGEGLETSASGVEDLSRLPSGEVLETSASGVGDISGLPSGGEVLEISASGVGD LSGLPSGGEGLETSASGVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDINGPASGDLDLGKLPSGTL GSGQAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTI GISGAGEISGLPSSELDISGEASGLPSGTELSGQASGSPDVSRETSGLFDVSGQPSGFPDTSGETSGVTE LSGLPSGQPGVSGEASGVPYGSSQPFGITDLSGETSGVPDLSGQRSGLPGFSGATSGVPDINSGATSGSG ESSDITFVDTSLVEVTPTTFKEEEGLGSVELSGLPSGEADLSGRSGMVDVSGQFSGTVDSSGFTSQTPEF SGLPIGIAEVSGESSGAETGSSLPSGAYYGSELPSGFPTVSLVDRTLVESVTQAPTAQEAGEGPPGILEL SGTHSGAPDMSGDHSGFLDVSGLQFGLVEPSGEPPSTPYFSGDFASTTDVSGESSAAMGTNGEASGLPEV TLITSEFMEGVTEPTVSQELGQRPPVTHTPQLFESSGEASAAGDISGATPVLPGSGVEVSSVPESSSETS AYPEAGVGASAAPETSGEDSGSPDLSETTSAFHEADLERSSGLGVSGSTLTFQEGEPSASPEVSGESTTT GDVGTEAPGLPSATPTASGXXXXXXPTRSCAEEPCGAGTCKETEGHVICLCPPGYTGEHCNIDQEVCEEG WTKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDG HPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGEPPMVQHARTFGQKK DRYEINSLVRYQCTEGFVQRHVPTIRCQPSGHWEEPRITCTDATAYKRRLQKRSSRHPRRSRPSTAH human 134 MGAPFVWALGLLMLQMLLFVAGEQGTQDITDASERGLHMQKLGSGSVQAALAELVALPCLFTLQ- PRPSAA neurocan RDAPRIKWTKVRTASGQRQDLPILVAKDNVVRVAKSWQGRVSLPSYPRRRANATLLLGPLRASD- SGLYRC QVVRGIEDEQDLVPLEVTGVVFHYRSARDRYALTFAEAQEACRLSSAIIAAPRHLQAAFEDGFDNCDAGW LSDRTVRYPITQSRPGCYGDRSSLPGVRSYGRRNPQELYDVYCFARELGGEVFYVGPARRLTLAGARAQC RRQGAALASVGQLHLAWHEGLOQCDPGWLADGSVRYPIQTPRRRCGGPAPGVRTVYRFANRTGFPSPAER FDAYCFRAHHPTSQHGDLETPSSGDEGEILSAEGPPVRELEPTLEEEEVVTPDFQEPLVSSGEEETLILE EKQESQQTLSPIPGDPMLASWPTGEVWLSTVAPSPEDMGAGTAASSHTEVAPTDPMPRRRGRFKGLNGRY FQQQEPEPGLQGGMEASAQPPTSEAAVNQMEPPLAMAVTEMLGSGQSRSPWADLTNEVDMPGAGSAGGKS SPEPWLWPPTMVPPSTSGHSRAPVLELEKAEGPSARPATPDLFWSPLEATVSAPSPAPWEAFPVATSPDL PMMAMLRGPKEWMLPHPTPISTEANRVEAHGEATATAPPSPAAETKVYSLPLSLTPTGQGGEAMPTTPES PRADFRETGETSPAQVNKAEHSSSSPWPSVNRNVAVGFVPTETATEPTGLRGIPGSESGVEDTAESPTSG LQATVDEVQDPWPSVYSKGLDASSPSAPLGSPGVFLVPKVTPNLEPWVATDEGPTVNPMDSTVTPAPSDA SGIWEPGSQVFEEAESTTLSPQVALDTSIVTPLTTLEQGDKVGVPAMSTLGSSSSQPHPEPEDQVETQGT SGASVPPHQSSPLGKPAVPPGTPTAASVGESASVSSGEPTVPWDPSSTLLPVTLGIEDFELEVLAGSPGV ESFWEEVASGEEPALPGTPMNAGABEVHSDPCENNPCLHGGTCNANGTMYGCSCDQGFAGENCEIDIDDC LCSPCENGGTCIDEVNGFVCLCLPSYGGSFCEKDTEGCDRGWHKFQGHCYRYFAHRRAWEDAEKDCRRRS GHLTSVHSPEEHSFINSFGHENTWIGLNDRIVERDFQWTDNTGLQFENWRENQPDNFFAGGEDCVVMVAH ESGRWNDVPCNYNLPYVCKKGTVLCGPPPAVENASLIGARKAKYNVHATVRYQCNEGFAQHHVATIRCRS NGKWDRPQIVCTKPRRSHRMRRHHHHHQHHHQHHHHKSRKERRKHKKHPTEDWEKDEGNFC human 135 MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLOGVLGGALTIPCHVHYLRP- PPSRRA brevican VIGSPRVKWTPLSRGREAEVLVARGVRVKVNEAYRFRVALPAYPASLTDVSLALSELRPNDSGI- YRCEVQ HGIDDSSDAVEVKVKGVVPLYREGSARYAFSFSGAQEACARIGAHIATPEQLYAAYLGGYEQCDAGWLSD QTVRYPIQTPREACYGDMDGFPGVRNYGVVDPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQER GAEIATTGQLYAAWDGGLDHCSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNV YCFRDSAQPSAIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSST PEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYEDEEEKEEEEEETEVEDEALWAWPSELSSPG PEASLPTEPAAQESSLSQAPARAVLQPGASPLPDGESEASRPPRVAGPPTETLPTPRERNLASPSPSTLV EAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRAPEGTRELEAPSEDNSGRTAPAGTSVQAQPV LPTDSASRGGVAVVPASGDCVPSPCHNGGTCLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACY KHFSTRRSWEEAETQCRMYGAHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWN PGQPDSYFLSGENCVVMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVL RYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKALLIPPSSPMPG P

TABLE-US-00032 TABLE C Serum albumin binding ISV sequences (''ID'' refers to the SEQ ID NO as used herein) Name ID Amino acid sequence Alb8 136 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 137 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTL YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 138 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDIATYYCTIGGSLSRSSQGTLVTVSSA Alb132 139 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTL YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIOGSLSRSSQGTLVTVSSA Alb11 140 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCIIGGSLSRSSQGTLVTVSS Alb11 141 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL (S112K)-A YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 142 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 143 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEMVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 144 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 145 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 146 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLOMNSLRPEDIALYYCTIGGSLSRSSQGTLVTVSSG Alb82-GG 147 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTTSRDNAKTTLYIQMNSLRPEDTALTYCITGGSLSRSSQGTLVTVSSGG Alb82-GGG 148 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALTYCTIGGSLSRSSQGTLVTVSSGGG Alb92 149 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTL YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 150 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTL YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA ALB-COR1 151 SFGMS ALB-CDR2 152 STSGSGSDTLYADSVKG ALB-CDR3 153 GGSLSR Alb135 171 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVKSA

TABLE-US-00033 TABLE D Linker sequences (''ID'' refers to the SEQ ID NO as used herein) Name ID Amino acid sequence 3A linker (Poly-A) 154 AAA 5GS linker 155 GGGGS 7GS linker 156 SGGSGGS 8GS linker 157 GGGGGGGS 9GS linker 158 GGGGSGGGS 10GS linker 159 GGGGSGGGGS 15GS linker 160 GGGGSGGGGSGGGGS 18GS linker 161 GGGGSGGGGSGGGGGGGS 20GS linker 162 GGGGSGGGGSGGGGSGGGGS 25GS linker 163 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS linker 164 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS linker 165 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS linker 166 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GS G1 hinge 167 EPKSCDKTHTCPPCP 9GS-G1 hinge 168 GGGGSGGGSEPKSCDKTHTCPPCP Llama upper long hinge 169 EPKTPKPQPAAA region G3 hinge 170 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCP RCPEPKSCDTPPPCPRCP

TABLE-US-00034 TABLE E-1 Polypeptides/constructs comprising a therapeutic ISV as indicated and an ISV binding Aggrecan as indicated Target (ISV binding) CAP (ISV) Cathepsin A 604F02 Cathepsin B 604F02 Cathepsin C 604F02 Cathepsin D 604F02 Cathepsin E 604F02 Cathepsin F 604F02 Cathepsin G 604F02 Cathepsin H 604F02 Cathepsin K 604F02 Cathepsin L1 604F02 Cathepsin L2 (or V) 604F02 Cathepsin 0 604F02 Cathepsin S 604F02 Cathepsin W 604F02 Cathepsin Z (or X) 604F02 ADAMTS1 604F02 ADAMTS2 604F02 ADAMTS3 604F02 ADAMTS4 604F02 ADAMTS5 604F02 ADAMTS6 604F02 ADAMTS7 604F02 ADAMTS8 604F02 ADAMTS9 604F02 ADAMTS10 604F02 ADAMTS11 604F02 ADAMTS12 604F02 ADAMTS13 604F02 ADAMTS14 604F02 ADAMTS15 604F02 ADAMTS16 604F02 ADAMTS17 604F02 ADAMTS18 604F02 ADAMTS19 604F02 ADAMTS20 604F02 MMP1 604F02 MMP2 604F02 MMP3 604F02 MMP7 604F02 MMP8 604F02 MMP9 604F02 MMP10 604F02 MMP11 604F02 MMP12 604F02 MMP13 604F02 MMP14 604F02 MMP15 604F02 MMP16 604F02 MMP17 604F02 MMP18 604F02 MMP19 604F02 MMP20 604F02 MMP21 604F02 MMP23A 604F02 MMP23B 604F02 MMP24 604F02 MMP25 604F02 MMP26 604F02 MMP27 604F02 MMP28 604F02

TABLE-US-00035 TABLE E-2 Polypeptides/constructs comprising a therapeutic ISV as indicated and two ISVs binding Aggrecan as indicated Target (ISV binding) CAP (ISV) Cathepsin A 114F08-114F08 Cathepsin B 114F08-114F08 Cathepsin C 114F08-114F08 Cathepsin D 114F08-114F08 Cathepsin E 114F08-114F08 Cathepsin F 114F08-114F08 Cathepsin G 114F08-114F08 Cathepsin H 114F08-114F08 Cathepsin K 114F08-114F08 Cathepsin L1 114F08-114F08 Cathepsin L2 (or V) 114F08-114F08 Cathepsin 0 114F08-114F08 Cathepsin S 114F08-114F08 Cathepsin W 114F08-114F08 Cathepsin Z (or X) 114F08-114F08 ADAMTS1 114F08-114F08 ADAMTS2 114F08-114F08 ADAMTS3 114F08-114F08 ADAMTS4 114F08-114F08 ADAMTS5 114F08-114F08 ADAMTS6 114F08-114F08 ADAMTS7 114F08-114F08 ADAMTS8 114F08-114F08 ADAMTS9 114F08-114F08 ADAMTS10 114F08-114F08 ADAMTS11 114F08-114F08 ADAMTS12 114F08-114F08 ADAMTS13 114F08-114F08 ADAMTS14 114F08-114F08 ADAMTS15 114F08-114F08 ADAMTS16 114F08-114F08 ADAMTS17 114F08-114F08 ADAMTS18 114F08-114F08 ADAMTS19 114F08-114F08 ADAMTS20 114F08-114F08 MMP1 114F08-114F08 MMP2 114F08-114F08 MMP3 114F08-114F08 MMP7 114F08-114F08 MMP8 114F08-114F08 MMP9 114F08-114F08 MMP10 114F08-114F08 MMP11 114F08-114F08 MMP12 114F08-114F08 MMP13 114F08-114F08 MMP14 114F08-114F08 MMP15 114F08-114F08 MMP16 114F08-114F08 MMP17 114F08-114F08 MMP18 114F08-114F08 MMP19 114F08-114F08 MMP20 114F08-114F08 MMP21 114F08-114F08 MMP23A 114F08-114F08 MMP23B 114F08-114F08 MMP24 114F08-114F08 MMP25 114F08-114F08 MMP26 114F08-114F08 MMP27 114F08-114F08 MMP28 114F08-114F08

Sequence CWU 1

1

1721124PRTArtificial SequenceNanobody Sequence 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Ser Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ser Ile Ile Ser Trp Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Gly Arg Leu Tyr Arg Ala Thr Pro Arg Pro Ala Asp Phe Gly 100 105 110Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 1202127PRTArtificial SequenceNanobody Sequence 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ala Phe Ser Asn Tyr 20 25 30Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val 35 40 45Ala Ala Ile Asn Trp Asn Gly Val Thr Thr His Tyr Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Ser Tyr65 70 75 80Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Ala Arg Gly Thr Val Tyr Ser Arg Thr Tyr Gly Val Ser Glu Glu 100 105 110Gly Tyr Met Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 1253115PRTArtificial SequenceNanobody Sequence 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Asn Arg 20 25 30Phe Met Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ser Ile Thr Leu Ser Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Thr Phe Leu Gln Asn Ser Phe Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110Val Ser Ser 1154115PRTArtificial SequenceNanobody Sequence 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30Trp Met Phe Trp Val Arg Gln Ala Pro Gly Lys Asp Tyr Glu Trp Val 35 40 45Ala Ser Ile Asn Ser Ser Gly Gly Arg Thr Tyr Tyr Asp Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95Ala Arg Ser Pro Arg Val Gly Ser Trp Gly Gln Gly Thr Gln Val Thr 100 105 110Val Ser Ser 1155121PRTArtificial SequenceNanobody Sequence 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn 20 25 30Val Val Arg Trp Tyr Arg Arg Thr Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg 50 55 60Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala Lys Asn Ala Val Asp Leu65 70 75 80Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 1206117PRTArtificial SequenceNanobody Sequence 6Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Asn Ser Ser Gly Gly Arg Thr Asn Tyr Ala Gly Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Asp Phe Leu Gly Gly Arg Asn Ser Arg Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser 1157123PRTArtificial SequenceNanobody Sequence 7Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Arg Thr Phe Asn Met Met 20 25 30Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Tyr 35 40 45Ile Thr Trp Asn Gly Gly Asp Thr Arg Tyr Ala Glu Ser Val Lys Gly 50 55 60Arg Phe Thr Val Ser Arg Asp Asp Val Lys Asn Thr Met Ala Leu Gln65 70 75 80Met Asn Arg Leu Asp Pro Leu Asp Thr Ala Val Tyr Tyr Cys Gly Val 85 90 95Arg Ile His Gly Ser Asn Trp Ser Thr Lys Ala Asp Asp Tyr Asp Asn 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 1208121PRTArtificial SequenceNanobody Sequence 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Ala Leu Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ser Arg 20 25 30Tyr Met Arg Trp Tyr Arg Gln Ala Pro Gly Arg Gln Arg Glu Leu Val 35 40 45Ala Ala Ile Ser Ser Gly Gly Arg Thr Asp Tyr Val Asp Ser Val Arg 50 55 60Gly Arg Phe Thr Leu Ser Ile Asn Asn Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95Arg Pro Arg Met Tyr Val Asp Gly Thr Tyr Glu Lys Glu Leu Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 1209124PRTArtificial SequenceNanobody Sequence 9Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Pro Thr Phe Ser Arg Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Thr Trp Ser Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser 50 55 60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Ala Ala Arg Ile Pro Val Arg Thr Tyr Thr Ser Glu Trp Asn 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12010122PRTArtificial SequenceNanobody Sequence 10Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Arg Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly Leu Ser Tyr Tyr Ser Pro His Ala Tyr Tyr Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12011123PRTArtificial SequenceNanobody Sequence 11Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Thr Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Tyr Arg Arg Pro Arg Tyr Ser Pro Thr Gly Thr Trp Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12012119PRTArtificial SequenceNanobody Sequence 12Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Ile Tyr 20 25 30Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Ser Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ala Tyr Thr Gly Pro Arg Ser Gly Tyr Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11513124PRTArtificial SequenceNanobody Sequence 13Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Tyr Arg Arg Arg Arg Ala Ser Ser Asn Arg Gly Leu Trp Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12014123PRTArtificial SequenceNanobody Sequence 14Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Tyr Arg Arg Val Arg Tyr Thr Asn Leu Glu Val Trp Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12015123PRTArtificial SequenceNanobody Sequence 15Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Thr Trp Ser Ser Ala Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ala Arg Ile Pro Val Gly Arg Arg Ser Glu Asn Trp Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12016120PRTArtificial SequenceNanobody Sequence 16Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Ile Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ala Tyr Thr Gly Arg Ser Tyr Gly Ser Tyr Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12017123PRTArtificial SequenceNanobody Sequence 17Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Ile Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Asn Gly Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Asp Arg Ser Gly Tyr Gly Thr Ser Leu Asp Trp Trp Tyr Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12018123PRTArtificial SequenceNanobody Sequence 18Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Pro Arg Tyr Tyr Tyr Tyr Ser Leu Tyr Ser Tyr Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 12019122PRTArtificial SequenceNanobody Sequence 19Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Phe Ser Ile Asn 20 25 30Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Thr Thr Gly Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Ala Glu Val Thr Thr Gly Trp Val Gly Tyr Ser Trp Tyr Asp Tyr Trp 100 105 110Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 1202010PRTArtificial SequenceCDR1 20Gly Arg Ser Phe Ser Ser Tyr Ala Met Gly1 5 102110PRTArtificial SequenceCDR1 21Gly Arg Ala Phe Ser Asn Tyr Ile Met Gly1 5 102210PRTArtificial SequenceCDR1 22Gly Ser Ile Phe Ser Asn Arg Phe Met Tyr1 5 102310PRTArtificial SequenceCDR1 23Gly Phe Thr Phe Ser Gly Ser Trp Met Phe1 5 102410PRTArtificial SequenceCDR1

