RU2771818C2 - Immunoglobulins binding aggrecan - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to new immunoglobulins and structures based on them that are capable of specifically binding aggrecan protein.

EFFECT: pharmaceutical compositions containing immunoglobulins as an active ingredient, according to the present invention, can be used in medical practice for preventive, therapeutic or diagnostical purposes, in particular in the treatment of various joint deceases, for example osteoarthritis.

19 cl, 15 tbl, 11 ex, 4 dwg

Description

The technical field to which the invention belongs

The present invention relates to immunoglobulins that bind aggrecan, and more specifically to polypeptides that include or essentially consist of one or more of such immunoglobulins (also referred to herein as "immunoglobulin(s) of the invention") and "polypeptides of the invention", respectively). The invention also relates to constructs containing such immunoglobulins or polypeptides, as well as to nucleic acids encoding such immunoglobulins or polypeptides (also referred to herein as "nucleic acid(s) of the invention"), to methods for producing such immunoglobulins, polypeptides and structures; host cells expressing or capable of expressing such immunoglobulins or polypeptides, to compositions and in particular to pharmaceutical compositions that include such immunoglobulins, polypeptides, constructs, nucleic acids and/or host cells, and to the use of immunoglobulins, polypeptides, constructs , nucleic acids, host cells and/or compositions, in particular for prophylactic and/or therapeutic purposes, such as the prophylactic and/or therapeutic purposes mentioned herein. Other aspects, embodiments, advantages and applications of the invention will become apparent from the further description provided herein.

Prior Art

Osteoarthritis is one of the most common causes of disability worldwide. It affects 30 million Americans and is the most common joint disease. In the US, it is expected to affect more than 20 percent of the population by 2025. The disease can occur in all joints, most often in the knees, hips, hands and spine. Osteoarthritis (OA) can be defined as a diverse group of conditions characterized by a combination of joint symptoms, signs due to articular cartilage defects, and changes in adjacent tissues, including bones, tendons, and muscles. OA is characterized by progressive erosion of the articular cartilage (the cartilage that covers the bones). Eventually, the disease leads to complete destruction of the articular cartilage, sclerosis of the underlying bone, formation of osteophytes, etc., leading to loss of mobility and pain. Pain is the most prominent symptom of OA, and it is for this reason that patients seek medical attention.

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

OA is characterized by 1) degradation of aggrecan, gradual release of the G3 and G2 domains (resulting in cartilage "deflation") and eventually release of the G1 domain, and 2) degradation of collagen, irreversibly destroying cartilage structure.

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 progression of the disease or heal the joints. For large joints, the drug may be injected into the joint to help limit potential side effects such as pain. Therapeutic strategies are primarily aimed at reducing pain and improving joint function. Fasinumab, a non-opioid anti-NGF pain medication, has been shown to improve a key pain score during phase II/III trials. Duloxetine has been approved for the treatment of chronic knee pain due to osteoarthritis and has been provisionally recommended by the American College of Rheumatology. Strontium ranelate was found to significantly reduce the rate of decrease in joint space width as well as improve pain scores compared to placebo in a large multicenter study in patients with symptomatic osteoarthritis of the knee. However, at the same time, biological agents, interleukin-1 receptor antagonists and anti-tumor necrosis factor antibodies, have not been shown to be effective or change the course of osteoarthritis (Smelter Hochberg, 2013, Current Opin. Rheumatol. 25:310). Consequently, many of these treatments are ineffective and/or associated with side effects. Ultimately, patients will undergo total knee or hip replacement therapy if the pain cannot be controlled.

Pharmacological therapy begins with oral administration of paracetamol in combination with NSAIDs or COX-2 inhibitors and a weak opioid. The main disadvantages of oral medications are limited bioavailability at the site of interest and the risk of side effects such as liver damage, gastrointestinal (GI) ulcers, gastrointestinal bleeding and constipation.

Since OA is localized, intra-articular drug administration offers an excellent opportunity to improve treatment. However, most newly developed disease-modifying osteoarthritis (DMOAD) drugs 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 was limited by their rapid clearance from the joint space and lack of retention in cartilage. The synovial retention time of the drug in the joint is often less than 24 hours. Due to the rapid elimination of most drugs administered by IA, frequent injections will be required to maintain an effective concentration (Owen et al., 1994, Br. J. Clin Pharmacol. 38:349-355). However, frequent intravenous injections are undesirable due to pain and discomfort, which can lead to impaired performance of the patient, and also because of the risk of joint infection.