24Gly Ser Thr Phe Ile Ile Asn Val Val Arg1 5 102510PRTArtificial SequenceCDR1 25Gly Phe Thr Phe Ser Met Tyr Ala Met Lys1 5 10268PRTArtificial SequenceCDR1 26Arg Arg Thr Phe Asn Met Met Gly1 52710PRTArtificial SequenceCDR1 27Gly Ile Thr Phe Ser Ser Arg Tyr Met Arg1 5 102810PRTArtificial SequenceCDR1 28Gly Pro Thr Phe Ser Arg Tyr Ala Met Gly1 5 102910PRTArtificial SequenceCDR1 29Gly Arg Thr Phe Ser Ser Tyr Ala Val Gly1 5 103010PRTArtificial SequenceCDR1 30Gly Leu Thr Phe Ser Thr Tyr Ala Met Gly1 5 103110PRTArtificial SequenceCDR1 31Gly Arg Thr Phe Ser Ile Tyr Thr Met Ala1 5 103210PRTArtificial SequenceCDR1 32Gly Arg Thr Phe Ser Ser Tyr Thr Met Gly1 5 103310PRTArtificial SequenceCDR1 33Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly1 5 103410PRTArtificial SequenceCDR1 34Gly Arg Thr Phe Ser Ile Tyr Thr Met Gly1 5 103510PRTArtificial SequenceCDR1 35Gly Arg Thr Phe Ser Ile Tyr Gly Met Gly1 5 103610PRTArtificial SequenceCDR1 36Gly Arg Thr Phe Ser Thr Tyr Thr Met Gly1 5 103710PRTArtificial SequenceCDR1 37Gly Thr Ile Phe Ser Ile Asn Val Met Gly1 5 103810PRTArtificial SequenceCDR2 38Ile Ile Ser Trp Ser Gly Gly Ser Thr Val1 5 103910PRTArtificial SequenceCDR2 39Ala Ile Asn Trp Asn Gly Val Thr Thr His1 5 10409PRTArtificial SequenceCDR2 40Ser Ile Thr Leu Ser Gly Ser Thr Asn1 54110PRTArtificial SequenceCDR2 41Ser Ile Asn Ser Ser Gly Gly Arg Thr Tyr1 5 10429PRTArtificial SequenceCDR2 42Thr Ile Ser Ser Gly Gly Asn Ala Asn1 54310PRTArtificial SequenceCDR2 43Gly Ile Asn Ser Ser Gly Gly Arg Thr Asn1 5 104410PRTArtificial SequenceCDR2 44Tyr Ile Thr Trp Asn Gly Gly Asp Thr Arg1 5 10459PRTArtificial SequenceCDR2 45Ala Ile Ser Ser Gly Gly Arg Thr Asp1 54611PRTArtificial SequenceCDR2 46Ala Ile Thr Trp Ser Ser Gly Gly Arg Thr Tyr1 5 104710PRTArtificial SequenceCDR2 47Ala Ile Ser Arg Ser Gly Arg Ser Thr Tyr1 5 104810PRTArtificial SequenceCDR2 48Ala Ile Ser Trp Ser Gly Ser Arg Thr Tyr1 5 104910PRTArtificial SequenceCDR2 49Ala Ile Ser Trp Ser Ser Gly Arg Thr Tyr1 5 105010PRTArtificial SequenceCDR2 50Ala Ile Ser Trp Ser Gly Gly Arg Thr Tyr1 5 105110PRTArtificial SequenceCDR2 51Ala Ile Ser Trp Ser Gly Arg Thr Thr Tyr1 5 105210PRTArtificial SequenceCDR2 52Ala Ile Thr Trp Ser Ser Ala Thr Thr Tyr1 5 10539PRTArtificial SequenceCDR2 53Ala Ile Asn Gly Gly Ser Arg Thr Tyr1 55410PRTArtificial SequenceCDR2 54Ala Ile Ser Trp Ser Gly Gly Thr Thr Tyr1 5 10559PRTArtificial SequenceCDR2 55Ala Ile Thr Thr Gly Gly Arg Thr Asn1 55615PRTArtificial SequenceCDR3 56Gly Arg Leu Tyr Arg Ala Thr Pro Arg Pro Ala Asp Phe Gly Ser1 5 10 155718PRTArtificial SequenceCDR3 57Arg Gly Thr Val Tyr Ser Arg Thr Tyr Gly Val Ser Glu Glu Gly Tyr1 5 10 15Met Tyr587PRTArtificial SequenceCDR3 58Phe Leu Gln Asn Ser Phe Tyr1 5596PRTArtificial SequenceCDR3 59Ser Pro Arg Val Gly Ser1 56013PRTArtificial SequenceCDR3 60Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr1 5 10618PRTArtificial SequenceCDR3 61Asp Phe Leu Gly Gly Arg Asn Ser1 56216PRTArtificial SequenceCDR3 62Arg Ile His Gly Ser Asn Trp Ser Thr Lys Ala Asp Asp Tyr Asp Asn1 5 10 156313PRTArtificial SequenceCDR3 63Pro Arg Met Tyr Val Asp Gly Thr Tyr Glu Lys Glu Leu1 5 106414PRTArtificial SequenceCDR3 64Ala Arg Ile Pro Val Arg Thr Tyr Thr Ser Glu Trp Asn Tyr1 5 106513PRTArtificial SequenceCDR3 65Gly Leu Ser Tyr Tyr Ser Pro His Ala Tyr Tyr Asp Tyr1 5 106614PRTArtificial SequenceCDR3 66Tyr Arg Arg Pro Arg Tyr Ser Pro Thr Gly Thr Trp Asp Tyr1 5 106710PRTArtificial SequenceCDR3 67Tyr Thr Gly Pro Arg Ser Gly Tyr Asp Tyr1 5 106815PRTArtificial SequenceCDR3 68Tyr Arg Arg Arg Arg Ala Ser Ser Asn Arg Gly Leu Trp Asp Tyr1 5 10 156914PRTArtificial SequenceCDR3 69Tyr Arg Arg Val Arg Tyr Thr Asn Leu Glu Val Trp Asp Tyr1 5 107014PRTArtificial SequenceCDR3 70Ala Arg Ile Pro Val Gly Arg Arg Ser Glu Asn Trp Asp Tyr1 5 107111PRTArtificial SequenceCDR3 71Tyr Thr Gly Arg Ser Tyr Gly Ser Tyr Asp Tyr1 5 107215PRTArtificial SequenceCDR3 72Asp Arg Ser Gly Tyr Gly Thr Ser Leu Asp Trp Trp Tyr Asp Tyr1 5 10 157314PRTArtificial SequenceCDR3 73Arg Pro Arg Tyr Tyr Tyr Tyr Ser Leu Tyr Ser Tyr Asp Tyr1 5 107414PRTArtificial SequenceCDR3 74Glu Val Thr Thr Gly Trp Val Gly Tyr Ser Trp Tyr Asp Tyr1 5 107525PRTArtificial SequenceFR1 75Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 257625PRTArtificial SequenceFR1 76Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser 20 257725PRTArtificial SequenceFR1 77Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 257825PRTArtificial SequenceFR1 78Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser 20 257925PRTArtificial SequenceFR1 79Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 258025PRTArtificial SequenceFR1 80Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 258125PRTArtificial SequenceFR1 81Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Ala Leu Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 258225PRTArtificial SequenceFR1 82Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 258325PRTArtificial SequenceFR1 83Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 258425PRTArtificial SequenceFR1 84Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser 20 258514PRTArtificial SequenceFR2 85Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser1 5 108614PRTArtificial SequenceFR2 86Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val Ala1 5 108714PRTArtificial SequenceFR2 87Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala1 5 108814PRTArtificial SequenceFR2 88Trp Val Arg Gln Ala Pro Gly Lys Asp Tyr Glu Trp Val Ala1 5 108914PRTArtificial SequenceFR2 89Trp Tyr Arg Arg Thr Pro Gly Lys Gln Arg Glu Leu Val Ala1 5 109014PRTArtificial SequenceFR2 90Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser1 5 109114PRTArtificial SequenceFR2 91Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala1 5 109214PRTArtificial SequenceFR2 92Trp Tyr Arg Gln Ala Pro Gly Arg Gln Arg Glu Leu Val Ala1 5 109314PRTArtificial SequenceFR2 93Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala1 5 109439PRTArtificial SequenceFR3 94Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Ile Tyr Tyr Cys Ala Ala 359539PRTArtificial SequenceFR3 95Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala1 5 10 15Lys Ser Thr Ser Tyr Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr 20 25 30Ala Val Tyr Phe Cys Ala Ala 359639PRTArtificial SequenceFR3 96Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Asn Thr 359739PRTArtificial SequenceFR3 97Tyr Asp Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala1 5 10 15Lys Asn Thr Leu Tyr Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr 20 25 30Ala Leu Tyr Phe Cys Ala Arg 359839PRTArtificial SequenceFR3 98Tyr Val Asp Ser Val Arg Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala1 5 10 15Lys Asn Ala Val Asp Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Asn Val 359939PRTArtificial SequenceFR3 99Tyr Ala Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala1 5 10 15Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Ala Thr 3510039PRTArtificial SequenceFR3 100Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Val1 5 10 15Lys Asn Thr Met Ala Leu Gln Met Asn Arg Leu Asp Pro Leu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Gly Val 3510139PRTArtificial SequenceFR3 101Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Leu Ser Ile Asn Asn Ala1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Tyr Arg 3510239PRTArtificial SequenceFR3 102Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Ala Ala 3510339PRTArtificial SequenceFR3 103Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Thr Ala 3510439PRTArtificial SequenceFR3 104Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Asn Ala 3510511PRTArtificial SequenceFR4 105Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser1 5 1010611PRTArtificial SequenceFR4 106Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser1 5 1010711PRTArtificial SequenceFR4 107Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 1010811PRTArtificial SequenceFR4 108Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser1 5 1010910PRTArtificial SequenceCDR1 109Gly Ser Thr Phe Ile Ile Ser Val Met Arg1 5 101109PRTArtificial SequenceCDR2 110Ala Ile Arg Thr Gly Gly Asn Thr Asp1 511113PRTArtificial SequenceCDR3 111Pro Thr Thr Arg Tyr Gly Gly Asp Tyr Tyr Gly Pro Tyr1 5 1011239PRTArtificial SequenceFR3 112Tyr Ala Gly Pro Val Arg Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala1 5 10 15Lys Asn Ala Val Asp Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Asn Val 3511339PRTArtificial SequenceFR3 113Tyr Ala Gly Pro Val Arg Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala1 5 10 15Lys Asp Ala Val Asp Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Asn Val 35114121PRTArtificial SequenceNanobody Sequence 114Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Ser 20 25 30Val Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ala Ile Arg Thr Gly Gly Asn Thr Asp Tyr Ala Gly Pro Val Arg 50 55 60Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala Lys Asn Ala Val Asp Leu65 70 75 80Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Val Pro Thr Thr Arg Tyr Gly Gly Asp Tyr Tyr Gly Pro Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 120115121PRTArtificial SequenceNanobody Sequence 115Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Ser 20 25 30Val Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ala Ile Arg Thr Gly Gly Asn Thr Asp Tyr Ala Gly Pro Val Arg 50 55 60Gly Arg Phe Ser Ile Ser Arg Asp Gly Ala Lys Asp Ala Val Asp Leu65 70 75 80Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Val Pro Thr Thr Arg Tyr Gly Gly Asp Tyr Tyr Gly Pro Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 120116121PRTArtificial SequenceNanobody Sequence 116Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn 20 25 30Val Val Arg Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120117122PRTArtificial SequenceNanobody Sequence 117Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ile Ile Asn 20 25 30Val Val Arg Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Thr Ile Ser Ser Gly Gly Asn Ala Asn Tyr Val Asp Ser Val Arg 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn 85 90 95Val Pro Thr Thr His Tyr Gly Gly Val Tyr Tyr Gly Pro Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120118125PRTArtificial SequenceNanobody Sequence 118Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly

Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Ala Tyr Arg Arg Arg Arg Ala Ser Ser Asn Arg Gly Leu Trp Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 12511925PRTArtificial SequenceFR1 119Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 2512025PRTArtificial SequenceFR1 120Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 2512114PRTArtificial SequenceFR2 121Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala1 5 1012239PRTArtificial SequenceFR3 122Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr Cys Asn Val 3512339PRTArtificial SequenceFR3 123Tyr Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Leu Tyr Tyr Cys Asn Val 3512439PRTArtificial SequenceFR3 124Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Leu Tyr Tyr Cys Ala Ala 351252415PRTHomo sapiens 125Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Thr Ala1 5 10 15Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser 20 25 30Ile Pro Gln Pro Ser Pro Leu Arg Val Leu Leu Gly Thr Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys65 70 75 80Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val 85 90 95Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Val Gln Ser Leu Arg Ser Asn Asp Ser 115 120 125Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala 130 135 140Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 260 265 270Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly His Val Tyr Leu 275 280 285Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315 320Asn Leu Leu Gly Val Arg Thr Val Tyr Val His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu 355 360 365Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu 370 375 380Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr385 390 395 400Val Lys Pro Ile Phe Glu Val Ser Pro Ser Pro Leu Glu Pro Glu Glu 405 410 415Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Ala Glu Val 420 425 430Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro 435 440 445Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln 450 455 460Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe465 470 475 480His Tyr Arg Pro Gly Pro Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala 485 490 495Gln Gln Ala Cys Pro Gly Thr Gly Ala Val Ile Ala Ser Pro Glu Gln 500 505 510Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp 515 520 525Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro 530 535 540Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val545 550 555 560Arg Pro Ser Thr Glu Thr Tyr Asp Val Tyr Cys Phe Val Asp Arg Leu 565 570 575Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln 580 585 590Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Ala Thr Thr Gly 595 600 605Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala Gly 610 615 620Trp Leu Ala Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr Pro Arg Pro625 630 635 640Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro 645 650 655Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe Cys 660 665 670Phe Arg Gly Ile Ser Ala Val Pro Ser Pro Gly Glu Glu Glu Gly Gly 675 680 685Thr Pro Thr Ser Pro Ser Gly Val Glu Glu Trp Ile Val Thr Gln Val 690 695 700Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala Val705 710 715 720Pro Ser Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro Glu 725 730 735Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Val Gly Thr Ser Pro 740 745 750Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Ala Glu Thr Glu 755 760 765Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Pro Ser Ala Ser Glu 770 775 780Glu Pro Ser Pro Ser Glu Val Pro Phe Pro Ser Glu Glu Pro Ser Pro785 790 795 800Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu Leu 805 810 815Phe Pro Ser Glu Glu Pro Phe Pro Ser Lys Glu Pro Ser Pro Ser Glu 820 825 830Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Glu Pro 835 840 845Ser Trp Thr Glu Leu Pro Ser Ser Gly Glu Glu Ser Gly Ala Pro Asp 850 855 860Val Ser Gly Asp Phe Thr Gly Ser Gly Asp Val Ser Gly His Leu Asp865 870 875 880Phe Ser Gly Gln Leu Ser Gly Asp Arg Ala Ser Gly Leu Pro Ser Gly 885 890 895Asp Leu Asp Ser Ser Gly Leu Thr Ser Thr Val Gly Ser Gly Leu Thr 900 905 910Val Glu Ser Gly Leu Pro Ser Gly Asp Glu Glu Arg Ile Glu Trp Pro 915 920 925Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu Glu 930 935 940Gly Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu945 950 955 960Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro 965 970 975Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser 980 985 990Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu 995 1000 1005Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala 1010 1015 1020Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu 1025 1030 1035Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser 1040 1045 1050Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser 1055 1060 1065Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val 1070 1075 1080Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala 1085 1090 1095Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val 1100 1105 1110Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro 1115 1120 1125Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile 1130 1135 1140Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly 1145 1150 1155Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr 1160 1165 1170Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu 1175 1180 1185Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu 1190 1195 1200Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp 1205 1210 1215Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro 1220 1225 1230Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu 1235 1240 1245Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly 1250 1255 1260Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly 1265 1270 1275Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu 1280 1285 1290Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala 1295 1300 1305Pro Gly Val Asp Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu 1310 1315 1320Glu Thr Thr Ala Pro Gly Val Glu Glu Ile Ser Gly Leu Pro Ser 1325 1330 1335Gly Glu Val Leu Glu Thr Ser Thr Ser Ala Val Gly Asp Leu Ser 1340 1345 1350Gly Leu Pro Ser Gly Gly Glu Val Leu Glu Ile Ser Val Ser Gly 1355 1360 1365Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Val Glu Thr 1370 1375 1380Ser Ala Ser Gly Ile Glu Asp Val Ser Glu Leu Pro Ser Gly Glu 1385 1390 1395Gly Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Leu Ser Arg Leu 1400 1405 1410Pro Ser Gly Glu Glu Val Leu Glu Ile Ser Ala Ser Gly Phe Gly 1415 1420 1425Asp Leu Ser Gly Val Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser 1430 1435 1440Ala Ser Glu Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Arg 1445 1450 1455Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser Gly 1460 1465 1470Leu Pro Ser Gly Lys Glu Asp Leu Val Gly Ser Ala Ser Gly Asp 1475 1480 1485Leu Asp Leu Gly Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln 1490 1495 1500Ala Pro Glu Thr Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr 1505 1510 1515Ser Gly Val Asp Leu Gly Ser Gly Pro Pro Ser Gly Leu Pro Asp 1520 1525 1530Phe Ser Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp 1535 1540 1545Ser Thr Leu Val Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu 1550 1555 1560Glu Gly Arg Gly Thr Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser 1565 1570 1575Gly Leu Pro Ser Ser Glu Leu Asp Ile Ser Gly Arg Ala Ser Gly 1580 1585 1590Leu Pro Ser Gly Thr Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro 1595 1600 1605Asp Val Ser Gly Glu Ile Pro Gly Leu Phe Gly Val Ser Gly Gln 1610 1615 1620Pro Ser Gly Phe Pro Asp Thr Ser Gly Glu Thr Ser Gly Val Thr 1625 1630 1635Glu Leu Ser Gly Leu Ser Ser Gly Gln Pro Gly Val Ser Gly Glu 1640 1645 1650Ala Ser Gly Val Leu Tyr Gly Thr Ser Gln Pro Phe Gly Ile Thr 1655 1660 1665Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp Leu Ser Gly Gln 1670 1675 1680Pro Ser Gly Leu Pro Gly Phe Ser Gly Ala Thr Ser Gly Val Pro 1685 1690 1695Asp Leu Val Ser Gly Thr Thr Ser Gly Ser Gly Glu Ser Ser Gly 1700 1705 1710Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Ala Pro Thr Thr 1715 1720 1725Phe Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu 1730 1735 1740Pro Ser Gly Glu Ala Asp Leu Ser Gly Lys Ser Gly Met Val Asp 1745 1750 1755Val Ser Gly Gln Phe Ser Gly Thr Val Asp Ser Ser Gly Phe Thr 1760 1765 1770Ser Gln Thr Pro Glu Phe Ser Gly Leu Pro Ser Gly Ile Ala Glu 1775 1780 1785Val Ser Gly Glu Ser Ser Arg Ala Glu Ile Gly Ser Ser Leu Pro 1790 1795 1800Ser Gly Ala Tyr Tyr Gly Ser Gly Thr Pro Ser Ser Phe Pro Thr 1805 1810 1815Val Ser Leu Val Asp Arg Thr Leu Val Glu Ser Val Thr Gln Ala 1820 1825 1830Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro Ser Gly Ile Leu Glu 1835 1840 1845Leu Ser Gly Ala His Ser Gly Ala Pro Asp Met Ser Gly Glu His 1850 1855 1860Ser Gly Phe Leu Asp Leu Ser Gly Leu Gln Ser Gly Leu Ile Glu 1865 1870 1875Pro Ser Gly Glu Pro Pro Gly Thr Pro Tyr Phe Ser Gly Asp Phe 1880 1885 1890Ala Ser Thr Thr Asn Val Ser Gly Glu Ser Ser Val Ala Met Gly 1895 1900 1905Thr Ser Gly Glu Ala Ser Gly Leu Pro Glu Val Thr Leu Ile Thr 1910 1915 1920Ser Glu Phe Val Glu Gly Val Thr Glu Pro Thr Ile Ser Gln Glu 1925 1930 1935Leu Gly Gln Arg Pro Pro Val Thr His Thr Pro Gln Leu Phe Glu 1940 1945 1950Ser Ser Gly Lys Val Ser Thr Ala Gly Asp Ile Ser Gly Ala Thr 1955 1960 1965Pro Val Leu Pro Gly Ser Gly Val Glu Val Ser Ser Val Pro Glu 1970 1975 1980Ser Ser Ser Glu Thr Ser Ala Tyr Pro Glu Ala Gly Phe Gly Ala 1985 1990 1995Ser Ala Ala Pro Glu Ala Ser Arg Glu Asp Ser Gly Ser Pro Asp 2000 2005 2010Leu Ser Glu Thr Thr Ser Ala Phe His Glu Ala Asn Leu Glu Arg 2015 2020 2025Ser Ser Gly Leu Gly Val Ser Gly Ser Thr Leu Thr Phe Gln Glu 2030 2035 2040Gly Glu Ala Ser Ala Ala Pro Glu Val Ser Gly Glu Ser Thr Thr 2045 2050 2055Thr Ser Asp Val Gly Thr Glu Ala Pro Gly Leu Pro Ser Ala Thr 2060 2065 2070Pro Thr Ala Ser Gly Asp Arg Thr Glu Ile Ser Gly Asp Leu Ser 2075 2080 2085Gly His Thr Ser Gln Leu Gly Val Val Ile Ser Thr Ser Ile Pro 2090 2095 2100Glu Ser Glu Trp Thr Gln Gln Thr Gln Arg Pro Ala Glu Thr His 2105 2110 2115Leu Glu Ile Glu Ser Ser Ser Leu Leu Tyr Ser Gly Glu Glu Thr 2120 2125 2130His Thr Val Glu Thr Ala Thr Ser Pro Thr Asp Ala Ser Ile Pro 2135 2140 2145Ala Ser Pro Glu Trp Lys Arg Glu Ser Glu Ser Thr Ala Ala Ala 2150 2155 2160Pro Ala Arg Ser Cys Ala Glu Glu Pro Cys Gly Ala Gly Thr Cys

2165 2170 2175Lys Glu Thr Glu Gly His Val Ile Cys Leu Cys Pro Pro Gly Tyr 2180 2185 2190Thr Gly Glu His Cys Asn Ile Asp Gln Glu Val Cys Glu Glu Gly 2195 2200 2205Trp Asn Lys Tyr Gln Gly His Cys Tyr Arg His Phe Pro Asp Arg 2210 2215 2220Glu Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln Gln Ser 2225 2230 2235His Leu Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe Val Asn 2240 2245 2250Asn Asn Ala Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp Arg Thr 2255 2260 2265Ile Glu Gly Asp Phe Arg Trp Ser Asp Gly His Pro Met Gln Phe 2270 2275 2280Glu Asn Trp Arg Pro Asn Gln Pro Asp Asn Phe Phe Ala Ala Gly 2285 2290 2295Glu Asp Cys Val Val Met Ile Trp His Glu Lys Gly Glu Trp Asn 2300 2305 2310Asp Val Pro Cys Asn Tyr His Leu Pro Phe Thr Cys Lys Lys Gly 2315 2320 2325Thr Val Ala Cys Gly Glu Pro Pro Val Val Glu His Ala Arg Thr 2330 2335 2340Phe Gly Gln Lys Lys Asp Arg Tyr Glu Ile Asn Ser Leu Val Arg 2345 2350 2355Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg His Met Pro Thr Ile 2360 2365 2370Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro Arg Ile Thr Cys 2375 2380 2385Thr Asp Ala Thr Thr Tyr Lys Arg Arg Leu Gln Lys Arg Ser Ser 2390 2395 2400Arg His Pro Arg Arg Ser Arg Pro Ser Thr Ala His 2405 2410 24151262333PRTCanis lupus 126Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Thr Ala1 5 10 15Ala Ser Ser Glu Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser 20 25 30Ile Pro Glu Pro Ser Pro Met Arg Val Leu Leu Gly Ser Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Ile Thr Lys65 70 75 80Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gln Val Arg Ile 85 90 95Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser 115 120 125Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala 130 135 140Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Leu 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 260 265 270Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 275 280 285Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315 320Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu 355 360 365Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu 370 375 380Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Asn Val Ile Leu Thr385 390 395 400Val Lys Pro Ile Phe Asp Leu Ser Pro Thr Ala Pro Glu Pro Glu Glu 405 410 415Pro Phe Thr Phe Val Pro Glu Pro Glu Lys Pro Phe Thr Phe Ala Thr 420 425 430Asp Val Gly Val Thr Ala Phe Pro Glu Ala Glu Asn Arg Thr Gly Glu 435 440 445Ala Thr Arg Pro Trp Gly Val Pro Glu Glu Ser Thr Pro Gly Pro Ala 450 455 460Phe Thr Ala Phe Thr Ser Glu Asp His Val Val Gln Val Thr Ala Val465 470 475 480Pro Gly Ala Ala Glu Val Pro Gly Gln Pro Arg Leu Pro Gly Gly Val 485 490 495Val Phe His Tyr Arg Pro Gly Ser Ala Arg Tyr Ser Leu Thr Phe Glu 500 505 510Glu Ala Gln Gln Ala Cys Leu Arg Thr Gly Ala Val Ile Ala Ser Pro 515 520 525Glu Gln Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala 530 535 540Gly Trp Leu Gln Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg545 550 555 560Thr Pro Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr 565 570 575Gly Val Arg Pro Pro Ser Glu Thr Tyr Asp Val Tyr Cys Tyr Val Asp 580 585 590Lys Leu Glu Gly Glu Val Phe Phe Ile Thr Arg Leu Glu Gln Phe Thr 595 600 605Phe Gln Glu Ala Leu Ala Phe Cys Glu Ser His Asn Ala Thr Leu Ala 610 615 620Ser Thr Gly Gln Leu Tyr Ala Ala Trp Arg Gln Gly Leu Asp Lys Cys625 630 635 640Tyr Ala Gly Trp Leu Ser Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr 645 650 655Pro Arg Pro Ser Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr 660 665 670Leu Tyr Pro Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His 675 680 685Val Phe Cys Phe Arg Gly Val Ser Gly Val Pro Ser Pro Gly Glu Glu 690 695 700Glu Gly Gly Thr Pro Thr Pro Ser Val Val Glu Asp Trp Ile Pro Thr705 710 715 720Gln Val Gly Pro Val Val Pro Ser Val Pro Met Gly Glu Glu Thr Thr 725 730 735Ala Ile Leu Asp Phe Thr Ile Glu Pro Glu Asn Gln Thr Glu Trp Glu 740 745 750Pro Ala Tyr Ser Pro Ala Gly Thr Ser Pro Leu Pro Gly Ile Pro Pro 755 760 765Thr Trp Pro Pro Thr Ser Thr Ala Thr Glu Glu Ser Thr Glu Gly Pro 770 775 780Ser Gly Thr Glu Val Pro Ser Val Ser Glu Glu Pro Ser Pro Ser Glu785 790 795 800Glu Pro Phe Pro Trp Glu Glu Leu Ser Thr Leu Ser Pro Pro Gly Pro 805 810 815Ser Gly Thr Glu Leu Pro Gly Ser Gly Glu Ala Ser Gly Val Pro Glu 820 825 830Val Ser Gly Asp Phe Thr Gly Ser Gly Glu Val Ser Gly His Pro Asp 835 840 845Ser Ser Gly Gln Leu Ser Gly Glu Ser Ala Ser Gly Leu Pro Ser Glu 850 855 860Asp Leu Asp Ser Ser Gly Leu Thr Ser Ala Val Gly Ser Gly Leu Ala865 870 875 880Ser Gly Asp Glu Asp Arg Ile Thr Leu Ser Ser Ile Pro Lys Val Glu 885 890 895Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Leu Ser Gly 900 905 910Leu Pro Ser Gly Arg Glu Gly Leu Glu Thr Ser Thr Ser Gly Val Gly 915 920 925Asp Leu Ser Gly Leu Pro Ser Gly Glu Gly Leu Glu Val Ser Ala Ser 930 935 940Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Gly Pro Glu Thr945 950 955 960Ser Thr Ser Gly Val Gly Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly 965 970 975Pro Glu Val Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser 980 985 990Gly Arg Glu Gly Leu Glu Thr Ser Thr Ser Gly Val Glu Asp Leu Ser 995 1000 1005Gly Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Thr Ser Gly Val 1010 1015 1020Gly Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val Ser 1025 1030 1035Ala Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Gly 1040 1045 1050Leu Glu Ala Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro 1055 1060 1065Ser Gly Glu Gly Pro Glu Ala Ser Ala Ser Gly Val Gly Asp Leu 1070 1075 1080Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser Gly 1085 1090 1095Val Glu Asp Leu Ser Gly Leu Ser Ser Gly Glu Ser Pro Glu Ala 1100 1105 1110Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Arg 1115 1120 1125Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly 1130 1135 1140Leu Pro Ser Gly Glu Gly Gln Glu Ala Ser Ala Ser Gly Val Glu 1145 1150 1155Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Ala 1160 1165 1170Ser Gly Val Gly Glu Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly 1175 1180 1185Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro 1190 1195 1200Ser Gly Glu Gly Pro Glu Ala Phe Ala Ser Gly Val Glu Asp Leu 1205 1210 1215Ser Ile Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Ala Ser Gly 1220 1225 1230Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu 1235 1240 1245Thr Ser Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly 1250 1255 1260Arg Glu Gly Leu Glu Thr Ser Thr Ser Gly Val Gly Asp Leu Ser 1265 1270 1275Gly Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Ala Ser Gly Ile 1280 1285 1290Gly Asp Ile Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu Thr 1295 1300 1305Ser Ser Ser Gly Val Glu Asp His Pro Glu Thr Ser Ala Ser Gly 1310 1315 1320Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Val Glu Gly His Pro 1325 1330 1335Glu Thr Ser Ala Ser Gly Val Glu Asp Leu Ser Asp Leu Ser Ser 1340 1345 1350Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu 1355 1360 1365Ser Gly Phe Pro Ser Gly Lys Glu Asp Leu Ile Gly Ser Ala Ser 1370 1375 1380Gly Ala Leu Asp Phe Gly Arg Ile Pro Ser Gly Thr Leu Gly Ser 1385 1390 1395Gly Gln Ala Pro Glu Ala Ser Ser Leu Pro Ser Gly Phe Ser Gly 1400 1405 1410Glu Tyr Ser Gly Val Asp Phe Gly Ser Gly Pro Ile Ser Gly Leu 1415 1420 1425Pro Asp Phe Ser Gly Leu Pro Ser Gly Phe Pro Thr Ile Ser Leu 1430 1435 1440Val Asp Thr Thr Leu Val Glu Val Ile Thr Thr Thr Ser Ala Ser 1445 1450 1455Glu Leu Glu Gly Arg Gly Thr Ile Gly Ile Ser Gly Ala Gly Glu 1460 1465 1470Thr Ser Gly Leu Pro Val Ser Glu Leu Asp Ile Ser Gly Ala Val 1475 1480 1485Ser Gly Leu Pro Ser Gly Ala Glu Leu Ser Gly Gln Ala Ser Gly 1490 1495 1500Ser Pro Asp Met Ser Gly Glu Thr Ser Gly Phe Phe Gly Val Ser 1505 1510 1515Gly Gln Pro Ser Gly Phe Pro Asp Ile Ser Gly Gly Thr Ser Gly 1520 1525 1530Leu Phe Glu Val Ser Gly Gln Pro Ser Gly Phe Ser Gly Glu Thr 1535 1540 1545Ser Gly Val Thr Glu Leu Ser Gly Leu Tyr Ser Gly Gln Pro Asp 1550 1555 1560Val Ser Gly Glu Ala Ser Gly Val Pro Ser Gly Ser Gly Gln Pro 1565 1570 1575Phe Gly Met Thr Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp 1580 1585 1590Ile Ser Gly Gln Pro Ser Gly Leu Pro Glu Phe Ser Gly Thr Thr 1595 1600 1605Ser Gly Ile Pro Asp Leu Val Ser Ser Thr Met Ser Gly Ser Gly 1610 1615 1620Glu Ser Ser Gly Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val 1625 1630 1635Thr Pro Thr Thr Phe Lys Glu Lys Lys Arg Leu Gly Ser Val Glu 1640 1645 1650Leu Ser Gly Leu Pro Ser Gly Glu Val Asp Leu Ser Gly Ala Ser 1655 1660 1665Gly Thr Met Asp Ile Ser Gly Gln Ser Ser Gly Ala Thr Asp Ser 1670 1675 1680Ser Gly Leu Thr Ser His Leu Pro Lys Phe Ser Gly Leu Pro Ser 1685 1690 1695Gly Ala Ala Glu Val Ser Gly Glu Ser Ser Gly Ala Glu Val Gly 1700 1705 1710Ser Ser Leu Pro Ser Gly Thr Tyr Glu Gly Ser Gly Asn Phe His 1715 1720 1725Pro Ala Phe Pro Thr Val Phe Leu Val Asp Arg Thr Leu Val Glu 1730 1735 1740Ser Val Thr Gln Ala Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro 1745 1750 1755Ser Gly Ile Leu Glu Leu Ser Gly Ala His Ser Gly Ala Pro Asp 1760 1765 1770Val Ser Gly Asp His Ser Gly Ser Leu Asp Leu Ser Gly Met Gln 1775 1780 1785Ser Gly Leu Val Glu Pro Ser Gly Glu Pro Ser Ser Thr Pro Tyr 1790 1795 1800Phe Ser Gly Asp Phe Ser Gly Thr Met Asp Val Thr Gly Glu Pro 1805 1810 1815Ser Thr Ala Met Ser Ala Ser Gly Glu Ala Ser Gly Leu Leu Glu 1820 1825 1830Val Thr Leu Ile Thr Ser Glu Phe Val Glu Gly Val Thr Glu Pro 1835 1840 1845Thr Val Ser Gln Glu Leu Ala Gln Arg Pro Pro Val Thr His Thr 1850 1855 1860Pro Gln Leu Phe Glu Ser Ser Gly Glu Ala Ser Ala Ser Gly Glu 1865 1870 1875Ile Ser Gly Ala Thr Pro Ala Phe Pro Gly Ser Gly Leu Glu Ala 1880 1885 1890Ser Ser Val Pro Glu Ser Ser Ser Glu Thr Ser Asp Phe Pro Glu 1895 1900 1905Arg Ala Val Gly Val Ser Ala Ala Pro Glu Ala Ser Gly Gly Ala 1910 1915 1920Ser Gly Ala Pro Asp Val Ser Glu Ala Thr Ser Thr Phe Pro Glu 1925 1930 1935Ala Asp Val Glu Gly Ala Ser Gly Leu Gly Val Ser Gly Gly Thr 1940 1945 1950Ser Ala Phe Pro Glu Ala Pro Arg Glu Gly Ser Ala Thr Pro Glu 1955 1960 1965Val Gln Glu Glu Pro Thr Thr Ser Tyr Asp Val Gly Arg Glu Ala 1970 1975 1980Leu Gly Trp Pro Ser Ala Thr Pro Thr Ala Ser Gly Asp Arg Ile 1985 1990 1995Glu Val Ser Gly Asp Leu Ser Gly His Thr Ser Gly Leu Asp Val 2000 2005 2010Val Ile Ser Thr Ser Val Pro Glu Ser Glu Trp Ile Gln Gln Thr 2015 2020 2025Gln Arg Pro Ala Glu Ala His Leu Glu Ile Glu Ala Ser Ser Pro 2030 2035 2040Leu His Ser Gly Glu Glu Thr Gln Thr Ala Glu Thr Ala Thr Ser 2045 2050 2055Pro Thr Asp Asp Ala Ser Ile Pro Thr Ser Pro Ser Gly Thr Asp 2060 2065 2070Glu Ser Ala Pro Ala Ile Pro Asp Ile Asp Glu Cys Leu Ser Ser 2075 2080 2085Pro Cys Leu Asn Gly Ala Thr Cys Val Asp Ala Ile Asp Ser Phe 2090 2095 2100Thr Cys Leu Cys Leu Pro Ser Tyr Arg Gly Asp Leu Cys Glu Ile 2105 2110 2115Asp Gln Glu Leu Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly His 2120 2125 2130Cys Tyr Arg Tyr Phe Pro Asp Arg Glu Ser Trp Val Asp Ala Glu 2135 2140 2145Ser Arg Cys Arg Ala Gln Gln Ser His Leu Ser Ser Ile Val Thr 2150 2155 2160Pro Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr Gln 2165 2170 2175Trp Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg Trp 2180 2185 2190Ser Asp Gly His Ser Leu Gln Phe Glu Asn Trp Arg Pro Asn Gln 2195

2200 2205Pro Asp Asn Phe Phe Val Ser Gly Glu Asp Cys Val Val Met Ile 2210 2215 2220Trp His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr Tyr 2225 2230 2235Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Asp Pro 2240 2245 2250Pro Val Val Glu His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg 2255 2260 2265Tyr Glu Ile Asn Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly Phe 2270 2275 2280Val Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Gly His 2285 2290 2295Trp Glu Lys Pro Arg Ile Thr Cys Thr Asp Pro Ser Thr Tyr Lys 2300 2305 2310Arg Arg Leu Gln Lys Arg Ser Ser Arg Ala Pro Arg Arg Ser Arg 2315 2320 2325Pro Ser Thr Ala His 23301272364PRTBos taurus 127Met Thr Thr Leu Leu Leu Val Phe Val Thr Leu Arg Val Ile Thr Ala1 5 10 15Ala Ile Ser Val Glu Val Ser Glu Pro Asp Asn Ser Leu Ser Val Ser 20 25 30Ile Pro Glu Pro Ser Pro Leu Arg Val Leu Leu Gly Ser Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Ile Ser Lys65 70 75 80Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val 85 90 95Asn Ser Ala Tyr Gln Asp Lys Val Thr Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Met Arg Ser Asn Asp Ser 115 120 125Gly Ile Leu Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Gln Ala 130 135 140Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 260 265 270Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 275 280 285Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315 320Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Val Asp Ile Pro Glu Ser Phe Phe Gly Val Gly Gly Glu Glu 355 360 365Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu 370 375 380Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr385 390 395 400Ala Lys Pro Asp Phe Glu Val Ser Pro Thr Ala Pro Glu Pro Glu Glu 405 410 415Pro Phe Thr Phe Val Pro Glu Val Arg Ala Thr Ala Phe Pro Glu Val 420 425 430Glu Asn Arg Thr Glu Glu Ala Thr Arg Pro Trp Ala Phe Pro Arg Glu 435 440 445Ser Thr Pro Gly Leu Gly Ala Pro Thr Ala Phe Thr Ser Glu Asp Leu 450 455 460Val Val Gln Val Thr Leu Ala Pro Gly Ala Ala Glu Val Pro Gly Gln465 470 475 480Pro Arg Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Ser Ser 485 490 495Arg Tyr Ser Leu Thr Phe Glu Glu Ala Lys Gln Ala Cys Leu Arg Thr 500 505 510Gly Ala Ile Ile Ala Ser Pro Glu Gln Leu Gln Ala Ala Tyr Glu Ala 515 520 525Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg 530 535 540Tyr Pro Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser545 550 555 560Ser Pro Gly Val Arg Thr Tyr Gly Val Arg Pro Pro Ser Glu Thr Tyr 565 570 575Asp Val Tyr Cys Tyr Val Asp Arg Leu Glu Gly Glu Val Phe Phe Ala 580 585 590Thr Arg Leu Glu Gln Phe Thr Phe Trp Glu Ala Gln Glu Phe Cys Glu 595 600 605Ser Gln Asn Ala Thr Leu Ala Thr Thr Gly Gln Leu Tyr Ala Ala Trp 610 615 620Ser Arg Gly Leu Asp Lys Cys Tyr Ala Gly Trp Leu Ala Asp Gly Ser625 630 635 640Leu Arg Tyr Pro Ile Val Thr Pro Arg Pro Ala Cys Gly Gly Asp Lys 645 650 655Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Leu 660 665 670Asp Pro Leu Ser Arg His His Ala Phe Cys Phe Arg Gly Val Ser Ala 675 680 685Ala Pro Ser Pro Glu Glu Glu Glu Gly Ser Ala Pro Thr Ala Gly Pro 690 695 700Asp Val Glu Glu Trp Met Val Thr Gln Val Gly Pro Gly Val Ala Ala705 710 715 720Val Pro Ile Gly Glu Glu Thr Thr Ala Ile Pro Gly Phe Thr Val Glu 725 730 735Pro Glu Asn Lys Thr Glu Trp Glu Leu Ala Tyr Thr Pro Ala Gly Thr 740 745 750Leu Pro Leu Pro Gly Ile Pro Pro Thr Trp Pro Pro Thr Gly Glu Ala 755 760 765Thr Glu Glu His Thr Glu Gly Pro Ser Ala Thr Glu Val Pro Ser Ala 770 775 780Ser Glu Lys Pro Phe Pro Ser Glu Glu Pro Phe Pro Pro Glu Glu Pro785 790 795 800Phe Pro Ser Glu Lys Pro Phe Pro Pro Glu Glu Leu Phe Pro Ser Glu 805 810 815Lys Pro Phe Pro Ser Glu Lys Pro Phe Pro Ser Glu Glu Pro Phe Pro 820 825 830Ser Glu Lys Pro Phe Pro Pro Glu Glu Leu Phe Pro Ser Glu Lys Pro 835 840 845Ile Pro Ser Glu Glu Pro Phe Pro Ser Glu Glu Pro Phe Pro Ser Glu 850 855 860Lys Pro Phe Pro Pro Glu Glu Pro Phe Pro Ser Glu Lys Pro Ile Pro865 870 875 880Ser Glu Glu Pro Phe Pro Ser Glu Lys Pro Phe Pro Ser Glu Glu Pro 885 890 895Phe Pro Ser Glu Glu Pro Ser Thr Leu Ser Ala Pro Val Pro Ser Arg 900 905 910Thr Glu Leu Pro Ser Ser Gly Glu Val Ser Gly Val Pro Glu Ile Ser 915 920 925Gly Asp Phe Thr Gly Ser Gly Glu Ile Ser Gly His Leu Asp Phe Ser 930 935 940Gly Gln Pro Ser Gly Glu Ser Ala Ser Gly Leu Pro Ser Glu Asp Leu945 950 955 960Asp Ser Ser Gly Leu Thr Ser Thr Val Gly Ser Gly Leu Pro Val Glu 965 970 975Ser Gly Leu Pro Ser Gly Glu Glu Glu Arg Ile Thr Trp Thr Ser Ala 980 985 990Pro Lys Val Asp Arg Leu Pro Ser Gly Gly Glu Gly Pro Glu Val Ser 995 1000 1005Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Gly Glu Val His 1010 1015 1020Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Ile Ser Gly Leu Pro 1025 1030 1035Ser Gly Gly Glu Val His Leu Glu Ile Ser Ala Ser Gly Val Glu 1040 1045 1050Asp Leu Ser Arg Ile Pro Ser Gly Glu Gly Pro Glu Ile Ser Ala 1055 1060 1065Ser Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Glu Gly 1070 1075 1080His Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Leu Ser Gly Ile 1085 1090 1095Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser Gly Val Glu Asp 1100 1105 1110Leu Ile Gly Leu Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser 1115 1120 1125Gly Val Glu Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu 1130 1135 1140Val Ser Ala Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly 1145 1150 1155Glu Gly Pro Glu Val Ser Val Ser Gly Val Glu Asp Leu Ser Arg 1160 1165 1170Leu Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser Gly Val Glu 1175 1180 1185Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Ile Ser Val 1190 1195 1200Ser Gly Val Glu Asp Ile Ser Ile Leu Pro Ser Gly Glu Gly Pro 1205 1210 1215Glu Val Ser Ala Ser Gly Val Glu Asp Leu Ser Val Leu Pro Ser 1220 1225 1230Gly Glu Gly His Leu Glu Ile Ser Thr Ser Gly Val Glu Asp Leu 1235 1240 1245Ser Val Leu Pro Ser Gly Glu Gly His Leu Glu Thr Ser Ser Gly 1250 1255 1260Val Glu Asp Ile Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val 1265 1270 1275Ser Ala Ser Gly Val Glu Asp Leu Ser Val Leu Pro Ser Gly Glu 1280 1285 1290Asp His Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Leu Gly Val 1295 1300 1305Leu Pro Ser Gly Glu Asp His Leu Glu Ile Ser Ala Ser Gly Val 1310 1315 1320Glu Asp Ile Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val Ser 1325 1330 1335Ala Ser Gly Val Glu Asp Leu Ser Val Leu Pro Ser Gly Glu Gly 1340 1345 1350His Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Leu Ser Arg Leu 1355 1360 1365Pro Ser Gly Gly Glu Asp His Leu Glu Thr Ser Ala Ser Gly Val 1370 1375 1380Gly Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu Ile 1385 1390 1395Ser Ala Ser Gly Ala Gly Asp Leu Ser Gly Leu Thr Ser Gly Lys 1400 1405 1410Glu Asp Leu Thr Gly Ser Ala Ser Gly Ala Leu Asp Leu Gly Arg 1415 1420 1425Ile Pro Ser Val Thr Leu Gly Ser Gly Gln Ala Pro Glu Ala Ser 1430 1435 1440Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly Val Asp Leu 1445 1450 1455Glu Ser Gly Pro Ser Ser Gly Leu Pro Asp Phe Ser Gly Leu Pro 1460 1465 1470Ser Gly Phe Pro Thr Val Ser Leu Val Asp Thr Thr Leu Val Glu 1475 1480 1485Val Val Thr Ala Thr Thr Ala Gly Glu Leu Glu Gly Arg Gly Thr 1490 1495 1500Ile Asp Ile Ser Gly Ala Gly Glu Thr Ser Gly Leu Pro Phe Ser 1505 1510 1515Glu Leu Asp Ile Ser Gly Gly Ala Ser Gly Leu Ser Ser Gly Ala 1520 1525 1530Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro Asp Ile Ser Gly Glu 1535 1540 1545Thr Ser Gly Leu Phe Gly Val Ser Gly Gln Pro Ser Gly Phe Pro 1550 1555 1560Asp Ile Ser Gly Glu Thr Ser Gly Leu Leu Glu Val Ser Gly Gln 1565 1570 1575Pro Ser Gly Phe Tyr Gly Glu Ile Ser Gly Val Thr Glu Leu Ser 1580 1585 1590Gly Leu Ala Ser Gly Gln Pro Glu Ile Ser Gly Glu Ala Ser Gly 1595 1600 1605Ile Leu Ser Gly Leu Gly Pro Pro Phe Gly Ile Thr Asp Leu Ser 1610 1615 1620Gly Glu Ala Pro Gly Ile Pro Asp Leu Ser Gly Gln Pro Ser Gly 1625 1630 1635Leu Pro Glu Phe Ser Gly Thr Ala Ser Gly Ile Pro Asp Leu Val 1640 1645 1650Ser Ser Ala Val Ser Gly Ser Gly Glu Ser Ser Gly Ile Thr Phe 1655 1660 1665Val Asp Thr Ser Leu Val Glu Val Thr Pro Thr Thr Phe Lys Glu 1670 1675 1680Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu Pro Ser Gly 1685 1690 1695Glu Leu Gly Val Ser Gly Thr Ser Gly Leu Ala Asp Val Ser Gly 1700 1705 1710Leu Ser Ser Gly Ala Ile Asp Ser Ser Gly Phe Thr Ser Gln Pro 1715 1720 1725Pro Glu Phe Ser Gly Leu Pro Ser Gly Val Thr Glu Val Ser Gly 1730 1735 1740Glu Ala Ser Gly Ala Glu Ser Gly Ser Ser Leu Pro Ser Gly Ala 1745 1750 1755Tyr Asp Ser Ser Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Phe 1760 1765 1770Val Asp Arg Thr Leu Val Glu Ser Val Thr Gln Ala Pro Thr Ala 1775 1780 1785Gln Glu Ala Gly Glu Gly Pro Ser Gly Ile Leu Glu Leu Ser Gly 1790 1795 1800Ala Pro Ser Gly Ala Pro Asp Met Ser Gly Asp His Leu Gly Ser 1805 1810 1815Leu Asp Gln Ser Gly Leu Gln Ser Gly Leu Val Glu Pro Ser Gly 1820 1825 1830Glu Pro Ala Ser Thr Pro Tyr Phe Ser Gly Asp Phe Ser Gly Thr 1835 1840 1845Thr Asp Val Ser Gly Glu Ser Ser Ala Ala Thr Ser Thr Ser Gly 1850 1855 1860Glu Ala Ser Gly Leu Pro Glu Val Thr Leu Ile Thr Ser Glu Leu 1865 1870 1875Val Glu Gly Val Thr Glu Pro Thr Val Ser Gln Glu Leu Gly Gln 1880 1885 1890Arg Pro Pro Val Thr Tyr Thr Pro Gln Leu Phe Glu Ser Ser Gly 1895 1900 1905Glu Ala Ser Ala Ser Gly Asp Val Pro Arg Phe Pro Gly Ser Gly 1910 1915 1920Val Glu Val Ser Ser Val Pro Glu Ser Ser Gly Glu Thr Ser Ala 1925 1930 1935Tyr Pro Glu Ala Glu Val Gly Ala Ser Ala Ala Pro Glu Ala Ser 1940 1945 1950Gly Gly Ala Ser Gly Ser Pro Asn Leu Ser Glu Thr Thr Ser Thr 1955 1960 1965Phe His Glu Ala Asp Leu Glu Gly Thr Ser Gly Leu Gly Val Ser 1970 1975 1980Gly Ser Pro Ser Ala Phe Pro Glu Gly Pro Thr Glu Gly Leu Ala 1985 1990 1995Thr Pro Glu Val Ser Gly Glu Ser Thr Thr Ala Phe Asp Val Ser 2000 2005 2010Val Glu Ala Ser Gly Ser Pro Ser Ala Thr Pro Leu Ala Ser Gly 2015 2020 2025Asp Arg Thr Asp Thr Ser Gly Asp Leu Ser Gly His Thr Ser Gly 2030 2035 2040Leu Asp Ile Val Ile Ser Thr Thr Ile Pro Glu Ser Glu Trp Thr 2045 2050 2055Gln Gln Thr Gln Arg Pro Ala Glu Ala Arg Leu Glu Ile Glu Ser 2060 2065 2070Ser Ser Pro Val His Ser Gly Glu Glu Ser Gln Thr Ala Asp Thr 2075 2080 2085Ala Thr Ser Pro Thr Asp Ala Ser Ile Pro Ala Ser Ala Gly Gly 2090 2095 2100Thr Asp Asp Ser Glu Ala Thr Thr Thr Asp Ile Asp Glu Cys Leu 2105 2110 2115Ser Ser Pro Cys Leu Asn Gly Ala Thr Cys Val Asp Ala Ile Asp 2120 2125 2130Ser Phe Thr Cys Leu Cys Leu Pro Ser Tyr Gln Gly Asp Val Cys 2135 2140 2145Glu Ile Gln Lys Leu Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly 2150 2155 2160His Cys Tyr Arg His Phe Pro Asp Arg Ala Thr Trp Val Asp Ala 2165 2170 2175Glu Ser Gln Cys Arg Lys Gln Gln Ser His Leu Ser Ser Ile Val 2180 2185 2190Thr Pro Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr 2195 2200 2205Gln Trp Ile Gly Leu Asn Asp Lys Thr Ile Glu Gly Asp Phe Arg 2210 2215 2220Trp Ser Asp Gly His Ser Leu Gln Phe Glu Asn Trp Arg Pro Asn 2225 2230 2235Gln Pro Asp Asn Phe Phe Ala Thr Gly Glu Asp Cys Val Val Met 2240 2245 2250Ile Trp His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr 2255 2260 2265Gln Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu 2270 2275 2280Pro Pro Val Val Glu His Ala Arg Ile Phe Gly Gln Lys Lys Asp 2285 2290 2295Arg Tyr Glu Ile Asn Ala Leu Val Arg Tyr Gln Cys Thr Glu Gly 2300 2305 2310Phe Ile Gln Gly His Val Pro Thr Ile Arg Cys Gln Pro