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

There remains a need for additional cartilage anchoring proteins (CAP).

The essence of the invention

The present inventors have hypothesized that the efficacy of a therapeutic drug can be significantly improved by combining the therapeutic drug with a component that "fixes" the drug in the joint and therefore increases drug retention, but which should not interfere with the efficacy of said therapeutic drug. drug (also referred to herein as "cartilage anchoring protein" or "CAP"). This concept of anchoring will not only increase drug efficacy but also functional specificity for the diseased joint by reducing toxicity and side effects, thus increasing the number of possible beneficial drugs. The present inventors also hypothesized that aggrecan binding agents could potentially function as such an anchor, although aggrecan is highly glycosylated and degraded in various disorders affecting cartilage in the joints. In addition, due to the cost and extensive testing in various animal models required for a drug to reach the clinic, such aggrecan binding agents should preferably have broad cross-reactivity, e.g., aggrecan binding agents should bind to aggrecan. various types.

Using various original methods of immunization, screening and characterization, the authors of the present invention were able to identify a number of agents that bind aggrecan, with excellent properties of selectivity, stability and/or specificity, which allowed long-term retention and activity in the joint.

Accordingly, the present invention provides 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), porcine aggrecan (core) (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 substantially bind to neurocan (SEQ ID NO : 134) and/or brevican (SEQ ID NO: 135).

In one aspect, the present invention relates to an ISV as described herein, wherein the ISV has greater than 10-fold, greater than 100-fold, preferably greater than 1000-fold binding selectivity for aggrecan over neurocan and/or brevican, and/ or said ISV preferably binds to cartilage tissue such as cartilage and/or meniscus and/or said ISV is stable for at least 7 days, such as 14 days, 21 days, 1 month, 2 months or even 3 months in synovial fluid (SF) at 37° C., and/or said ISV has a cartilage retention of at least 2, e.g., at least 3, 4, 5, or 6 RU in a cartilage retention assay, and/or said ISV penetrates cartilage at least 5 µm, e.g. at least 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, or even more, and/or said ISV essentially consists of a domain antibody, immunoglobulin , which is suitable for use as a domain antibody, single domain antibody, immunoglobulin, which is suitable for use as a single domain antibody, a dAb, an immunoglobulin which is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been obtained by affinity maturation.

In one aspect, the present invention relates to an ISV described herein, which essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which: CDR1 is selected from the group consisting from SEQ ID NOs: 24, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 and 109; CDR2 is selected from the group consisting of SEQ ID NOs: 42, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 110; and CDR3 is selected from the group consisting of SEQ ID NOs: 60, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 111.

In one aspect, the present invention provides an ISV as described herein, wherein said ISV binds to the G1 domain of aggrecan, preferably said ISV has a pI greater than 8 and/or said ISV has a K _off less than 2×10 ^-2 s ^-1 , and/ or said ISV has an EC _{50 of} less than 1⋅10 ^-6 M.

In one aspect, the present invention relates to an ISV described herein, which essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 24, 20 or 21; or b) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 24, where at position 2 S is changed to R, F, I or T; in position 3 T changed to I; in position 5 I changed to S; in position 6 I changed to S, T or M; in position 7 N changed to Y or R; in position 8 V changed to A, Y, T or G; in position 9 V changed to M; and/or at position 10 R is changed to G, K or A; and/or

ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 42, 38 or 39; or d) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 42, where at position 1 T is changed to A or G; S or N is inserted between position 3 and position 4 (position 2a, table 1.3B); in position 3 S changed to R, W, N or T; in position 4 S changed to T or G; in position 5 G changed to S; in position 6 G changed to S or R; in position 7 N changed to S, T or R; in position 8 A changed to T; and/or at position 9, N is changed to D or Y; and/or

iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 60, 56 or 57; or f) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 60, where at position 1 P is changed to G, R, D, or E, or is missing; in position 2 T changed to R, L, P or V or absent; in position 3, T is changed to M, S or R or absent; in position 4 H changed to D, Y, G or T; in position 5 Y changed to F, V, T or G; in position 6 G changed to L, D, S, Y or W; R, T, Y or V are inserted between position 6 and position 7 (position 6a, Table 1.3C); in position 7 G changed to P or S; in position 8 V changed to G, T, H, R, L or Y; at position 9, Y is changed to R, A, S, D, or G; at position 10 Y changed to N, E, G, W or S; W inserted between position 10 and position 11 (position 10a, table 1.3C); in position 11 G changed to S, K or Y; in position 12, P changed to E or D or absent; and/or at position 13 Y changed to L or missing.