Ser Gly 2315 2320 2325His Trp Glu Glu Pro Arg Ile Thr Cys Thr Asp Pro Ala Thr Tyr 2330 2335 2340Lys Arg Arg Leu Gln Lys Arg Ser Ser Arg Pro Leu Arg Arg Ser 2345 2350 2355His Pro Ser Thr Ala His 23601282124PRTRattus norvegicus 128Met Thr Thr Leu Leu Leu Val Phe Val Thr Leu Arg Val Ile Ala Ala1 5 10 15Val Ile Ser Glu Glu Val Pro Asp His Asp Asn Ser Leu Ser Val Ser 20 25 30Ile Pro Gln Pro Ser Pro Leu Lys Ala Leu Leu Gly Thr Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Thr Pro Arg Ile Lys Trp Ser Arg Val Ser Lys65 70 75 80Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gln Val Arg Val 85 90 95Asn Ser Ile Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser 115 120 125Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala 130 135 140Thr Leu Glu Val Ile Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 260 265 270Cys Arg Thr Val Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 275 280 285Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315 320Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu 355 360 365Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Leu Glu Leu Pro Leu 370 375 380Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Asn Val Ile Leu Thr385 390 395 400Ala Lys Pro Ile Phe Asp Met Ser Pro Thr Val Ser Glu Pro Gly Glu 405 410 415Ala Leu Thr Leu Ala Pro Glu Val Gly Thr Thr Val Phe Pro Glu Ala 420 425 430Gly Glu Arg Thr Glu Lys Thr Thr Arg Pro Trp Gly Phe Pro Glu Glu 435 440 445Ala Thr Arg Gly Pro Asp Ser Ala Thr Ala Phe Ala Ser Glu Asp Leu 450 455 460Val Val Arg Val Thr Ile Ser Pro Gly Ala Val Glu Val Pro Gly Gln465 470 475 480Pro Arg Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Ser Thr 485 490 495Arg Tyr Ser Leu Thr Phe Glu Glu Ala Gln Gln Ala Cys Ile Arg Thr 500 505 510Gly Ala Ala Ile Ala Ser Pro Glu Gln Leu Gln Ala Ala Tyr Glu Ala 515 520 525Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg 530 535 540Tyr Pro Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser545 550 555 560Ser Pro Gly Val Arg Thr Tyr Gly Val Arg Pro Ser Ser Glu Thr Tyr 565 570 575Asp Val Tyr Cys Tyr Val Asp Lys Leu Glu Gly Glu Val Phe Phe Ala 580 585 590Thr Gln Met Glu Gln Phe Thr Phe Gln Glu Ala Gln Ala Phe Cys Ala 595 600 605Ala Gln Asn Ala Thr Leu Ala Ser Thr Gly Gln Leu Tyr Ala Ala Trp 610 615 620Ser Gln Gly Leu Asp Lys Cys Tyr Ala Gly Trp Leu Ala Asp Gly Thr625 630 635 640Leu Arg Tyr Pro Ile Val Asn Pro Arg Pro Ala Cys Gly Gly Asp Lys 645 650 655Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Pro 660 665 670Asp Pro Leu Ser Lys His His Ala Phe Cys Phe Arg Gly Val Ser Val 675 680 685Val Pro Ser Pro Gly Gly Thr Pro Thr Ser Pro Ser Asp Ile Glu Asp 690 695 700Trp Ile Val Thr Arg Val Glu Pro Gly Val Asp Ala Val Pro Leu Glu705 710 715 720Pro Glu Thr Thr Glu Val Pro Tyr Phe Thr Thr Glu Pro Glu Lys Gln 725 730 735Thr Glu Trp Glu Pro Ala Tyr Thr Pro Val Gly Thr Ser Pro Leu Pro 740 745 750Gly Ile Pro Pro Thr Trp Leu Pro Thr Val Pro Ala Ala Glu Glu His 755 760 765Thr Glu Ser Pro Ser Ala Ser Gln Glu Pro Ser Ala Ser Gln Val Pro 770 775 780Ser Thr Ser Glu Glu Pro Tyr Thr Pro Ser Leu Ala Val Pro Ser Gly785 790 795 800Thr Glu Leu Pro Ser Ser Gly Asp Thr Ser Gly Ala Pro Asp Leu Ser 805 810 815Gly Asp Phe Thr Gly Ser Thr Asp Thr Ser Gly Arg Leu Asp Ser Ser 820 825 830Gly Glu Pro Ser Gly Gly Ser Glu Ser Gly Leu Pro Ser Gly Asp Leu 835 840 845Asp Ser Ser Gly Leu Gly Pro Thr Val Ser Ser Gly Leu Pro Val Glu 850 855 860Ser Gly Ser Ala Ser Gly Asp Gly Glu Ile Pro Trp Ser Ser Thr Pro865 870 875 880Thr Val Asp Arg Leu Pro Ser Gly Gly Glu Ser Leu Glu Gly Ser Ala 885 890 895Ser Ala Ser Gly Thr Gly Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu 900 905 910Ile Thr Glu Thr Ser Ala Ser Gly Thr Glu Glu Ile Ser Gly Leu Pro 915 920 925Ser Gly Gly Asp Asp Leu Glu Thr Ser Thr Ser Gly Ile Asp Gly Ala 930 935 940Ser Val Leu Pro Thr Gly Arg Gly Gly Leu Glu Thr Ser Ala Ser Gly945 950 955 960Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Glu Gly Ser Glu Thr 965 970 975Ser Thr Ser Gly Ile Glu Asp Ile Ser Val Leu Pro Thr Gly Glu Ser 980 985 990Pro Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser 995 1000 1005Gly Gly Glu Ser Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Val 1010 1015 1020Thr Gln Leu Pro Thr Glu Arg Gly Gly Leu Glu Thr Ser Ala Ser 1025 1030 1035Gly Ile Glu Asp Ile Thr Val Leu Pro Thr Gly Arg Glu Asn Leu 1040 1045 1050Glu Thr Ser Ala Ser Gly Val Glu Asp Val Ser Gly Leu Pro Ser 1055 1060 1065Gly Lys Glu Gly Leu Glu Thr Ser Ala Ser Gly Ile Glu Asp Ile 1070 1075 1080Ser Val Phe Pro Thr Glu Ala Glu Gly Leu Glu Thr Ser Ala Ser 1085 1090 1095Gly Gly Tyr Val Ser Gly Ile Pro Ser Gly Glu Asp Gly Thr Glu 1100 1105 1110Thr Ser Thr Ser Gly Val Glu Gly Val Ser Gly Leu Pro Ser Gly 1115 1120 1125Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Leu Gly 1130 1135 1140Leu Pro Thr Arg Asp Ser Leu Glu Thr Ser Ala Ser Gly Val Asp 1145 1150 1155Val Thr Gly Tyr Pro Ser Gly Arg Glu Asp Thr Glu Thr Ser Val 1160 1165 1170Pro Gly Val Gly Asp Asp Leu Ser Gly Leu Pro Ser Gly Gln Glu 1175 1180 1185Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu Gly Gly Leu 1190 1195 1200Pro Ser Gly Lys Glu Asp Leu Val Gly Ser Ala Ser Gly Ala Leu 1205 1210 1215Asp Phe Gly Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln Thr 1220 1225 1230Pro Glu Ala Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser 1235 1240 1245Gly Val Asp Ile Gly Ser Gly Pro Ser Ser Gly Leu Pro Asp Phe 1250 1255 1260Ser Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser 1265 1270 1275Thr Leu Val Glu Val Ile Thr Ala Thr Thr Ala Ser Glu Leu Glu 1280 1285 1290Gly Arg Gly Thr Ile Ser Val Ser Gly Ser Gly Glu Glu Ser Gly 1295 1300 1305Pro Pro Leu Ser Glu Leu Asp Ser Ser Ala Asp Ile Ser Gly Leu 1310 1315 1320Pro Ser Gly Thr Glu Leu Ser Gly Gln Thr Ser Gly Ser Leu Asp 1325 1330 1335Val Ser Gly Glu Thr Ser Gly Phe Phe Asp Val Ser Gly Gln Pro 1340 1345 1350Phe Gly Ser Ser Gly Thr Gly Glu Gly Thr Ser Gly Ile Pro Glu 1355 1360 1365Val Ser Gly Gln Ala Val Arg Ser Pro Asp Thr Thr Glu Ile Ser 1370 1375 1380Glu Leu Ser Gly Leu Ser Ser Gly Gln Pro Asp Val Ser Gly Glu 1385 1390 1395Gly Ser Gly Ile Leu Phe Gly Ser Gly Gln Ser Ser Gly Ile Thr 1400 1405 1410Ser Val Ser Gly Glu Thr Ser Gly Ile Ser Asp Leu Ser Gly Gln 1415 1420 1425Pro Ser Gly Phe Pro Val Leu Ser Gly Thr Thr Pro Gly Thr Pro 1430 1435 1440Asp Leu Ala Ser Gly Ala Met Ser Gly Ser Gly Asp Ser Ser Gly 1445 1450 1455Ile Thr Phe Val Asp Thr Ser Leu Ile Glu Val Thr Pro Thr Thr 1460 1465 1470Phe Arg Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu 1475 1480 1485Pro Ser Gly Glu Thr Asp Leu Ser Gly Thr Ser Gly Met Val Asp 1490 1495 1500Val Ser Gly Gln Ser Ser Gly Ala Ile Asp Ser Ser Gly Leu Ile 1505 1510 1515Ser Pro Thr Pro Glu Phe Ser Gly Leu Pro Ser Gly Val Ala Glu 1520 1525 1530Val Ser Gly Glu Val Ser Gly Val Glu Thr Gly Ser Ser Leu Ser 1535 1540 1545Ser Gly Ala Phe Asp Gly Ser Gly Leu Val Ser Gly Phe Pro Thr 1550 1555 1560Val Ser Leu Val Asp Arg Thr Leu Val Glu Ser Ile Thr Leu Ala 1565 1570 1575Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro Ser Ser Ile Leu Glu 1580 1585 1590Phe Ser Gly Ala His Ser Gly Thr Pro Asp Ile Ser Gly Asp Leu 1595 1600 1605Ser Gly Ser Leu Asp Gln Ser Thr Trp Gln Pro Gly Trp Thr Glu 1610 1615 1620Ala Ser Thr Glu Pro Pro Ser Ser Pro Tyr Phe Ser Gly Asp Phe 1625 1630 1635Ser Ser Thr Thr Asp Ala Ser Gly Glu Ser Ile Thr Ala Pro Thr 1640 1645 1650Gly Ser Gly Glu Thr Ser Gly Leu Pro Glu Val Thr Leu Ile Thr 1655 1660 1665Ser Glu Leu Val Glu Gly Val Thr Glu Pro Thr Val Ser Gln Glu 1670 1675 1680Leu Gly His Gly Pro Ser Met Thr Tyr Thr Pro Arg Leu Phe Glu 1685 1690 1695Ala Ser Gly Glu Ala Ser Ala Ser Gly Asp Leu Gly Gly Pro Val 1700 1705 1710Thr Ile Phe Pro Gly Ser Gly Val Glu Ala Ser Val Pro Glu Gly 1715 1720 1725Ser Ser Asp Pro Ser Ala Tyr Pro Glu Ala Gly Val Gly Val Ser 1730 1735 1740Ala Ala Pro Glu Ala Ser Ser Gln Leu Ser Glu Phe Pro Asp Leu 1745 1750 1755His Gly Ile Thr Ser Ala Ser Arg Glu Thr Asp Leu Glu Met Thr 1760 1765 1770Thr Pro Gly Thr Glu Val Ser Ser Asn Pro Trp Thr Phe Gln Glu 1775 1780 1785Gly Thr Arg Glu Gly Ser Ala Ala Pro Glu Val Ser Gly Glu Ser 1790 1795 1800Ser Thr Thr Ser Asp Ile Asp Ala Gly Thr Ser Gly Val Pro Phe 1805 1810 1815Ala Thr Pro Met Thr Ser Gly Asp Arg Thr Glu Ile Ser Gly Glu 1820 1825 1830Trp Ser Asp His Thr Ser Glu Val Asn Val Thr Val Ser Thr Thr 1835 1840 1845Val Pro Glu Ser Arg Trp Ala Gln Ser Thr Gln His Pro Thr Glu 1850 1855 1860Thr Leu Gln Glu Ile Gly Ser Pro Asn Pro Ser Tyr Ser Gly Glu 1865 1870 1875Glu Thr Gln Thr Ala Glu Thr Ala Lys Ser Leu Thr Asp Thr Pro 1880 1885 1890Thr Leu Ala Ser Pro Glu Gly Ser Gly Glu Thr Glu Ser Thr Ala 1895 1900 1905Ala Asp Gln Glu Gln Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly 1910 1915 1920His Cys Tyr Arg His Phe Pro Asp Arg Glu Thr Trp Val Asp Ala 1925 1930 1935Glu Arg Arg Cys Arg Glu Gln Gln Ser His Leu Ser Ser Ile Val 1940 1945 1950Thr Pro Glu Glu Gln Glu Phe Val Asn Lys Asn Ala Gln Asp Tyr 1955 1960 1965Gln Trp Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg 1970 1975 1980Trp Ser Asp Gly His Ser Leu Gln Phe Glu Lys Trp Arg Pro Asn 1985 1990 1995Gln Pro Asp Asn Phe Phe Ala Thr Gly Glu Asp Cys Val Val Met 2000 2005 2010Ile Trp His Glu Arg Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr 2015 2020 2025Gln Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu 2030 2035 2040Pro Pro Ala Val Glu His Ala Arg Thr Leu Gly Gln Lys Lys Asp 2045 2050 2055Arg Tyr Glu Ile Ser Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly 2060 2065 2070Phe Val Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Ala 2075 2080 2085Asp Trp Glu Glu Pro Arg Ile Thr Cys Thr Asp Pro Asn Thr Tyr 2090 2095 2100Lys His Arg Leu Gln Lys Arg Thr Met Arg Pro Thr Arg Arg Ser 2105 2110 2115Arg Pro Ser Met Ala His 2120129537PRTSus scrofaVARIANT(45)..(45)Xaa can be any naturally occurring amino acidVARIANT(49)..(49)Xaa can be any naturally occurring amino acidVARIANT(146)..(146)Xaa can be any naturally occurring amino acid 129Ala Ile Ser Val Glu Val Ser Glu Pro Asp Asn Ser Leu Ser Val Ser1 5 10 15Ile Pro Gln Pro Ser Pro Leu Arg Val Leu Leu Gly Gly Ser Leu Thr 20 25 30Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Xaa Thr Ala Pro 35 40 45Xaa Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys 50 55 60Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gln Val Arg Val65 70 75 80Asn Ser Ala Tyr Gln Asp Arg Val Thr Leu Pro Asn Tyr Pro Ala Ile 85 90 95Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser 100 105 110Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala 115 120 125Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile 130 135 140Ser Xaa Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu145 150 155 160Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 165 170 175Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 180 185 190Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 195 200 205Asp Glu Phe Pro Gly Val Ile Thr Tyr Gly Ile Arg Asp Thr Asn Glu 210 215 220Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe225 230 235 240Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 245 250

255Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 260 265 270Ala Trp Arg Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 275 280 285Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly 290 295 300Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly305 310 315 320Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 325 330 335Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu 340 345 350Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu 355 360 365Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Thr Val Ile Leu Thr 370 375 380Val Lys Pro Val Phe Glu Phe Ser Pro Thr Ala Pro Glu Pro Glu Glu385 390 395 400Pro Phe Thr Phe Ala Pro Gly Thr Gly Ala Thr Ala Phe Pro Glu Ala 405 410 415Glu Asn Arg Thr Gly Glu Ala Thr Arg Pro Trp Ala Phe Pro Glu Glu 420 425 430Ser Thr Pro Gly Leu Gly Ala Pro Thr Ala Phe Thr Ser Glu Asp Leu 435 440 445Val Val Gln Val Thr Ser Ala Ala Thr Glu Glu Gly Thr Glu Gly Pro 450 455 460Ser Ala Thr Glu Ala Pro Ser Thr Ser Glu Glu Pro Phe Pro Ser Glu465 470 475 480Lys Pro Phe Pro Ser Glu Glu Pro Phe Pro Ser Glu Glu Pro Phe Pro 485 490 495Ser Glu Lys Pro Ser Ala Ser Glu Glu Pro Phe Pro Ser Glu Gln Pro 500 505 510Ser Thr Leu Ser Ala Pro Val Pro Ser Arg Thr Glu Leu Pro Gly Ser 515 520 525Gly Glu Val Ser Gly Ala Pro Glu Val 530 5351302132PRTMus musculus 130Met Thr Thr Leu Leu Leu Val Phe Val Thr Leu Arg Val Ile Ala Ala1 5 10 15Val Ile Ser Glu Glu Val Pro Asp His Asp Asn Ser Leu Ser Val Ser 20 25 30Ile Pro Gln Pro Ser Pro Leu Lys Val Leu Leu Gly Ser Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Thr Pro Arg Ile Lys Trp Ser Arg Val Ser Lys65 70 75 80Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gln Val Arg Val 85 90 95Asn Ser Ile Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser 115 120 125Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala 130 135 140Thr Leu Glu Val Ile Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 260 265 270Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 275 280 285Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315 320Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Asp 355 360 365Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Leu Glu Leu Pro Leu 370 375 380Pro Arg Asn Val Thr Glu Gly Glu Ala Leu Gly Ser Val Ile Leu Thr385 390 395 400Ala Lys Pro Ile Phe Asp Leu Ser Pro Thr Ile Ser Glu Pro Gly Glu 405 410 415Ala Leu Thr Leu Ala Pro Glu Val Gly Ser Thr Ala Phe Pro Glu Ala 420 425 430Glu Glu Arg Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Ala Glu 435 440 445Val Thr Arg Gly Pro Asp Ser Ala Thr Ala Phe Ala Ser Glu Asp Leu 450 455 460Val Val Arg Val Thr Ile Ser Pro Gly Ala Ala Glu Val Pro Gly Gln465 470 475 480Pro Arg Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Ser Thr 485 490 495Arg Tyr Ser Leu Thr Phe Glu Glu Ala Gln Gln Ala Cys Met His Thr 500 505 510Gly Ala Val Ile Ala Ser Pro Glu Gln Leu Gln Ala Ala Tyr Glu Ala 515 520 525Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg 530 535 540Tyr Pro Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser545 550 555 560Ser Pro Gly Val Arg Thr Tyr Gly Val Arg Pro Ser Ser Glu Thr Tyr 565 570 575Asp Val Tyr Cys Tyr Val Asp Lys Leu Glu Gly Glu Val Phe Phe Ala 580 585 590Thr Arg Leu Glu Gln Phe Thr Phe Gln Glu Ala Arg Ala Phe Cys Ala 595 600 605Ala Gln Asn Ala Thr Leu Ala Ser Thr Gly Gln Leu Tyr Ala Ala Trp 610 615 620Ser Gln Gly Leu Asp Lys Cys Tyr Ala Gly Trp Leu Ala Asp Gly Thr625 630 635 640Leu Arg Tyr Pro Ile Ile Thr Pro Arg Pro Ala Cys Gly Gly Asp Lys 645 650 655Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Pro 660 665 670Asp Pro Leu Ser Lys His His Ala Phe Cys Phe Arg Gly Val Ser Val 675 680 685Ala Pro Ser Pro Gly Glu Glu Gly Gly Ser Thr Pro Thr Ser Pro Ser 690 695 700Asp Ile Glu Asp Trp Ile Val Thr Gln Val Gly Pro Gly Val Asp Ala705 710 715 720Val Pro Leu Glu Pro Lys Thr Thr Glu Val Pro Tyr Phe Thr Thr Glu 725 730 735Pro Arg Lys Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Val Gly Thr 740 745 750Ser Pro Gln Pro Gly Ile Pro Pro Thr Trp Leu Pro Thr Leu Pro Ala 755 760 765Ala Glu Glu His Thr Glu Ser Pro Ser Ala Ser Glu Glu Pro Ser Ala 770 775 780Ser Ala Val Pro Ser Thr Ser Glu Glu Pro Tyr Thr Ser Ser Phe Ala785 790 795 800Val Pro Ser Met Thr Glu Leu Pro Gly Ser Gly Glu Ala Ser Gly Ala 805 810 815Pro Asp Leu Ser Gly Asp Phe Thr Gly Ser Gly Asp Ala Ser Gly Arg 820 825 830Leu Asp Ser Ser Gly Gln Pro Ser Gly Gly Ile Glu Ser Gly Leu Pro 835 840 845Ser Gly Asp Leu Asp Ser Ser Gly Leu Ser Pro Thr Val Ser Ser Gly 850 855 860Leu Pro Val Glu Ser Gly Ser Ala Ser Gly Asp Gly Glu Val Pro Trp865 870 875 880Ser His Thr Pro Thr Val Gly Arg Leu Pro Ser Gly Gly Glu Ser Pro 885 890 895Glu Gly Ser Ala Ser Ala Ser Gly Thr Gly Asp Leu Ser Gly Leu Pro 900 905 910Ser Gly Gly Glu Ile Thr Glu Thr Ser Thr Ser Gly Ala Glu Glu Thr 915 920 925Ser Gly Leu Pro Ser Gly Gly Asp Gly Leu Glu Thr Ser Thr Ser Gly 930 935 940Val Asp Asp Val Ser Gly Ile Pro Thr Gly Arg Glu Gly Leu Glu Thr945 950 955 960Ser Ala Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Glu 965 970 975Gly Ser Glu Thr Ser Thr Ser Gly Ile Glu Asp Ile Ser Val Leu Pro 980 985 990Thr Gly Gly Glu Ser Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu 995 1000 1005Ser Gly Leu Pro Ser Gly Gly Glu Ser Leu Glu Thr Ser Ala Ser 1010 1015 1020Gly Ala Glu Asp Val Thr Gln Leu Pro Thr Glu Arg Gly Gly Leu 1025 1030 1035Glu Thr Ser Ala Ser Gly Val Glu Asp Ile Thr Val Leu Pro Thr 1040 1045 1050Gly Arg Glu Ser Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Val 1055 1060 1065Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu Thr Ser Ala Ser 1070 1075 1080Gly Ile Glu Asp Ile Ser Val Phe Pro Thr Glu Ala Glu Gly Leu 1085 1090 1095Asp Thr Ser Ala Ser Gly Gly Tyr Val Ser Gly Ile Pro Ser Gly 1100 1105 1110Gly Asp Gly Thr Glu Thr Ser Ala Ser Gly Val Glu Asp Val Ser 1115 1120 1125Gly Leu Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly 1130 1135 1140Val Glu Asp Leu Gly Pro Ser Thr Arg Asp Ser Leu Glu Thr Ser 1145 1150 1155Ala Ser Gly Val Asp Val Thr Gly Phe Pro Ser Gly Arg Gly Asp 1160 1165 1170Pro Glu Thr Ser Val Ser Gly Val Gly Asp Asp Phe Ser Gly Leu 1175 1180 1185Pro Ser Gly Lys Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu 1190 1195 1200Asp Leu Ser Gly Leu Pro Ser Gly Lys Glu Asp Leu Val Gly Ser 1205 1210 1215Ala Ser Gly Ala Leu Asp Phe Gly Lys Leu Pro Pro Gly Thr Leu 1220 1225 1230Gly Ser Gly Gln Thr Pro Glu Val Asn Gly Phe Pro Ser Gly Phe 1235 1240 1245Ser Gly Glu Tyr Ser Gly Ala Asp Ile Gly Ser Gly Pro Ser Ser 1250 1255 1260Gly Leu Pro Asp Phe Ser Gly Leu Pro Ser Gly Phe Pro Thr Val 1265 1270 1275Ser Leu Val Asp Ser Thr Leu Val Glu Val Ile Thr Ala Thr Thr 1280 1285 1290Ser Ser Glu Leu Glu Gly Arg Gly Thr Ile Gly Ile Ser Gly Ser 1295 1300 1305Gly Glu Val Ser Gly Leu Pro Leu Gly Glu Leu Asp Ser Ser Ala 1310 1315 1320Asp Ile Ser Gly Leu Pro Ser Gly Thr Glu Leu Ser Gly Gln Ala 1325 1330 1335Ser Gly Ser Pro Asp Ser Ser Gly Glu Thr Ser Gly Phe Phe Asp 1340 1345 1350Val Ser Gly Gln Pro Phe Gly Ser Ser Gly Val Ser Glu Glu Thr 1355 1360 1365Ser Gly Ile Pro Glu Ile Ser Gly Gln Pro Ser Gly Thr Pro Asp 1370 1375 1380Thr Thr Ala Thr Ser Gly Val Thr Glu Leu Asn Glu Leu Ser Ser 1385 1390 1395Gly Gln Pro Asp Val Ser Gly Asp Gly Ser Gly Ile Leu Phe Gly 1400 1405 1410Ser Gly Gln Ser Ser Gly Ile Thr Ser Val Ser Gly Glu Thr Ser 1415 1420 1425Gly Ile Ser Asp Leu Ser Gly Gln Pro Ser Gly Phe Pro Val Phe 1430 1435 1440Ser Gly Thr Ala Thr Arg Thr Pro Asp Leu Ala Ser Gly Thr Ile 1445 1450 1455Ser Gly Ser Gly Glu Ser Ser Gly Ile Thr Phe Val Asp Thr Ser 1460 1465 1470Phe Val Glu Val Thr Pro Thr Thr Phe Arg Glu Glu Glu Gly Leu 1475 1480 1485Gly Ser Val Glu Leu Ser Gly Phe Pro Ser Gly Glu Thr Glu Leu 1490 1495 1500Ser Gly Thr Ser Gly Thr Val Asp Val Ser Glu Gln Ser Ser Gly 1505 1510 1515Ala Ile Asp Ser Ser Gly Leu Thr Ser Pro Thr Pro Glu Phe Ser 1520 1525 1530Gly Leu Pro Ser Gly Val Ala Glu Val Ser Gly Glu Phe Ser Gly 1535 1540 1545Val Glu Thr Gly Ser Ser Leu Pro Ser Gly Ala Phe Asp Gly Ser 1550 1555 1560Gly Leu Val Ser Gly Phe Pro Thr Val Ser Leu Val Asp Arg Thr 1565 1570 1575Leu Val Glu Ser Ile Thr Gln Ala Pro Thr Ala Gln Glu Ala Gly 1580 1585 1590Glu Gly Pro Ser Gly Ile Leu Glu Phe Ser Gly Ala His Ser Gly 1595 1600 1605Thr Pro Asp Ile Ser Gly Glu Leu Ser Gly Ser Leu Asp Leu Ser 1610 1615 1620Thr Leu Gln Ser Gly Gln Met Glu Thr Ser Thr Glu Thr Pro Ser 1625 1630 1635Ser Pro Tyr Phe Ser Gly Asp Phe Ser Ser Thr Thr Asp Val Ser 1640 1645 1650Gly Glu Ser Ile Ala Ala Thr Thr Gly Ser Gly Glu Ser Ser Gly 1655 1660 1665Leu Pro Glu Val Thr Leu Asn Thr Ser Glu Leu Val Glu Gly Val 1670 1675 1680Thr Glu Pro Thr Val Ser Gln Glu Leu Gly His Gly Pro Ser Met 1685 1690 1695Thr Tyr Thr Pro Arg Leu Phe Glu Ala Ser Gly Asp Ala Ser Ala 1700 1705 1710Ser Gly Asp Leu Gly Gly Ala Val Thr Asn Phe Pro Gly Ser Gly 1715 1720 1725Ile Glu Ala Ser Val Pro Glu Ala Ser Ser Asp Leu Ser Ala Tyr 1730 1735 1740Pro Glu Ala Gly Val Gly Val Ser Ala Ala Pro Glu Ala Ser Ser 1745 1750 1755Lys Leu Ser Glu Phe Pro Asp Leu His Gly Ile Thr Ser Ala Phe 1760 1765 1770His Glu Thr Asp Leu Glu Met Thr Thr Pro Ser Thr Glu Val Asn 1775 1780 1785Ser Asn Pro Trp Thr Phe Gln Glu Gly Thr Arg Glu Gly Ser Ala 1790 1795 1800Ala Pro Glu Val Ser Gly Glu Ser Ser Thr Thr Ser Asp Ile Asp 1805 1810 1815Thr Gly Thr Ser Gly Val Pro Ser Ala Thr Pro Met Ala Ser Gly 1820 1825 1830Asp Arg Thr Glu Ile Ser Gly Glu Trp Ser Asp His Thr Ser Glu 1835 1840 1845Val Asn Val Ala Ile Ser Ser Thr Ile Thr Glu Ser Glu Trp Ala 1850 1855 1860Gln Pro Thr Arg Tyr Pro Thr Glu Thr Leu Gln Glu Ile Glu Ser 1865 1870 1875Pro Asn Pro Ser Tyr Ser Gly Glu Glu Thr Gln Thr Ala Glu Thr 1880 1885 1890Thr Met Ser Leu Thr Asp Ala Pro Thr Leu Ser Ser Ser Glu Gly 1895 1900 1905Ser Gly Glu Thr Glu Ser Thr Val Ala Asp Gln Glu Gln Cys Glu 1910 1915 1920Glu Gly Trp Thr Lys Phe Gln Gly His Cys Tyr Arg His Phe His 1925 1930 1935Asp Arg Glu Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln 1940 1945 1950Gln Ser His Leu Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe 1955 1960 1965Val Asn Lys Asn Ala Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp 1970 1975 1980Arg Thr Ile Glu Gly Asp Phe Arg Trp Ser Asp Gly His Ser Leu 1985 1990 1995Gln Phe Glu Lys Trp Arg Pro Asn Gln Pro Asp Asn Phe Phe Ala 2000 2005 2010Thr Gly Glu Asp Cys Val Val Met Ile Trp His Glu Arg Gly Glu 2015 2020 2025Trp Asn Asp Val Pro Cys Asn Tyr Gln Leu Pro Phe Thr Cys Lys 2030 2035 2040Lys Gly Thr Val Ala Cys Gly Asp Pro Pro Val Val Glu His Ala 2045 2050 2055Arg Thr Leu Gly Gln Lys Lys Asp Arg Tyr Glu Ile Ser Ser Leu 2060 2065 2070Val Arg Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg His Val Pro 2075 2080 2085Thr Ile Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro Arg Ile 2090 2095 2100Thr Cys Thr Asp Pro Asn Thr Tyr Lys His Arg Leu Gln Lys Arg 2105 2110 2115Ser Met Arg Pro Thr Arg Arg Ser Arg Pro Ser Met Ala His 2120 2125 21301312167PRTOryctolagus cuniculus 131Met Thr Thr Leu Leu Leu Val Leu Val Ala Leu Arg Val Ile Ala Ala1 5 10 15Ala Ile Ser Gly Asp Val Ser Asp Leu Asp Asn Ala Leu Ser Val Ser 20 25 30Ile Pro

Gln Pro Ser Pro Val Arg Ala Leu Leu Gly Thr Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Val His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Thr Pro Arg Ile Lys Trp Ser Arg Ile Ser Lys65 70 75 80Asp Lys Glu Val Val Leu Leu Val Ala Asn Glu Gly Arg Val Arg Ile 85 90 95Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Ile Gln Ser Leu Arg Ser Asn Asp Ser 115 120 125Gly Ile Tyr Arg Cys Glu Val Met His Gly Leu Glu Asp Ser Glu Ala 130 135 140Thr Leu Glu Val Val Val Lys Gly Val Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Ser Glu 260 265 270Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 275 280 285Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315 320Asn Leu Leu Gly Val Arg Thr Val Tyr Val His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Met Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu 355 360 365Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Val 370 375 380Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val Val Leu Thr385 390 395 400Ala Lys Pro Val Leu Asp Val Ser Pro Thr Ala Pro Gln Pro Glu Glu 405 410 415Thr Phe Ala Pro Gly Val Gly Ala Thr Ala Phe Pro Gly Val Glu Asn 420 425 430Gly Thr Glu Glu Ala Thr Arg Pro Arg Gly Phe Ala Asp Glu Ala Thr 435 440 445Leu Gly Pro Ser Ser Ala Thr Ala Phe Thr Ser Ala Asp Leu Val Val 450 455 460Gln Val Thr Ala Ala Pro Gly Val Ala Glu Val Pro Gly Gln Pro Arg465 470 475 480Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Pro Thr Arg Tyr 485 490 495Ser Leu Thr Phe Glu Glu Ala Gln Gln Ala Cys Leu Arg Thr Gly Ala 500 505 510Ala Met Ala Ser Ala Glu Gln Leu Gln Ala Ala Tyr Glu Ala Gly Tyr 515 520 525Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg Tyr Pro 530 535 540Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser Ser Pro545 550 555 560Gly Val Arg Thr Tyr Gly Val Arg Pro Pro Ser Glu Thr Tyr Asp Val 565 570 575Tyr Cys Tyr Val Asp Arg Leu Glu Gly Glu Val Phe Phe Ala Thr Arg 580 585 590Leu Glu Gln Phe Thr Phe Gln Glu Ala Leu Glu Phe Cys Glu Ser His 595 600 605Asn Ala Thr Leu Ala Ser Thr Gly Gln Leu Tyr Ala Ala Trp Ser Arg 610 615 620Gly Leu Asp Arg Cys Tyr Ala Gly Trp Leu Ala Asp Gly Ser Leu Arg625 630 635 640Tyr Pro Ile Val Thr Pro Arg Pro Ala Cys Gly Gly Asp Lys Pro Gly 645 650 655Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Pro Asp Pro 660 665 670Leu Ser Arg His His Ala Phe Cys Phe Arg Gly Thr Ser Glu Ala Pro 675 680 685Ser Pro Gly Pro Glu Glu Gly Gly Thr Ala Thr Pro Ala Ser Gly Leu 690 695 700Glu Asp Trp Ile Val Thr Gln Val Gly Pro Gly Val Ala Ala Thr Pro705 710 715 720Arg Ala Glu Glu Arg Thr Ala Val Pro Ser Phe Ala Thr Glu Pro Gly 725 730 735Asn Gln Thr Gly Trp Glu Ala Ala Ser Ser Pro Val Gly Thr Ser Leu 740 745 750Leu Pro Gly Ile Pro Pro Thr Trp Pro Pro Thr Gly Thr Ala Ala Glu 755 760 765Gly Thr Thr Glu Gly Leu Ser Thr Ala Ala Met Pro Ser Ala Ser Glu 770 775 780Gly Pro Tyr Thr Pro Ser Ser Leu Val Ala Arg Glu Thr Glu Leu Pro785 790 795 800Gly Leu Gly Val Thr Ser Val Pro Pro Asp Ile Ser Gly Asp Leu Thr 805 810 815Ser Ser Gly Glu Ala Ser Gly Leu Phe Gly Pro Thr Gly Gln Pro Leu 820 825 830Gly Gly Ser Ala Ser Gly Leu Pro Ser Gly Glu Leu Asp Ser Gly Ser 835 840 845Leu Thr Pro Thr Val Gly Ser Gly Leu Pro Ile Gly Ser Gly Leu Ala 850 855 860Ser Gly Asp Glu Asp Arg Ile Gln Trp Ser Ser Ser Thr Glu Val Gly865 870 875 880Gly Val Thr Ser Gly Ala Glu Ile Pro Glu Thr Ser Ala Ser Gly Val 885 890 895Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Pro Glu Thr 900 905 910Phe Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala 915 920 925Glu Ile Pro Glu Thr Phe Ala Ser Gly Val Gly Thr Asp Leu Ser Gly 930 935 940Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser Ala Ser Gly Val Gly945 950 955 960Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser 965 970 975Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu 980 985 990Ile Leu Glu Thr Ser Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu 995 1000 1005Pro Ser Gly Ala Glu Ile Pro Glu Thr Phe Ala Ser Gly Val Gly 1010 1015 1020Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr 1025 1030 1035Ser Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly 1040 1045 1050Ala Glu Ile Pro Glu Thr Ser Ala Ser Gly Val Gly Thr Asp Leu 1055 1060 1065Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser Ala Ser 1070 1075 1080Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile 1085 1090 1095Leu Glu Thr Ser Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu 1100 1105 1110Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser Ala Ser Gly Val Gly 1115 1120 1125Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr 1130 1135 1140Ser Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly 1145 1150 1155Ala Glu Ile Leu Glu Thr Ser Ala Ser Gly Val Gly Thr Asp Leu 1160 1165 1170Ser Gly Leu Pro Ser Gly Gly Glu Ile Pro Glu Thr Phe Ala Ser 1175 1180 1185Gly Val Gly Asp Leu Ser Gly Leu Pro Pro Gly Arg Glu Asp Leu 1190 1195 1200Glu Thr Leu Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Ser Ser 1205 1210 1215Gly Lys Asp Gly Leu Val Gly Ser Ala Ser Gly Ala Leu Asp Phe 1220 1225 1230Gly Gly Thr Leu Gly Ser Gly Gln Ile Pro Glu Thr Ser Gly Leu 1235 1240 1245Pro Ser Gly Tyr Ser Gly Glu Tyr Ser Glu Val Asp Leu Gly Ser 1250 1255 1260Gly Pro Ser Ser Gly Leu Pro Asp Phe Ser Gly Leu Pro Ser Gly 1265 1270 1275Phe Pro Thr Val Ser Leu Val Asp Thr Pro Leu Val Glu Val Val 1280 1285 1290Thr Ala Thr Thr Ala Arg Glu Leu Glu Gly Arg Gly Thr Ile Gly 1295 1300 1305Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser Ser Glu Leu 1310 1315 1320Asp Val Ser Gly Gly Thr Ser Gly Ala Asp Ile Ser Gly Glu Ala 1325 1330 1335Asp Val Gly Gly Glu Ala Ser Gly Leu Ile Val Arg Gly Gln Pro 1340 1345 1350Ser Gly Phe Pro Asp Thr Ser Gly Glu Ala Phe Gly Val Thr Glu 1355 1360 1365Val Ser Gly Leu Ser Ser Gly Gln Pro Asp Leu Ser Gly Glu Ala 1370 1375 1380Ser Gly Val Leu Phe Gly Ser Gly Pro Pro Phe Gly Ile Thr Asp 1385 1390 1395Leu Ser Gly Glu Pro Ser Gly Gln Pro Ser Gly Leu Pro Glu Phe 1400 1405 1410Ser Gly Thr Thr His Arg Ile Pro Asp Leu Val Ser Gly Ala Thr 1415 1420 1425Ser Gly Ser Gly Glu Ser Ser Gly Ile Ala Phe Val Asp Thr Ser 1430 1435 1440Val Val Glu Val Thr Pro Thr Thr Leu Arg Glu Glu Glu Gly Leu 1445 1450 1455Gly Ser Val Glu Phe Ser Gly Phe Pro Ser Gly Glu Thr Gly Leu 1460 1465 1470Ser Gly Thr Pro Glu Thr Ile Asp Val Ser Gly Gln Ser Ser Gly 1475 1480 1485Thr Ile Asp Ser Ser Gly Phe Thr Ser Leu Ala Pro Glu Val Ser 1490 1495 1500Gly Ser Pro Ser Gly Val Ala Glu Val Ser Gly Glu Ala Ser Gly 1505 1510 1515Thr Glu Ile Thr Ser Gly Leu Pro Ser Gly Val Phe Asp Ser Ser 1520 1525 1530Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Arg Thr 1535 1540 1545Leu Val Glu Ser Val Thr Gln Ala Pro Thr Ala Gln Glu Ala Glu 1550 1555 1560Gly Pro Ser Asp Ile Leu Glu Leu Ser Gly Val His Ser Gly Leu 1565 1570 1575Pro Asp Val Ser Gly Ala His Ser Gly Phe Leu Asp Pro Ser Gly 1580 1585 1590Leu Gln Ser Gly Leu Val Glu Pro Ser Gly Glu Pro Pro Arg Thr 1595 1600 1605Pro Tyr Phe Ser Gly Asp Phe Pro Ser Thr Pro Asp Val Ser Gly 1610 1615 1620Glu Ala Ser Ala Ala Thr Ser Ser Ser Gly Asp Ile Ser Gly Leu 1625 1630 1635Pro Glu Val Thr Leu Val Thr Ser Glu Phe Met Glu Gly Val Thr 1640 1645 1650Arg Pro Thr Val Ser Gln Glu Leu Gly Gln Gly Pro Pro Met Thr 1655 1660 1665His Val Pro Lys Leu Phe Glu Ser Ser Gly Glu Ala Leu Ala Ser 1670 1675 1680Gly Asp Thr Ser Gly Ala Ala Pro Ala Phe Pro Gly Ser Gly Leu 1685 1690 1695Glu Ala Ser Ser Val Pro Glu Ser His Gly Glu Thr Ser Ala Tyr 1700 1705 1710Ala Glu Pro Gly Thr Lys Ala Ala Ala Ala Pro Asp Ala Ser Gly 1715 1720 1725Glu Ala Ser Gly Ser Pro Asp Ser Gly Glu Ile Thr Ser Val Phe 1730 1735 1740Arg Glu Ala Ala Gly Glu Gly Ala Ser Gly Leu Glu Val Ser Ser 1745 1750 1755Ser Ser Leu Ala Ser Gln Gln Gly Pro Arg Glu Gly Ser Ala Ser 1760 1765 1770Pro Glu Val Ser Gly Glu Ser Thr Thr Ser Tyr Glu Ile Gly Thr 1775 1780 1785Glu Thr Ser Gly Leu Pro Leu Ala Thr Pro Ala Ala Ser Glu Asp 1790 1795 1800Arg Ala Glu Val Ser Gly Asp Leu Ser Gly Arg Thr Pro Val Pro 1805 1810 1815Val Asp Val Val Thr Asn Val Pro Glu Ala Glu Trp Ile Gln His 1820 1825 1830Ser Gln Arg Pro Ala Glu Met Trp Pro Glu Thr Lys Ser Ser Ser 1835 1840 1845Pro Ser Tyr Ser Gly Glu Asp Thr Ala Gly Thr Ala Ala Ser Pro 1850 1855 1860Ala Ser Ala Asp Thr Pro Gly Glu Pro Gly Pro Thr Thr Ala Ala 1865 1870 1875Pro Arg Ser Cys Ala Glu Glu Pro Cys Gly Pro Gly Thr Cys Gln 1880 1885 1890Glu Thr Glu Gly Arg Val Thr Cys Leu Cys Pro Pro Gly His Thr 1895 1900 1905Gly Glu Tyr Cys Asp Ile Asp Ile Asp Glu Cys Leu Ser Ser Pro 1910 1915 1920Cys Val Asn Gly Ala Thr Cys Val Asp Ala Ser Asp Ser Phe Thr 1925 1930 1935Cys Leu Cys Leu Pro Ser Tyr Gly Gly Asp Leu Cys Glu Thr Asp 1940 1945 1950Gln Glu Val Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly His Cys 1955 1960 1965Tyr Arg His Phe Pro Asp Arg Glu Thr Trp Val Asp Ala Glu Gly 1970 1975 1980Arg Cys Arg Glu Gln Gln Ser His Leu Ser Ser Ile Val Thr Pro 1985 1990 1995Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr Gln Trp 2000 2005 2010Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg Trp Ser 2015 2020 2025Asp Gly His Pro Leu Gln Phe Glu Asn Trp Arg Pro Asn Gln Pro 2030 2035 2040Asp Asn Phe Phe Ala Thr Gly Glu Asp Cys Val Val Met Ile Trp 2045 2050 2055His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr His Leu 2060 2065 2070Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Asp Pro Pro 2075 2080 2085Val Val Glu His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg Tyr 2090 2095 2100Glu Ile Asn Ser Leu Val Arg Tyr Gln Cys Ala Glu Gly Phe Thr 2105 2110 2115Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Gly His Trp 2120 2125 2130Glu Glu Pro Arg Ile Thr Cys Thr His Pro Thr Thr Tyr Lys Arg 2135 2140 2145Arg Val Gln Lys Arg Ser Ser Arg Thr Leu Gln Arg Ser Gln Ala 2150 2155 2160Ser Ser Ala Pro 21651322266PRTMacaca fascicularis 132Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Ala Ala1 5 10 15Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser 20 25 30Ile Pro Gln Pro Ser Pro Leu Arg Val Leu Leu Gly Thr Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys65 70 75 80Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val 85 90 95Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Ile Gln Ser Leu Arg Ser Asn Asp Ser 115 120 125Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala 130 135 140Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 260 265 270Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 275 280 285Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315

320Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu 355 360 365Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu 370 375 380Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr385 390 395 400Val Lys Pro Ile Phe Asp Val Ser Pro Ser Pro Leu Glu Pro Glu Glu 405 410 415Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Pro Glu Val 420 425 430Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro 435 440 445Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln 450 455 460Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe465 470 475 480His Tyr Arg Pro Gly Ser Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala 485 490 495Gln Gln Ala Cys Leu Arg Thr Gly Ala Val Ile Ala Ser Pro Glu Gln 500 505 510Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp 515 520 525Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro 530 535 540Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val545 550 555 560Arg Pro Ser Thr Glu Thr Tyr Asp Val Tyr Cys Tyr Val Asp Arg Leu 565 570 575Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln 580 585 590Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Leu Ala Thr Thr 595 600 605Gly Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala 610 615 620Gly Trp Leu Ala Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr Pro Arg625 630 635 640Pro Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr 645 650 655Pro Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe 660 665 670Cys Phe Arg Gly Val Ser Ala Val Pro Ser Pro Gly Glu Glu Glu Gly 675 680 685Gly Thr Pro Thr Ser Pro Ser Gly Val Glu Asp Trp Ile Ala Thr Gln 690 695 700Val Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala705 710 715 720Val Pro Leu Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro 725 730 735Glu Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Met Gly Thr Ser 740 745 750Pro Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Thr Ala Thr 755 760 765Glu Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Leu Thr Ala Ser 770 775 780Lys Glu Pro Ser Pro Pro Glu Val Pro Phe Pro Ser Glu Glu Pro Ser785 790 795 800Pro Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu 805 810 815Pro Ser Pro Ser Glu Glu Pro Phe Pro Ser Val Glu Pro Ser Pro Ser 820 825 830Glu Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Val 835 840 845Pro Ser Trp Thr Glu Leu Pro Gly Ser Gly Glu Glu Ser Gly Ala Pro 850 855 860Asp Val Ser Gly Asp Phe Ile Gly Ser Gly Asp Val Ser Gly His Leu865 870 875 880Asp Phe Ser Gly Gln Leu Ser Gly Asp Arg Ile Ser Gly Leu Pro Ser 885 890 895Gly Asp Leu Asp Ser Ser Gly Leu Thr Ser Thr Val Gly Ser Gly Leu 900 905 910Pro Val Asp Ser Gly Leu Ala Ser Gly Asp Glu Glu Arg Ile Glu Trp 915 920 925Ser Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu 930 935 940Glu Gly Ser Ala Ser Glu Val Gly Asp Leu Ser Gly Leu Pro Ser Gly945 950 955 960Asp Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu 965 970 975Pro Ser Gly Glu Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu 980 985 990Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val 995 1000 1005Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser 1010 1015 1020Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Ala Gly Glu 1025 1030 1035Val Leu Glu Thr Thr Ala Ser Gly Val Glu Asp Ile Ser Gly Leu 1040 1045 1050Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser Gly Val Glu Asp 1055 1060 1065Ile Ser Gly Phe Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser 1070 1075 1080Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu 1085 1090 1095Thr Thr Ala Ser Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly 1100 1105 1110Glu Val Leu Glu Thr Thr Ala Ser Gly Val Gly Asp Leu Gly Gly 1115 1120 1125Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val Gly 1130 1135 1140Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Val Glu Thr Ser Thr 1145 1150 1155Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu Val 1160 1165 1170Leu Glu Thr Ser Thr Ser Gly Val Glu Asp Ile Ser Gly Leu Pro 1175 1180 1185Ser Gly Glu Val Leu Glu Thr Thr Ala Ser Gly Ile Glu Asp Val 1190 1195 1200Ser Glu Leu Pro Ser Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly 1205 1210 1215Val Glu Asp Leu Ser Arg Leu Pro Ser Gly Glu Val Leu Glu Thr 1220 1225 1230Ser Ala Ser Gly Val Gly Asp Ile Ser Gly Leu Pro Ser Gly Gly 1235 1240 1245Glu Val Leu Glu Ile Ser Ala Ser Gly Val Gly Asp Leu Ser Gly 1250 1255 1260Leu Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val 1265 1270 1275Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu 1280 1285 1290Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser Gly Leu Pro Ser Gly 1295 1300 1305Lys Glu Asp Leu Val Gly Pro Ala Ser Gly Asp Leu Asp Leu Gly 1310 1315 1320Lys Leu Pro Ser Gly Thr Leu Arg Ser Gly Gln Ala Pro Glu Thr 1325 1330 1335Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly Val Asp 1340 1345 1350Leu Gly Ser Gly Pro Pro Ser Gly Leu Pro Asp Phe Ser Gly Leu 1355 1360 1365Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser Thr Leu Val 1370 1375 1380Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu Glu Gly Arg Gly 1385 1390 1395Thr Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser 1400 1405 1410Ser Glu Leu Asp Ile Ser Gly Glu Ala Ser Gly Leu Pro Ser Gly 1415 1420 1425Thr Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro Asp Val Ser Arg 1430 1435 1440Glu Thr Pro Gly Leu Phe Asp Val Ser Gly Gln Pro Ser Gly Phe 1445 1450 1455Pro Asp Ile Ser Gly Gly Thr Ser Gly Ile Ser Glu Val Ser Gly 1460 1465 1470Gln Pro Ser Gly Phe Pro Asp Thr Ser Gly Glu Thr Ser Gly Val 1475 1480 1485Thr Glu Leu Ser Gly Leu Pro Ser Gly Gln Pro Gly Val Ser Gly 1490 1495 1500Glu Ala Ser Gly Val Pro Tyr Gly Ser Ser Gln Pro Phe Gly Ile 1505 1510 1515Thr Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp Leu Ser Gly 1520 1525 1530Gln Pro Ser Gly Leu Pro Gly Phe Ser Gly Ala Thr Ser Gly Val 1535 1540 1545Pro Asp Leu Val Ser Gly Ala Thr Ser Gly Ser Gly Glu Ser Ser 1550 1555 1560Gly Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Thr Pro Thr 1565 1570 1575Thr Phe Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly 1580 1585 1590Leu Pro Ser Gly Glu Ala Asp Leu Ser Gly Arg Ser Gly Met Val 1595 1600 1605Asp Val Ser Gly Gln Phe Ser Gly Thr Val Asp Ser Ser Gly Phe 1610 1615 1620Thr Ser Gln Thr Pro Glu Phe Ser Gly Leu Pro Ile Gly Ile Ala 1625 1630 1635Glu Val Ser Gly Glu Ser Ser Gly Ala Glu Thr Gly Ser Ser Leu 1640 1645 1650Pro Ser Gly Ala Tyr Tyr Gly Ser Gly Leu Pro Ser Gly Phe Pro 1655 1660 1665Thr Val Ser Leu Val Asp Arg Thr Leu Val Glu Ser Val Thr Gln 1670 1675 1680Ala Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro Pro Gly Ile Leu 1685 1690 1695Glu Leu Ser Gly Thr His Ser Gly Ala Pro Asp Met Ser Gly Asp 1700 1705 1710His Ser Gly Phe Leu Asp Val Ser Gly Leu Gln Phe Gly Leu Val 1715 1720 1725Glu Pro Ser Gly Glu Pro Pro Ser Thr Pro Tyr Phe Ser Gly Asp 1730 1735 1740Phe Ala Ser Thr Thr Asp Val Ser Gly Glu Ser Ser Ala Ala Met 1745 1750 1755Gly Thr Ser Gly Glu Ala Ser Gly Leu Pro Gly Val Thr Leu Ile 1760 1765 1770Thr Ser Glu Phe Met Glu Gly Val Thr Glu Pro Thr Val Ser Gln 1775 1780 1785Glu Leu Gly Gln Arg Pro Pro Val Thr His Thr Pro Gln Leu Phe 1790 1795 1800Glu Ser Ser Gly Glu Ala Ser Ala Ala Gly Asp Ile Ser Gly Ala 1805 1810 1815Thr Pro Val Leu Pro Gly Ser Gly Val Glu Val Ser Ser Val Pro 1820 1825 1830Glu Ser Ser Ser Glu Thr Ser Ala Tyr Pro Glu Ala Gly Val Gly 1835 1840 1845Ala Ser Ala Ala Pro Glu Thr Ser Gly Glu Asp Ser Gly Ser Pro 1850 1855 1860Asp Leu Ser Glu Thr Thr Ser Ala Phe His Glu Ala Asp Leu Glu 1865 1870 1875Arg Ser Ser Gly Leu Gly Val Ser Gly Ser Thr Leu Thr Phe Gln 1880 1885 1890Glu Gly Glu Pro Ser Ala Ser Pro Glu Val Ser Gly Glu Ser Thr 1895 1900 1905Thr Thr Gly Asp Val Gly Thr Glu Ala Pro Gly Leu Pro Ser Ala 1910 1915 1920Thr Pro Thr Ala Ser Gly Asp Arg Thr Glu Ile Ser Gly Asp Leu 1925 1930 1935Ser Gly His Thr Ser Gly Leu Gly Val Val Ile Ser Thr Ser Ile 1940 1945 1950Pro Glu Ser Glu Trp Thr Gln Gln Thr Gln Arg Pro Ala Glu Ala 1955 1960 1965His Leu Glu Thr Glu Ser Ser Ser Leu Leu Tyr Ser Gly Glu Glu 1970 1975 1980Thr His Thr Ala Glu Thr Ala Thr Ser Pro Thr Asp Ala Ser Ile 1985 1990 1995Pro Ala Ser Pro Glu Trp Thr Gly Glu Ser Glu Ser Thr Val Ala 2000 2005 2010Asp Ile Asp Glu Cys Leu Ser Ser Pro Cys Leu Asn Gly Ala Thr 2015 2020 2025Cys Val Asp Ala Ile Asp Ser Phe Thr Cys Leu Cys Leu Pro Ser 2030 2035 2040Tyr Gly Gly Asp Leu Cys Glu Ile Asp Gln Glu Val Cys Glu Glu 2045 2050 2055Gly Trp Thr Lys Tyr Gln Gly His Cys Tyr Arg His Phe Pro Asp 2060 2065 2070Arg Glu Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln Gln 2075 2080 2085Ser His Leu Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe Val 2090 2095 2100Asn Asn Asn Ala Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp Arg 2105 2110 2115Thr Ile Glu Gly Asp Phe Arg Trp Ser Asp Gly His Pro Met Gln 2120 2125 2130Phe Glu Asn Trp Arg Pro Asn Gln Pro Asp Asn Phe Phe Ala Ala 2135 2140 2145Gly Glu Asp Cys Val Val Met Ile Trp His Glu Lys Gly Glu Trp 2150 2155 2160Asn Asp Val Pro Cys Asn Tyr His Leu Pro Phe Thr Cys Lys Lys 2165 2170 2175Gly Thr Val Ala Cys Gly Glu Pro Pro Met Val Gln His Ala Arg 2180 2185 2190Thr Phe Gly Gln Lys Lys Asp Arg Tyr Glu Ile Asn Ser Leu Val 2195 2200 2205Arg Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg His Val Pro Thr 2210 2215 2220Ile Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro Arg Ile Thr 2225 2230 2235Cys Thr Asp Ala Thr Ala Tyr Lys Arg Arg Leu Gln Lys Arg Ser 2240 2245 2250Ser Arg His Pro Arg Arg Ser Arg Pro Ser Thr Ala His 2255 2260 22651332167PRTMacaca mulattaVARIANT(1910)..(1915)Xaa can be any naturally occurring amino acid 133Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Ala Ala1 5 10 15Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser 20 25 30Ile Pro Gln Pro Ser Pro Leu Arg Val Leu Leu Gly Thr Ser Leu Thr 35 40 45Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro 50 55 60Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys65 70 75 80Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val 85 90 95Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile 100 105 110Pro Ser Asp Ala Thr Leu Glu Ile Gln Ser Leu Arg Ser Asn Asp Ser 115 120 125Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala 130 135 140Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile145 150 155 160Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu 165 170 175Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr 180 185 190Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr 195 200 205Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys 210 215 220Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu225 230 235 240Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe 245 250 255Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu 260 265 270Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu 275 280 285Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp 290 295 300Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly305 310 315 320Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly 325 330 335Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala Ile Cys Tyr Thr Gly Glu 340 345 350Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu 355 360 365Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu 370 375 380Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr385 390 395 400Val Lys Pro Ile Phe Asp Val Ser Pro Ser Pro Leu Glu Pro Glu Glu 405 410 415Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Pro Glu Val 420 425 430Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro 435 440 445Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln 450 455 460Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe465 470 475 480His Tyr Arg Pro Gly Ser Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala