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

In an aspect, the present invention relates to an ISV described herein, wherein said ISV is selected from the group of ISVs in which:

- 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.

In one aspect, the present invention relates to an ISV described herein, which essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of: a) SEQ ID NOs: 24 and 109; or b) amino acid sequences that have a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 24, where at position 7 N is changed to S; and/or at position 9 V is changed to M; and/or

ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 42 and 110; or d) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 42, where at position 1 T is changed to A; in position 3 S changed to R; in position 4 S changed to T; in position 8 A changed to T; and/or at position 9, N is changed to D; and/or

iii) CDR3 is selected from the group consisting of: e) SEQ ID NOs: 60 and 111; or f) amino acid sequences that have a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 60, where at position 4 H is changed to R; and/or in position 8 V changed to D.

In an aspect, the present invention relates to an ISV described herein, in which the specified ISV is selected from the group ISV, where CDR1 is selected from the group consisting of SEQ ID NO: 24 and 109; CDR2 is selected from the group consisting of SEQ ID NOs: 42 and 110; and CDR3 is selected from the group consisting of SEQ ID NOs: 60 and 111.

In one aspect, the present invention relates to an ISV described herein, wherein said ISV belongs to epitope group 1 or epitope group 4, preferably said ISV essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 regions, determining complementarity (CDR1-CDR3, respectively), in which:

i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 36; and b) amino acid sequences that have a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 36, where at position 3 T is changed to S; in position 6 T changed to S; in position 8 T changed to A; and/or at position 9, M is changed to V; and/or

ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 54; and d) amino acid sequences that differ by 5, 4, 3, 2, or 1 amino acid from the amino acid sequence of SEQ ID NO: 54, where at position 1 A is changed to I; in position 4 W changed to R; in position 7 G changed to R; and/or in position 8, T is changed to S; and/or

iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 73; and f) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 73, where at position 1, R is changed to G; in position 2 P changed to R or L; in position 3 R changed to L or S; in position 5 Y changed to R; in position 6 Y changed to S or A; in position 7 Y changed to T or absent; in position 8 S changed to P; in position 9 L changed to H or R; in position 10 Y changed to P or A; in position 11 S changed to A or Y; in position 12 Y changed to D; in position 13 D changed to F; in position 14 Y changed to G or missing; and/or an S is inserted after position 14.

In one aspect, the present invention relates to an ISV described herein, where the specified ISV is selected from the group ISV, where: CDR1 is selected from the group consisting of SEQ ID NOS: 20, 29 and 36; CDR2 is selected from the group consisting of SEQ ID NOs: 38, 47 and 54; and CDR3 is selected from the group consisting of SEQ ID NOs: 56, 65 and 73.

In one aspect, the present invention provides an ISV as described herein, wherein said ISV cross-blocks the binding 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, dAb, an immunoglobulin which is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation for the G1 domain of aggrecan.

In one aspect, the present invention provides 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, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation and that binds to epitope group 1 of the aggrecan G1 domain, and which competes for binding to the aggrecan G1 domain to the ISV described herein.

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

In one aspect, the present invention provides 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, an overblown VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to epitope group 4 of the aggrecan G1 domain and that competes for binding to the aggrecan G1 domain of ISV, described in this document.

In one aspect, the present invention provides an ISV described herein, wherein said ISV is selected from the group consisting of ISVs with SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19, and ISVs that have greater than 80%, e.g., 90% or 95% sequence identity with any of SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12, 16, 17, 18, and 19.