485 490 495Gln Gln Ala Cys Leu Arg Thr Gly Ala Val Ile Ala Ser Pro Glu Gln 500 505 510Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp 515 520 525Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro 530 535 540Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val545 550 555 560Arg Pro Ser Thr Glu Thr Tyr Asp Val Tyr Cys Tyr Val Asp Arg Leu 565 570 575Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln 580 585 590Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Leu Ala Thr Thr 595 600 605Gly Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala 610 615 620Gly Trp Leu Ala Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr Pro Arg625 630 635 640Pro Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr 645 650 655Pro Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe 660 665 670Cys Phe Arg Gly Val Ser Ala Val Pro Ser Pro Gly Glu Glu Glu Gly 675 680 685Gly Thr Pro Thr Ser Pro Ser Gly Val Glu Asp Trp Ile Ala Thr Gln 690 695 700Val Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala705 710 715 720Val Pro Leu Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro 725 730 735Glu Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Met Gly Thr Ser 740 745 750Pro Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Thr Ala Thr 755 760 765Glu Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Leu Thr Ala Ser 770 775 780Lys Glu Pro Ser Pro Pro Glu Val Pro Phe Pro Ser Glu Glu Pro Ser785 790 795 800Pro Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu 805 810 815Pro Ser Pro Ser Glu Glu Pro Phe Pro Ser Val Glu Pro Ser Pro Ser 820 825 830Glu Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Val 835 840 845Pro Ser Trp Thr Glu Leu Pro Gly Ser Gly Glu Glu Ser Gly Ala Pro 850 855 860Asp Val Ser Gly Asp Phe Ile Gly Ser Gly Asp Val Ser Gly His Leu865 870 875 880Asp Phe Ser Gly Gln Leu Ser Gly Asp Arg Ile Ser Gly Leu Pro Ser 885 890 895Gly Asp Leu Asp Ser Ser Gly Leu Thr Ser Thr Val Gly Ser Gly Leu 900 905 910Pro Val Asp Ser Gly Leu Ala Ser Gly Asp Glu Glu Arg Ile Glu Trp 915 920 925Ser Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu 930 935 940Glu Gly Ser Ala Ser Glu Val Gly Asp Leu Ser Gly Leu Pro Ser Gly945 950 955 960Asp Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu 965 970 975Pro Ser Gly Glu Val Leu Glu Thr Ser Val Ser Gly Val Gly Asp Leu 980 985 990Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val 995 1000 1005Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser 1010 1015 1020Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Ala Gly Glu 1025 1030 1035Val Leu Glu Thr Thr Ala Ser Gly Val Glu Asp Ile Ser Gly Leu 1040 1045 1050Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser Gly Val Glu Asp 1055 1060 1065Ile Ser Gly Phe Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser 1070 1075 1080Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu 1085 1090 1095Thr Thr Ala Ser Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly 1100 1105 1110Glu Val Leu Glu Thr Thr Ala Ser Gly Val Gly Asp Leu Gly Gly 1115 1120 1125Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val Gly 1130 1135 1140Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Val Glu Thr Ser Thr 1145 1150 1155Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu Val 1160 1165 1170Leu Glu Thr Ser Thr Ser Gly Val Glu Asp Ile Ser Gly Leu Pro 1175 1180 1185Ser Gly Glu Val Leu Glu Thr Thr Ala Ser Gly Ile Glu Asp Val 1190 1195 1200Ser Glu Leu Pro Ser Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly 1205 1210 1215Val Glu Asp Leu Ser Arg Leu Pro Ser Gly Glu Val Leu Glu Thr 1220 1225 1230Ser Ala Ser Gly Val Gly Asp Ile Ser Gly Leu Pro Ser Gly Gly 1235 1240 1245Glu Val Leu Glu Ile Ser Ala Ser Gly Val Gly Asp Leu Ser Gly 1250 1255 1260Leu Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val 1265 1270 1275Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu 1280 1285 1290Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser Gly Leu Pro Ser Gly 1295 1300 1305Lys Glu Asp Leu Val Gly Pro Ala Ser Gly Asp Leu Asp Leu Gly 1310 1315 1320Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln Ala Pro Glu Thr 1325 1330 1335Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly Val Asp 1340 1345 1350Leu Gly Ser Gly Pro Pro Ser Gly Leu Pro Asp Phe Ser Gly Leu 1355 1360 1365Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser Thr Leu Val 1370 1375 1380Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu Glu Gly Arg Gly 1385 1390 1395Thr Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser 1400 1405 1410Ser Glu Leu Asp Ile Ser Gly Glu Ala Ser Gly Leu Pro Ser Gly 1415 1420 1425Thr Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro Asp Val Ser Arg 1430 1435 1440Glu Thr Ser Gly Leu Phe Asp Val Ser Gly Gln Pro Ser Gly Phe 1445 1450 1455Pro Asp Thr Ser Gly Glu Thr Ser Gly Val Thr Glu Leu Ser Gly 1460 1465 1470Leu Pro Ser Gly Gln Pro Gly Val Ser Gly Glu Ala Ser Gly Val 1475 1480 1485Pro Tyr Gly Ser Ser Gln Pro Phe Gly Ile Thr Asp Leu Ser Gly 1490 1495 1500Glu Thr Ser Gly Val Pro Asp Leu Ser Gly Gln Pro Ser Gly Leu 1505 1510 1515Pro Gly Phe Ser Gly Ala Thr Ser Gly Val Pro Asp Leu Val Ser 1520 1525 1530Gly Ala Thr Ser Gly Ser Gly Glu Ser Ser Asp Ile Thr Phe Val 1535 1540 1545Asp Thr Ser Leu Val Glu Val Thr Pro Thr Thr Phe Lys Glu Glu 1550 1555 1560Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu Pro Ser Gly Glu 1565 1570 1575Ala Asp Leu Ser Gly Arg Ser Gly Met Val Asp Val Ser Gly Gln 1580 1585 1590Phe Ser Gly Thr Val Asp Ser Ser Gly Phe Thr Ser Gln Thr Pro 1595 1600 1605Glu Phe Ser Gly Leu Pro Ile Gly Ile Ala Glu Val Ser Gly Glu 1610 1615 1620Ser Ser Gly Ala Glu Thr Gly Ser Ser Leu Pro Ser Gly Ala Tyr 1625 1630 1635Tyr Gly Ser Glu Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val 1640 1645 1650Asp Arg Thr Leu Val Glu Ser Val Thr Gln Ala Pro Thr Ala Gln 1655 1660 1665Glu Ala Gly Glu Gly Pro Pro Gly Ile Leu Glu Leu Ser Gly Thr 1670 1675 1680His Ser Gly Ala Pro Asp Met Ser Gly Asp His Ser Gly Phe Leu 1685 1690 1695Asp Val Ser Gly Leu Gln Phe Gly Leu Val Glu Pro Ser Gly Glu 1700 1705 1710Pro Pro Ser Thr Pro Tyr Phe Ser Gly Asp Phe Ala Ser Thr Thr 1715 1720 1725Asp Val Ser Gly Glu Ser Ser Ala Ala Met Gly Thr Asn Gly Glu 1730 1735 1740Ala Ser Gly Leu Pro Glu Val Thr Leu Ile Thr Ser Glu Phe Met 1745 1750 1755Glu Gly Val Thr Glu Pro Thr Val Ser Gln Glu Leu Gly Gln Arg 1760 1765 1770Pro Pro Val Thr His Thr Pro Gln Leu Phe Glu Ser Ser Gly Glu 1775 1780 1785Ala Ser Ala Ala Gly Asp Ile Ser Gly Ala Thr Pro Val Leu Pro 1790 1795 1800Gly Ser Gly Val Glu Val Ser Ser Val Pro Glu Ser Ser Ser Glu 1805 1810 1815Thr Ser Ala Tyr Pro Glu Ala Gly Val Gly Ala Ser Ala Ala Pro 1820 1825 1830Glu Thr Ser Gly Glu Asp Ser Gly Ser Pro Asp Leu Ser Glu Thr 1835 1840 1845Thr Ser Ala Phe His Glu Ala Asp Leu Glu Arg Ser Ser Gly Leu 1850 1855 1860Gly Val Ser Gly Ser Thr Leu Thr Phe Gln Glu Gly Glu Pro Ser 1865 1870 1875Ala Ser Pro Glu Val Ser Gly Glu Ser Thr Thr Thr Gly Asp Val 1880 1885 1890Gly Thr Glu Ala Pro Gly Leu Pro Ser Ala Thr Pro Thr Ala Ser 1895 1900 1905Gly Xaa Xaa Xaa Xaa Xaa Xaa Pro Thr Arg Ser Cys Ala Glu Glu 1910 1915 1920Pro Cys Gly Ala Gly Thr Cys Lys Glu Thr Glu Gly His Val Ile 1925 1930 1935Cys Leu Cys Pro Pro Gly Tyr Thr Gly Glu His Cys Asn Ile Asp 1940 1945 1950Gln Glu Val Cys Glu Glu Gly Trp Thr Lys Tyr Gln Gly His Cys 1955 1960 1965Tyr Arg His Phe Pro Asp Arg Glu Thr Trp Val Asp Ala Glu Arg 1970 1975 1980Arg Cys Arg Glu Gln Gln Ser His Leu Ser Ser Ile Val Thr Pro 1985 1990 1995Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr Gln Trp 2000 2005 2010Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg Trp Ser 2015 2020 2025Asp Gly His Pro Met Gln Phe Glu Asn Trp Arg Pro Asn Gln Pro 2030 2035 2040Asp Asn Phe Phe Ala Ala Gly Glu Asp Cys Val Val Met Ile Trp 2045 2050 2055His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr His Leu 2060 2065 2070Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu Pro Pro 2075 2080 2085Met Val Gln His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg Tyr 2090 2095 2100Glu Ile Asn Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly Phe Val 2105 2110 2115Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Gly His Trp 2120 2125 2130Glu Glu Pro Arg Ile Thr Cys Thr Asp Ala Thr Ala Tyr Lys Arg 2135 2140 2145Arg Leu Gln Lys Arg Ser Ser Arg His Pro Arg Arg Ser Arg Pro 2150 2155 2160Ser Thr Ala His 21651341321PRTHomo sapiens 134Met Gly Ala Pro Phe Val Trp Ala Leu Gly Leu Leu Met Leu Gln Met1 5 10 15Leu Leu Phe Val Ala Gly Glu Gln Gly Thr Gln Asp Ile Thr Asp Ala 20 25 30Ser Glu Arg Gly Leu His Met Gln Lys Leu Gly Ser Gly Ser Val Gln 35 40 45Ala Ala Leu Ala Glu Leu Val Ala Leu Pro Cys Leu Phe Thr Leu Gln 50 55 60Pro Arg Pro Ser Ala Ala Arg Asp Ala Pro Arg Ile Lys Trp Thr Lys65 70 75 80Val Arg Thr Ala Ser Gly Gln Arg Gln Asp Leu Pro Ile Leu Val Ala 85 90 95Lys Asp Asn Val Val Arg Val Ala Lys Ser Trp Gln Gly Arg Val Ser 100 105 110Leu Pro Ser Tyr Pro Arg Arg Arg Ala Asn Ala Thr Leu Leu Leu Gly 115 120 125Pro Leu Arg Ala Ser Asp Ser Gly Leu Tyr Arg Cys Gln Val Val Arg 130 135 140Gly Ile Glu Asp Glu Gln Asp Leu Val Pro Leu Glu Val Thr Gly Val145 150 155 160Val Phe His Tyr Arg Ser Ala Arg Asp Arg Tyr Ala Leu Thr Phe Ala 165 170 175Glu Ala Gln Glu Ala Cys Arg Leu Ser Ser Ala Ile Ile Ala Ala Pro 180 185 190Arg His Leu Gln Ala Ala Phe Glu Asp Gly Phe Asp Asn Cys Asp Ala 195 200 205Gly Trp Leu Ser Asp Arg Thr Val Arg Tyr Pro Ile Thr Gln Ser Arg 210 215 220Pro Gly Cys Tyr Gly Asp Arg Ser Ser Leu Pro Gly Val Arg Ser Tyr225 230 235 240Gly Arg Arg Asn Pro Gln Glu Leu Tyr Asp Val Tyr Cys Phe Ala Arg 245 250 255Glu Leu Gly Gly Glu Val Phe Tyr Val Gly Pro Ala Arg Arg Leu Thr 260 265 270Leu Ala Gly Ala Arg Ala Gln Cys Arg Arg Gln Gly Ala Ala Leu Ala 275 280 285Ser Val Gly Gln Leu His Leu Ala Trp His Glu Gly Leu Asp Gln Cys 290 295 300Asp Pro Gly Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Gln Thr305 310 315 320Pro Arg Arg Arg Cys Gly Gly Pro Ala Pro Gly Val Arg Thr Val Tyr 325 330 335Arg Phe Ala Asn Arg Thr Gly Phe Pro Ser Pro Ala Glu Arg Phe Asp 340 345 350Ala Tyr Cys Phe Arg Ala His His Pro Thr Ser Gln His Gly Asp Leu 355 360 365Glu Thr Pro Ser Ser Gly Asp Glu Gly Glu Ile Leu Ser Ala Glu Gly 370 375 380Pro Pro Val Arg Glu Leu Glu Pro Thr Leu Glu Glu Glu Glu Val Val385 390 395 400Thr Pro Asp Phe Gln Glu Pro Leu Val Ser Ser Gly Glu Glu Glu Thr 405 410 415Leu Ile Leu Glu Glu Lys Gln Glu Ser Gln Gln Thr Leu Ser Pro Thr 420 425 430Pro Gly Asp Pro Met Leu Ala Ser Trp Pro Thr Gly Glu Val Trp Leu 435 440 445Ser Thr Val Ala Pro Ser Pro Ser Asp Met Gly Ala Gly Thr Ala Ala 450 455 460Ser Ser His Thr Glu Val Ala Pro Thr Asp Pro Met Pro Arg Arg Arg465 470 475 480Gly Arg Phe Lys Gly Leu Asn Gly Arg Tyr Phe Gln Gln Gln Glu Pro 485 490 495Glu Pro Gly Leu Gln Gly Gly Met Glu Ala Ser Ala Gln Pro Pro Thr 500 505 510Ser Glu Ala Ala Val Asn Gln Met Glu Pro Pro Leu Ala Met Ala Val 515 520 525Thr Glu Met Leu Gly Ser Gly Gln Ser Arg Ser Pro Trp Ala Asp Leu 530 535 540Thr Asn Glu Val Asp Met Pro Gly Ala Gly Ser Ala Gly Gly Lys Ser545 550 555 560Ser Pro Glu Pro Trp Leu Trp Pro Pro Thr Met Val Pro Pro Ser Ile 565 570 575Ser Gly His Ser Arg Ala Pro Val Leu Glu Leu Glu Lys Ala Glu Gly 580 585 590Pro Ser Ala Arg Pro Ala Thr Pro Asp Leu Phe Trp Ser Pro Leu Glu 595 600 605Ala Thr Val Ser Ala Pro Ser Pro Ala Pro Trp Glu Ala Phe Pro Val 610 615 620Ala Thr Ser Pro Asp Leu Pro Met Met Ala Met Leu Arg Gly Pro Lys625 630 635 640Glu Trp Met Leu Pro His Pro Thr Pro Ile Ser Thr Glu Ala Asn Arg 645 650 655Val Glu Ala His Gly Glu Ala Thr Ala Thr Ala Pro Pro Ser Pro Ala 660 665 670Ala Glu Thr Lys Val Tyr Ser Leu Pro Leu Ser Leu Thr Pro Thr Gly 675 680 685Gln Gly Gly Glu Ala Met Pro Thr Thr Pro Glu Ser Pro Arg Ala Asp 690 695 700Phe Arg Glu Thr Gly Glu Thr Ser Pro Ala Gln Val Asn Lys Ala Glu705 710 715 720His Ser Ser Ser Ser Pro Trp Pro Ser Val Asn Arg Asn Val Ala Val 725 730 735Gly Phe Val Pro Thr Glu Thr Ala Thr Glu Pro Thr Gly Leu Arg Gly 740 745 750Ile Pro Gly Ser Glu Ser Gly Val Phe Asp Thr Ala Glu Ser Pro Thr 755 760 765Ser Gly Leu Gln Ala Thr Val Asp Glu Val Gln Asp Pro Trp

Pro Ser 770 775 780Val Tyr Ser Lys Gly Leu Asp Ala Ser Ser Pro Ser Ala Pro Leu Gly785 790 795 800Ser Pro Gly Val Phe Leu Val Pro Lys Val Thr Pro Asn Leu Glu Pro 805 810 815Trp Val Ala Thr Asp Glu Gly Pro Thr Val Asn Pro Met Asp Ser Thr 820 825 830Val Thr Pro Ala Pro Ser Asp Ala Ser Gly Ile Trp Glu Pro Gly Ser 835 840 845Gln Val Phe Glu Glu Ala Glu Ser Thr Thr Leu Ser Pro Gln Val Ala 850 855 860Leu Asp Thr Ser Ile Val Thr Pro Leu Thr Thr Leu Glu Gln Gly Asp865 870 875 880Lys Val Gly Val Pro Ala Met Ser Thr Leu Gly Ser Ser Ser Ser Gln 885 890 895Pro His Pro Glu Pro Glu Asp Gln Val Glu Thr Gln Gly Thr Ser Gly 900 905 910Ala Ser Val Pro Pro His Gln Ser Ser Pro Leu Gly Lys Pro Ala Val 915 920 925Pro Pro Gly Thr Pro Thr Ala Ala Ser Val Gly Glu Ser Ala Ser Val 930 935 940Ser Ser Gly Glu Pro Thr Val Pro Trp Asp Pro Ser Ser Thr Leu Leu945 950 955 960Pro Val Thr Leu Gly Ile Glu Asp Phe Glu Leu Glu Val Leu Ala Gly 965 970 975Ser Pro Gly Val Glu Ser Phe Trp Glu Glu Val Ala Ser Gly Glu Glu 980 985 990Pro Ala Leu Pro Gly Thr Pro Met Asn Ala Gly Ala Glu Glu Val His 995 1000 1005Ser Asp Pro Cys Glu Asn Asn Pro Cys Leu His Gly Gly Thr Cys 1010 1015 1020Asn Ala Asn Gly Thr Met Tyr Gly Cys Ser Cys Asp Gln Gly Phe 1025 1030 1035Ala Gly Glu Asn Cys Glu Ile Asp Ile Asp Asp Cys Leu Cys Ser 1040 1045 1050Pro Cys Glu Asn Gly Gly Thr Cys Ile Asp Glu Val Asn Gly Phe 1055 1060 1065Val Cys Leu Cys Leu Pro Ser Tyr Gly Gly Ser Phe Cys Glu Lys 1070 1075 1080Asp Thr Glu Gly Cys Asp Arg Gly Trp His Lys Phe Gln Gly His 1085 1090 1095Cys Tyr Arg Tyr Phe Ala His Arg Arg Ala Trp Glu Asp Ala Glu 1100 1105 1110Lys Asp Cys Arg Arg Arg Ser Gly His Leu Thr Ser Val His Ser 1115 1120 1125Pro Glu Glu His Ser Phe Ile Asn Ser Phe Gly His Glu Asn Thr 1130 1135 1140Trp Ile Gly Leu Asn Asp Arg Ile Val Glu Arg Asp Phe Gln Trp 1145 1150 1155Thr Asp Asn Thr Gly Leu Gln Phe Glu Asn Trp Arg Glu Asn Gln 1160 1165 1170Pro Asp Asn Phe Phe Ala Gly Gly Glu Asp Cys Val Val Met Val 1175 1180 1185Ala His Glu Ser Gly Arg Trp Asn Asp Val Pro Cys Asn Tyr Asn 1190 1195 1200Leu Pro Tyr Val Cys Lys Lys Gly Thr Val Leu Cys Gly Pro Pro 1205 1210 1215Pro Ala Val Glu Asn Ala Ser Leu Ile Gly Ala Arg Lys Ala Lys 1220 1225 1230Tyr Asn Val His Ala Thr Val Arg Tyr Gln Cys Asn Glu Gly Phe 1235 1240 1245Ala Gln His His Val Ala Thr Ile Arg Cys Arg Ser Asn Gly Lys 1250 1255 1260Trp Asp Arg Pro Gln Ile Val Cys Thr Lys Pro Arg Arg Ser His 1265 1270 1275Arg Met Arg Arg His His His His His Gln His His His Gln His 1280 1285 1290His His His Lys Ser Arg Lys Glu Arg Arg Lys His Lys Lys His 1295 1300 1305Pro Thr Glu Asp Trp Glu Lys Asp Glu Gly Asn Phe Cys 1310 1315 1320135911PRTHomo sapiens 135Met Ala Gln Leu Phe Leu Pro Leu Leu Ala Ala Leu Val Leu Ala Gln1 5 10 15Ala Pro Ala Ala Leu Ala Asp Val Leu Glu Gly Asp Ser Ser Glu Asp 20 25 30Arg Ala Phe Arg Val Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val 35 40 45Leu Gly Gly Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro 50 55 60Pro Pro Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr65 70 75 80Phe Leu Ser Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly Val 85 90 95Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro Ala 100 105 110Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Ala Leu Ser Glu Leu Arg 115 120 125Pro Asn Asp Ser Gly Ile Tyr Arg Cys Glu Val Gln His Gly Ile Asp 130 135 140Asp Ser Ser Asp Ala Val Glu Val Lys Val Lys Gly Val Val Phe Leu145 150 155 160Tyr Arg Glu Gly Ser Ala Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln 165 170 175Glu Ala Cys Ala Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu 180 185 190Tyr Ala Ala Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu 195 200 205Ser Asp Gln Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala Cys 210 215 220Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr Gly Val Val225 230 235 240Asp Pro Asp Asp Leu Tyr Asp Val Tyr Cys Tyr Ala Glu Asp Leu Asn 245 250 255Gly Glu Leu Phe Leu Gly Asp Pro Pro Glu Lys Leu Thr Leu Glu Glu 260 265 270Ala Arg Ala Tyr Cys Gln Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly 275 280 285Gln Leu Tyr Ala Ala Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly 290 295 300Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln305 310 315 320Arg Cys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro 325 330 335Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val Tyr Cys 340 345 350Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu Ala Ser Asn Pro 355 360 365Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu Ala Ile Val Thr Val Thr 370 375 380Glu Thr Leu Glu Glu Leu Gln Leu Pro Gln Glu Ala Thr Glu Ser Glu385 390 395 400Ser Arg Gly Ala Ile Tyr Ser Ile Pro Ile Met Glu Asp Gly Gly Gly 405 410 415Gly Ser Ser Thr Pro Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu 420 425 430Glu Phe Glu Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser Glu Glu 435 440 445Glu Gly Lys Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu Glu 450 455 460Lys Glu Glu Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala Leu Trp465 470 475 480Ala Trp Pro Ser Glu Leu Ser Ser Pro Gly Pro Glu Ala Ser Leu Pro 485 490 495Thr Glu Pro Ala Ala Gln Glu Glu Ser Leu Ser Gln Ala Pro Ala Arg 500 505 510Ala Val Leu Gln Pro Gly Ala Ser Pro Leu Pro Asp Gly Glu Ser Glu 515 520 525Ala Ser Arg Pro Pro Arg Val His Gly Pro Pro Thr Glu Thr Leu Pro 530 535 540Thr Pro Arg Glu Arg Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val545 550 555 560Glu Ala Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly 565 570 575Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly Ala Pro 580 585 590Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg Glu Leu Glu 595 600 605Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr Ala Pro Ala Gly Thr Ser 610 615 620Val Gln Ala Gln Pro Val Leu Pro Thr Asp Ser Ala Ser Arg Gly Gly625 630 635 640Val Ala Val Val Pro Ala Ser Gly Asp Cys Val Pro Ser Pro Cys His 645 650 655Asn Gly Gly Thr Cys Leu Glu Glu Glu Glu Gly Val Arg Cys Leu Cys 660 665 670Leu Pro Gly Tyr Gly Gly Asp Leu Cys Asp Val Gly Leu Arg Phe Cys 675 680 685Asn Pro Gly Trp Asp Ala Phe Gln Gly Ala Cys Tyr Lys His Phe Ser 690 695 700Thr Arg Arg Ser Trp Glu Glu Ala Glu Thr Gln Cys Arg Met Tyr Gly705 710 715 720Ala His Leu Ala Ser Ile Ser Thr Pro Glu Glu Gln Asp Phe Ile Asn 725 730 735Asn Arg Tyr Arg Glu Tyr Gln Trp Ile Gly Leu Asn Asp Arg Thr Ile 740 745 750Glu Gly Asp Phe Leu Trp Ser Asp Gly Val Pro Leu Leu Tyr Glu Asn 755 760 765Trp Asn Pro Gly Gln Pro Asp Ser Tyr Phe Leu Ser Gly Glu Asn Cys 770 775 780Val Val Met Val Trp His Asp Gln Gly Gln Trp Ser Asp Val Pro Cys785 790 795 800Asn Tyr His Leu Ser Tyr Thr Cys Lys Met Gly Leu Val Ser Cys Gly 805 810 815Pro Pro Pro Glu Leu Pro Leu Ala Gln Val Phe Gly Arg Pro Arg Leu 820 825 830Arg Tyr Glu Val Asp Thr Val Leu Arg Tyr Arg Cys Arg Glu Gly Leu 835 840 845Ala Gln Arg Asn Leu Pro Leu Ile Arg Cys Gln Glu Asn Gly Arg Trp 850 855 860Glu Ala Pro Gln Ile Ser Cys Val Pro Arg Arg Pro Ala Arg Ala Leu865 870 875 880His Pro Glu Glu Asp Pro Glu Gly Arg Gln Gly Arg Leu Leu Gly Arg 885 890 895Trp Lys Ala Leu Leu Ile Pro Pro Ser Ser Pro Met Pro Gly Pro 900 905 910136115PRTArtificial SequenceNanobody Sequence 136Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 115137115PRTArtificial SequenceNanobody Sequence 137Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 115138116PRTArtificial SequenceNanobody Sequence 138Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala 115139116PRTArtificial SequenceNanobody Sequence 139Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala 115140115PRTArtificial SequenceNanobody Sequence 140Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 115141116PRTArtificial SequenceNanobody Sequence 141Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Lys 100 105 110Val Ser Ser Ala 115142115PRTArtificial SequenceNanobody Sequence 142Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 115143116PRTArtificial SequenceNanobody Sequence 143Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala 115144117PRTArtificial SequenceNanobody Sequence 144Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ala 115145118PRTArtificial SequenceNanobody Sequence 145Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ala Ala 115146116PRTArtificial SequenceNanobody Sequence 146Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Gly 115147117PRTArtificial SequenceNanobody Sequence 147Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Gly Gly 115148118PRTArtificial SequenceNanobody Sequence 148Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Gly Gly Gly 115149115PRTArtificial SequenceNanobody Sequence 149Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 115150116PRTArtificial SequenceNanobody Sequence 150Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala 1151515PRTArtificial SequenceNanobody Sequence 151Ser Phe Gly Met Ser1 515217PRTArtificial SequenceNanobody Sequence 152Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys1 5 10 15Gly1536PRTArtificial SequenceNanobody Sequence 153Gly Gly Ser Leu Ser Arg1 51543PRTArtificial SequenceLinker Sequence 154Ala Ala Ala11555PRTArtificial SequenceLinker Sequence 155Gly Gly Gly Gly Ser1 51567PRTArtificial SequenceLinker Sequence 156Ser Gly Gly Ser Gly Gly Ser1 51578PRTArtificial SequenceLinker Sequence 157Gly Gly Gly Gly Gly Gly Gly Ser1 51589PRTArtificial SequenceLinker Sequence 158Gly Gly Gly Gly Ser Gly Gly Gly Ser1 515910PRTArtificial SequenceLinker Sequence 159Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 1016015PRTArtificial SequenceLinker Sequence 160Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 1516118PRTArtificial SequenceLinker Sequence 161Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly1 5 10 15Gly Ser16220PRTArtificial SequenceLinker Sequence 162Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser 2016325PRTArtificial SequenceLinker Sequence 163Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 2516430PRTArtificial SequenceLinker Sequence 164Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 3016535PRTArtificial SequenceLinker Sequence 165Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly Gly Ser 3516640PRTArtificial SequenceLinker Sequence 166Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly Gly Ser Gly Gly Gly Gly Ser 35 4016715PRTArtificial SequenceLinker Sequence 167Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10 1516824PRTArtificial SequenceLinker Sequence 168Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys1 5 10 15Thr His Thr Cys Pro Pro Cys Pro 2016912PRTArtificial SequenceLinker Sequence 169Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala1 5 1017062PRTArtificial SequenceLinker Sequence 170Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys1 5 10 15Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60171116PRTArtificial SequenceNanobody Sequence 171Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110Val Lys Ser Ala 11517217PRTArtificial Sequencemyc tag 172Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala1 5 10 15Ala