In one aspect, the present invention provides an ISV as described herein, wherein said ISV binds to the G1-IGD-G2 domain of aggrecan, preferably wherein said ISV has a pI greater than 8 and/or has a K _off less than 2×10 ^{- 2} s ^-1 and/or has an EC _{50 of} less than 1⋅10 ^-6 M.

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

In one aspect, the present invention relates to an ISV described herein, in which the specified ISV is selected from the group consisting of ISV with SEQ ID NO: 13, 4, 11 and 14 and ISV, which have more than 80%, for example, 90% or 95% sequence identity to any of SEQ ID NOs: 13, 4, 11 and 14.

In one aspect, the present invention provides an ISV as described herein, wherein said ISV cross-blocks the binding 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, dAb, an immunoglobulin which is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation for the G1-IGD-G2 domain of aggrecan. In one aspect, the present invention provides 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, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation and that binds to the G1-IGD-G2 domain of aggrecan and that competes for binding to the G1-IGD-G2 domain of aggrecan to the ISV described in this document.

In one aspect, the present invention provides an ISV as described herein, wherein said ISV binds to the G2 domain of aggrecan, preferably wherein said ISV has a pI greater than 8 and/or has a K _off less than 2×10 ^-2 s ^-1 and/or has an EC ₅₀ less than 1⋅10 ^-6 M.

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

In one aspect, the present invention relates to an ISV described herein, where the specified ISV is selected from the group consisting of ISV with SEQ ID NO: 9, 3, 7 and 15 and ISV, which have more than 80%, for example, 90% or 95% sequence identity with any of SEQ ID NOs: 9, 3, 7 and 15.

In one aspect, the present invention provides an ISV as described herein, wherein said ISV cross-blocks the binding 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, dAb, an immunoglobulin which is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation for the G2 domain of aggrecan. In one aspect, the present invention provides 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, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation and that binds to the G2 domain of aggrecan and competes for binding to the G2 domain of aggrecan to the ISV described herein.

In one aspect, the present invention relates to an ISV described herein, where the specified ISV is selected from the group consisting of SEQ ID NO: 1-19 and 114-118 and ISV, which have more than 80%, for example, 90% or 95% sequence identities with any of SEQ ID NOs: 1-19 and 114-118.

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

Preferably, in one aspect, the polypeptide of the invention comprises at least one additional ISV, eg, a therapeutic ISV. Preferably, said at least one additional ISV binds to a member of the serine protease family, cathepsins, matrix metalloproteinases (MMP)/matrixins or disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2 ), ADAMTS4 (aggrecanase-1) and/or ADAMTS11; moreover, the specified at least one additional ISV, for example, therapeutic ISV, preferably retains activity. Even more preferably, said at least one additional ISV, such as a therapeutic ISV, inhibits the activity of a member of the serine protease family, cathepsins, matrix metalloproteinases (MMP)/matrixins, or disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19 , MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and/or ADAMTS11.

In one aspect, the present invention relates to a polypeptide described herein, where the specified 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° C. and/or has a cartilage retention of at least 2, e.g., at least 3, 4, 5, or 6 RU in a cartilage retention assay, and/or penetrates cartilage by at least 5 µm, e.g., at least least 10 µm, 20 µm, 30 µm, 40 µm, 50 µm or even more.

In one aspect, the present invention provides a polypeptide as described herein, further comprising a serum protein binding protein or serum protein, preferably said serum protein binding fragment binds serum albumin; even more preferably, said serum protein binding moiety is an ISV that binds serum albumin; even more preferably, said serum albumin binding ISV essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively), in which CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG and CDR3 is GGSLSR; even more preferably said ISV binding serum albumin includes Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG , Alb82-GGG (see also Table C). In one aspect, the present invention provides a polypeptide as described herein further comprising a serum binding protein or whey protein, wherein said serum protein binding component is a non-antibody based polypeptide. In one aspect, the present invention provides a polypeptide as described herein further comprising PEG.

In one aspect, the present invention relates to a polypeptide described in this document, where these ISV directly linked to each other or connected through a linker. In one aspect, the present invention relates to a polypeptide described herein, where the first ISV and/or the second ISV, and/or possibly the third ISV, and/or possibly the fourth ISV, and/or possibly the specified ISV, binding serum albumin, connected through the linker(s); preferably said linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS linkers (see also Table D).

In one aspect, the present invention provides a polypeptide as described herein, wherein said polypeptide is selected from the group of polypeptides and/or constructs comprising an ISV that binds a target as indicated and one or two ISVs that bind aggrecan as indicated in Table E- 1 and Table E-2, respectively.

In one aspect, the present invention relates to a construct that contains or essentially consists of an ISV described herein or a polypeptide described herein, and which optionally additionally includes one or more other groups, residues, fragments or linking units , optionally linked via one or more peptide linkers; preferably, said one or more other groups, residues, fragments, or linkers are selected 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.

In one aspect, the present invention provides a nucleic acid encoding an ISV described herein, a polypeptide described herein, or a construct described herein.

In one aspect, the present invention provides an expression vector containing the nucleic acid described herein.

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

In one aspect, the present invention provides a method for producing an ISV described herein, or a polypeptide described herein, said method at least comprising the steps of: a) expression in a suitable host cell or host organism, or other suitable system expression of the nucleic acid described herein; optionally followed by: b) isolation and/or purification of the ISV described herein or the polypeptide described herein.

In one aspect, the present invention provides a composition comprising at least one ISV described herein, a polypeptide described herein, a construct described herein, or a nucleic acid 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 contains one or more other pharmaceutically active polypeptides and/or compounds.

In one aspect, the present invention provides a composition described herein, an ISV described herein, a polypeptide described herein, or a construct described herein for use as a drug. Preferably, the composition, ISV, polypeptide or construct described herein is for the prevention or treatment of arthropathies and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costochondritis, spondyloepimetaphyseal dysplasia , spinal disc herniation, degenerative lumbar disc disease, degenerative joint disease, and relapsing polychondritis.

In one aspect, the present invention relates to a method for the prevention or treatment of arthropathies and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costochondritis, spondyloepimetaphyseal dysplasia, spinal herniation, lumbar disc degeneration, degenerative joint diseases; 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 described herein to a person in need thereof.

In one aspect, the present invention provides a method for reducing and/or inhibiting efflux of a compound, polypeptide, or cartilage construct, wherein said method comprises administering a pharmaceutically active amount of at least one polypeptide described herein, a compound, or construct described herein. , or the composition described herein, to a person in need.

In one aspect, the present invention provides 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 described herein, a construct described herein, or a composition, described in this document to the person in need.

In one aspect, the present invention relates to the use of an ISV as described herein, a polypeptide as described herein, a construct as described herein, or a composition as described herein, for the preparation of a pharmaceutical composition for the treatment or prevention of arthropathy. and chondrodystrophy, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costochondritis, spondyloepimetaphyseal dysplasia, spinal herniation, lumbar disc degeneration, degenerative joint disease, and relapsing polychondritis.

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

Drawing captions

Figure 1: Exemplary autoradiographic images of sections of rat joints 2 or 4 weeks after administration of ¹²⁵ I-labeled ALB26-CAP constructs. For each of the results at 2 weeks post-injection and 4 weeks post-injection: left panel: histological section; Right panel: autoradiography.

Figure 2: Representative images of MARG. Specific MARG staining appears as black grains on images and is indicated by arrows.

Figure 3: Inhibition of cartilage degradation by nanobodies in a rat model of MMT using an anti-MMP13-CAP nanobody (C010100754) or an anti-ADAMTS5-CAP nanobody (C010100954). Treatment started 3 days after surgery with IA injection. Histopathology was performed 42 days after surgery. The medial and overall actual width of cartilage degeneration was determined, as well as the percentage reduction in cartilage degeneration. 20 animals were used per group.

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

Detailed description

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

Unless otherwise indicated, all methods, steps, techniques and manipulations, which are not particularly described in detail, can be performed and have been performed in a manner known per se, as will be understood by a person skilled in the art. For example, reference may again be made to the standard reference books and general art referenced herein and to additional sources cited therein; and, for example, to the following reviews Presta (Adv. Drug Deliv. Rev. 58 (5-6): 640-56, 2006), Levin and Weiss (Mol. Biosyst. 2 (1): 49-57, 2006), Irving et al. (J. Immunol. Methods 248(1-2): 31-45, 2001), Schmitz et al. (Placenta 21 Suppl. A: S106-12, 2000), Gonzales et al. (Tumour Biol. 26 (1): 31-43, 2005), which describe protein engineering techniques such as affinity maturation and other techniques for improving specificity and other desirable properties in proteins such as immunoglobulins.