Claims

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

2. The ISV of claim 1, wherein said ISV specifically binds to human Aggrecan [SEQ ID NO: 125].

3. The ISV of claim 1 or 2, wherein said ISV specifically binds to dog Aggrecan (SEQ ID NO: 126), bovine Aggrecan (SEQ ID NO: 127), rat Aggrecan (SEQ ID NO: 128); pig (core) Aggrecan (SEQ ID NO: 129); mouse Aggrecan (SEQ ID NO: 130), rabbit Aggrecan (SEQ ID NO: 131); cynomolgus Aggrecan (SEQ ID NO: 132) and/or rhesus Aggrecan (SEQ ID NO: 133).

4. The ISV according to any one of claims 1-3, wherein said ISV does not bind substantially to Neurocan (SEQ ID NO: 134) and/or Brevican (SEQ ID NO: 135).

5. The ISV according to any one of claims 1 to 3, wherein the ISV has more than 10 fold, more than 100 fold, preferably more than 1000 fold selectivity over Neurocan and/or Brevican for binding to Aggrecan.

6. The ISV according to any one of claims 1-4, wherein said ISV preferably binds to cartilaginous tissue such as cartilage and/or meniscus.

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

8. The ISV according to any one of claims 1-6, wherein said ISV has a cartilage retention of at least 2, such as at least, 3, 4, 5 or 6 RU in a cartilage retention assay.

9. The ISV of any one of claims 1-8, wherein said ISV penetrates into the cartilage by at least 5 .mu.m, such as at least 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m or even more.

10. The ISV according to any one of claims 1 to 9, wherein said ISV essentially consists of a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation.

11. The ISV according to any one of claims 1 to 10, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: CDR1 is chosen 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 chosen 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 chosen 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.

12. The ISV according to any one of claims 1 to 11, wherein said ISV binds to the G1 domain of Aggrecan.

13. The ISV according to claim 12, wherein said ISV has a pI of more than 8.

14. The ISV according to any one of claims 12-13, wherein said ISV has a Koff of less than 2*10.sup.-2 s.sup.-1.

15. The ISV according to any one of claims 12-14, wherein said ISV has an EC.sub.50 of less than 1*10.sup.-6M.

16. The ISV according to any one of claims 1-15, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NOs: 24, 20, or 21; or b) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein at position 2 the S has been changed into R, F, I, or T; at position 3 the T has been changed into I; at position 5 the I has been changed into S; at position 6 the I has been changed into S, T, or M; at position 7 the N has been changed into Y, or R; at position 8 the V has been changed into A, Y, T, or G; at position 9 the V has been changed into M; and/or at position 10 the R has been changed into G, K, or A; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NOs: 42, 38, or 39; or d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein at position 1 the T has been changed into A, or G; an S or N is inserted between position 3 and position 4 (position 2a Table 1.3 B); at position 3 the S has been changed into R, W, N, or T; at position 4 the S has been changed into T or G; at position 5 the G has been changed into S; at position 6 the G has been changed into S, or R; at position 7 the N has been changed into S, T, or R; at position 8 the A has been changed into T; and/or at position 9 the N has been changed into D or Y; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 60, 56 or 57; or f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, wherein at position 1 the P has been changed into G, R, D, or E, or is absent; at position 2 the T has been changed into R, L, P, or V, or is absent; at position 3 the T has been changed into M, S, or R, or is absent; at position 4 the H has been changed into D, Y, G, or T; at position 5 the Y has been changed into F, V, T or G; at position 6 the G has been changed into L, D, S, Y, or W; an R, T, Y or V is inserted between position 6 and position 7 (position 6a Table 1.3C); at position 7 the G has been changed into P, or S; at position 8 the V has been changed into G, T, H, R, L, or Y; at position 9 the Y has been changed into R, A, S, D or G; at position 10 the Y has been changed into N, E, G, W, or S; a W is inserted between position 10 and position 11 (position 10a Table 1.3C); at position 11 the G has been changed into S, K, or Y; at position 12 the P has been changed into E, or D, or is absent; and/or at position 13 the Y has been changed into L, or is absent.

17. The ISV according to any one of claims 1 to 16, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, 37 and 109; CDR2 is chosen from the group consisting of SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, 55, and 110; and CDR3 is chosen from the group consisting of SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73, 74, and 111.

18. The ISV according to claim 17, wherein said ISV is chosen from the group of ISVs, 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: 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.

19. The ISV according to any one of claims 1-16, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 24 and 109; or b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein at position 7 the N has been changed into S; and/or at position 9 the V has been changed into M; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 42 and 110; or d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein at position 1 the T has been changed into A; at position 3 the S has been changed into R; at position 4 the S has been changed into T; at position 8 the A has been changed into T; and/or at position 9 the N has been changed into D; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 60 and 111; or f) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, wherein at position 4 the H has been changed into R; and/or at position 8 the V has been changed into D.

20. The ISV according to any one of claims 1 to 16, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 24 and 109; CDR2 is chosen from the group consisting of SEQ ID NOs: 42 and 110; and CDR3 is chosen from the group consisting of SEQ ID NOs: 60 and 111.

21. The ISV according to any one of claims 12-20, wherein said ISV belongs to epitope bin 1 or epitope bin 4.

22. The ISV according to claim 21, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 36; and b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 36, wherein at position 3 the T has been changed into S; at position 6 the T has been changed into S; at position 8 the T has been changed into A; and/or at position 9 the M has been changed into V; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 54; and d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 54, wherein at position 1 the A has been changed into I; at position 4 the W has been changed into R; at position 7 the G has been changed into R; and/or at position 8 the T has been changed into S; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 73; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 73, wherein at position 1 the R has been changed into G; at position 2 the P has been changed into R or L; at position 3 the R has been changed into L or S; at position 5 the Y has been changed into R; at position 6 the Y has been changed into S or A; at position 7 the Y has been changed into T, or is absent; at position 8 the S has been changed into P; at position 9 the L has been changed into H or R; at position 10 the Y has been changed into P or A; at position 11 the S has been changed into A or Y; at position 12 the Y has been changed into D; at position 13 the D has been changed into F; at position 14 the Y has been changed into G, or is absent; and/or after position 14 an S is inserted.

23. The ISV according to claim 22, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 20, 29, and 36; CDR2 is chosen from the group consisting of SEQ ID NOs: 38, 47, and 54; and CDR3 is chosen from the group consisting of SEQ ID NOs: 56, 65, and 73.

24. The ISV according to any one of claims 22-23, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation, to the G1 domain of Aggrecan.

25. An ISV, a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to epitope bin 1 of the G1-domain of Aggrecan, and which competes for binding to the G1 domain of Aggrecan with the ISV according to any one of claims 22-23.

26. The ISV according to claim 21, that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 24; and b) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 24, wherein at position 2 the S has been changed into I or F; at position 5 the I has been changed into S; at position 6 the I has been changed into S or M; at position 7 the N has been changed into R or Y; at position 8 the V has been changed into A or Y; at position 9 the V has been changed into M; and/or at position 10 the R has been changed into K; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 42; and d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 42, wherein at position 1 the T has been changed into A or G; an N is inserted between position 2 and position 3 (position 2a Table 2.3B); at position 7 the N has been changed into R; at position 8 the A has been changed into T; and/or at position 9 the N has been changed into D; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 60; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 60, wherein at position 1 the P is absent; at position 2 the T has been changed into R or is absent; at position 3 the T has been changed into M or is absent; at position 4 the H has been changed into D or Y; at position 5 the Y has been changed into F or V; at position 6 the G has been changed into L or D; at position 8 the V has been changed into G or T; at position 9 the Y has been changed into R; at position 10 the Y has been changed into N or E; at position 11 the G has been changed into S or K; at position 12 the P has been changed into E or is absent; and/or at position 13 the Y has been changed into L or is absent.

27. The ISV according to claim 26, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 24, 25, and 27; CDR2 is chosen from the group consisting of SEQ ID NOs: 42, 43, and 45; and CDR3 is chosen from the group consisting of SEQ ID NOs: 60, 61, and 63.

28. The ISV according to any one of claims 26-27, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation, to the G1 domain of Aggrecan.

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

30. The ISV according to any one of claims 1 to 29, wherein said ISV is chosen from the group consisting of ISVs with SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19, and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19.

31. The ISV according to any one of claims 1 to 11, wherein said ISV binds to the G1-IGD-G2 domain of Aggrecan.

32. The ISV according to claim 31, wherein said ISV has a pI of more than 8.

33. The ISV according to any one of claims 31-32, wherein said ISV has a Koff of less than 2*10.sup.-2 s.sup.-1.

34. The ISV according to any one of claims 31-33, wherein said ISV has an EC50 of less than 1*10.sup.-6M.

35. The ISV according to any one of claims 31-34, in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 32, 30 and 23; and b) amino acid sequences that have 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 32, wherein at position 2 the R has been changed into L; at position 6 the S has been changed into T; and/or at position 8 the T has been changed into A; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 50, 41, 48 and 51; and d) amino acid sequences that have 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 50, wherein at position 7 the G has been changed into S or R; and/or at position 8 the R has been changed into T; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 68, 59, 66 and 69; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 68, wherein at position 4 the R has been changed into V, or P; at position 6 the A has been changed into Y; at position 7 the S has been changed into T; at position 8 the S is absent; at position 9 the N has been changed into P; at position 10 the R has been changed into T or L; at position 11 the G has been changed into E; and/or at position 12 the L has been changed into T or V.

36. The ISV according to any one of claims 31-35, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 32, 30 and 23; CDR2 is chosen from the group consisting of SEQ ID NOs: 50, 41, 48 and 51; and CDR3 is chosen from the group consisting of SEQ ID NOs: 68, 59, 66 and 69.

37. The ISV according to claim 36, wherein said ISV is chosen 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.

38. The ISV according to any one of claims 31-37, wherein said ISV is chosen from the group consisting of ISVs with SEQ ID NOs: 13, 4, 11 and 14, and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 13, 4, 11 and 14.

39. The ISV according to any one of claims 31-38, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation, to the G1-IGD-G2 domain of Aggrecan.

40. An ISV, a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to the G1-IGD-G2 domain of Aggrecan, and which competes for binding to the G1-IGD-G2 domain of Aggrecan with the ISV according to any one of claims 31-38.

41. The ISV according to any one of claims 1 to 11, wherein said ISV binds to the G2 domain of Aggrecan.

42. The ISV according to claim 41, wherein said ISV has a pI of more than 8.

43. The ISV according to any one of claims 41-42, wherein said ISV has a Koff of less than 2*10.sup.-2 s.sup.-1.

44. The ISV according to any one of claims 41-43, wherein said ISV has an EC50 of less than 1*10.sup.-6M

45. The ISV according to any one of claims 41-44, in which: i) CDR1 is chosen from the group consisting of: a) SEQ ID NO: 28; and b) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 28, wherein at position 1 the G has been changed into R; at position 2 the P has been changed into S or R; at position 3 the T has been changed into I; at position 5 the S has been changed into N; at position 6 the R has been changed into N, M, or S; at position 7 the Y has been changed into R or is absent; at position 8 the A has been changed into F or is absent; and/or at position 10 the G has been changed into Y; and/or ii) CDR2 is chosen from the group consisting of: c) SEQ ID NO: 46; and d) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 46, wherein at position 1 the A has been changed into S, or Y; at position 4 the W has been changed into L; at position 5 the S has been changed into N; at position 6 the S is absent; at position 7 the G is absent; at position 8 the G has been changed into A; at position 9 the R has been changed into S, D, or T; and/or at position 11 the Y has been changed into N or R; and/or iii) CDR3 is chosen from the group consisting of: e) SEQ ID NO: 64; and f) amino acid sequences that have 5, 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence of SEQ ID NO: 64, wherein at position 1 the A has been changed into R, or F; at position 2 the R has been changed into I, or L; at position 3 the I has been changed into H, or Q; at position 4 the P has been changed into G, or N; at position 5 the V has been changed into S; at position 6 the R has been changed into G, N, or F; at position 7 the T has been changed into R, W, or Y; at position 8 the Y has been changed into R, or S, or is absent; at position 9 the T has been changed into S, or is absent; at position 10 the S has been changed into E, K or is absent; at position 11 the E has been changed into N, A, or is absent; at position 12 the W has been changed into D, or is absent; at position 13 the N has been changed into D, or is absent; at position 14 the Y is absent; and/or D and/or N are added after position 14 of SEQ ID NO: 64.

46. The ISV according to any one of claims 41 to 45, wherein said ISV is chosen from the group of ISVs, wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 28, 22, 26, and 33; CDR2 is chosen from the group consisting of SEQ ID NOs: 46, 40, 44, and 52; and CDR3 is chosen from the group consisting of SEQ ID NOs: 64, 58, 62, and 70.

47. The ISV according to claim 46, wherein said ISV is chosen from the group of ISVs, wherein: 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.

48. The ISV according to any one of claims 41 to 47, wherein said ISV is chosen from the group consisting of ISVs with SEQ ID NOs: 9, 3, 7 and 15, and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 9, 3, 7 and 15.

49. The ISV according to any one of claims 41-48, wherein said ISV cross-blocks the binding of domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation, to the G2 domain of Aggrecan.

50. An ISV domain antibody, an immunoglobulin that is suitable for use as a domain antibody, a single domain antibody, an immunoglobulin that is suitable for use as a single domain antibody, a dAb, an immunoglobulin that is suitable for use as a dAb, a Nanobody, a VHH sequence, a humanized VHH sequence, a camelized VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to the G2-domain of Aggrecan, and which competes for binding to the G2 domain of Aggrecan with the ISV according to any one of claims 41-48.

51. The ISV according to any of the preceding claims, wherein said ISV is chosen from the group consisting of SEQ ID NO:s 1-19 and 114-118 and ISVs which have more than 80%, such as 90% or 95% sequence identity with any one of SEQ ID NOs: 1-19 and 114-118.

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

53. The polypeptide according to claim 52, that comprises at least two ISVs according to any one of claims 1 to 51, and possibly a third ISV, and possibly a fourth ISV.

54. The polypeptide according to claim 53, wherein said at least two ISVs can be the same or different.

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

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

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

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

59. The polypeptide according to claim 58, wherein said at least one further ISV binds to a member 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.

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

61. The polypeptide according to any one of claims 58-60, wherein said at least one further ISV inhibits an activity of a member 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.

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

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

64. The polypeptide according to any one of claims 52-63, wherein said polypeptide penetrates into the cartilage by at least 5 .mu.m, such as at least 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m or even more.

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

66. The polypeptide according to claim 65, wherein said serum protein binding moiety binds serum albumin.

67. The polypeptide according to claim 65 or 66, wherein said serum protein binding moiety is an ISV binding serum albumin.

68. The polypeptide according to claim 67, wherein said ISV binding serum albumin essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG and CDR3 is GGSLSR.

69. The polypeptide according to claim 68, wherein said ISV binding serum albumin comprises Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG.

70. The polypeptide according to claim 65 or 66, wherein said serum protein binding moiety is a non-antibody based polypeptide.

71. The polypeptide according to any of the claims 52-64, further comprising PEG.

72. The polypeptide according to any one of the preceding claims, wherein said ISVs are directly linked to each other or are linked via a linker.

73. The polypeptide according to any one of the claims 52-69, wherein a first ISV and/or a second ISV and/or possibly a third ISV and/or possibly fourth ISV and/or possibly said ISV binding serum albumin are linked via a linker(s).

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

75. The polypeptide according to any one of claims 52-74, wherein said polypeptide is chosen from the group of polypeptides and/or constructs comprising an ISV binding a target as indicated and one or two ISVs binding Aggrecan as indicated in Table E-1 and Table E-2, respectively.

76. A construct that comprises or essentially consists of an ISV according to any one of claims 1 to 51 or a polypeptide according to any one of claims 52-75, and which optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers.

77. The construct according to claim 76, in which said one or more other groups, residues, moieties or binding units is chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

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

79. An expression vector comprising a nucleic acid according to claim 78.

80. A host or host cell comprising a nucleic acid according to claim 78, or an expression vector according to claim 79.

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

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

83. The composition according to claim 82, which is a pharmaceutical composition.

84. The composition according to claim 83, which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.

85. The composition according to any one of claims 82-84, the ISV according to any one of claims 1 to 51, the polypeptide according to any one of claims 52-75, the construct according to any one of claims 76-77 for use as a medicament.

86. The composition, the ISV, the polypeptide, or the construct according to claim 85, for use in preventing or treating arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis.

87. A method for preventing or treating arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of at least a composition, the ISV, the polypeptide, or the compound or construct according to claim 85 to a person in need thereof.

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

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

90. Use of an ISV according to any one of claims 1 to 51, a polypeptide according to any one of claims 52-75, a construct according to any one of claims 76-77, or a composition according to any one of claims 82-86, in the preparation of a pharmaceutical composition for treating or preventing arthropathies and chondrodystrophies, arthritic disease, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar disk degeneration disease, degenerative joint disease, and relapsing polychondritis.