As used herein, the term "sequence" (for example, in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence", or "protein sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acids or nucleotide sequences encoding it, unless the context requires more limited interpretation.

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

Amino acids are those L-amino acids that are commonly found in natural proteins. Amino acid residues will be specified according to the standard three-letter or one-letter amino acid code. The source can be, for example, table A-2 on page 48 of WO 08/020079. These amino acid sequences containing D-amino acids are not intended to be covered by this definition. Any amino acid sequence that contains post-translationally modified amino acids can be described as an amino acid sequence that is originally translated using the symbols shown in this Table A-2, with positions changed; for example, hydroxylation or glycosylation, but these modifications do not have to be explicitly shown in the amino acid sequence. Any peptide or protein that can be expressed as sequential modified linkages, crosslinks and caps, non-peptidyl linkages, etc. is covered by this definition.

The terms "protein", "peptide", "protein/peptide" and "polypeptide" are used interchangeably throughout the disclosure, and each of them has the same meaning for the purposes of this disclosure. Each term refers to an organic compound consisting of a linear chain of two or more amino acids. A compound may contain 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. One skilled in the art will appreciate that polypeptides typically contain fewer amino acids than proteins, although there is no generally accepted limit in the art on the number of amino acids that distinguish a polypeptide from a protein; that polypeptides can be obtained by chemical synthesis or recombinant methods; and that proteins are generally produced in vitro or in vivo by recombinant methods, all of which are known in the art.

A nucleic acid or amino acid sequence is considered to be "(in) (substantially) isolated (in form)" - e.g., compared to the reaction medium or culture medium from which it was derived when it was separated from at least one other component with which it is usually associated in the specified source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or minor component. In particular, a nucleic acid or amino acid sequence is considered "(substantially) isolated" when it is at least 2-fold purified, in particular at least 10-fold, more specifically at least 100-fold, and up to 1000-fold or greater. . A nucleic acid or amino acid that is "(substantially) in isolated form" is preferably substantially homogeneous as determined by a suitable method such as a suitable chromatographic method such as polyacrylamide gel electrophoresis.

When a nucleotide sequence or amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or "essentially consist of" another nucleotide sequence or amino acid sequence, this may mean that the last nucleotide sequence or amino acid sequence was included in the first nucleotide sequence mentioned. sequence or amino acid sequence, respectively, but more commonly, this usually means that the first mentioned nucleotide sequence or amino acid sequence contains in its sequence a section of nucleotides or amino acid residues, respectively, which has the same nucleotide sequence or amino acid sequence, respectively, as the last sequence , regardless of how the first mentioned sequence was actually created or obtained (which can t be, for example, any suitable method described in this document). By way of 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 included in the sequence of the polypeptide of the invention, but more commonly it usually means that the polypeptide of the invention of the invention contains in its sequence the sequence of ISV, regardless of how the specified polypeptide according to the invention was created or obtained. In addition, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, the first nucleic acid or nucleotide sequence mentioned is preferably such that, when expressed in an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, the last nucleotide sequence is in the same reading frame as the first mentioned large nucleic acid or nucleotide sequence). In addition, when a construct of the invention is said to contain a polypeptide or ISV, this may mean that said construct at least encompasses said polypeptide or ISV, respectively, but more commonly it means that said construct includes groups, residues (e.g., amino acid residues ), fragments, and/or linking units in addition to said polypeptide or ISV, regardless of how said polypeptide or ISV is linked to said groups, residues (e.g., amino acid residues), fragments, and/or linking units, and regardless of how the specified construct has been created or obtained.

"Essentially consists of" means that the ISV used in the method of the invention is either exactly the same as the ISV of the invention or corresponds to an ISV of the invention that has a limited number of amino acid residues, e.g. 1-20 amino acid residues, e.g. , 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-terminus, at the carboxy-terminus or both at the amino-terminus and at carboxy-terminal ISV.

For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between the first nucleotide sequence and the second nucleotide sequence can be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in of the first nucleotide sequence] and multiplying by [100%], wherein 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 in one nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using a known computer algorithm for sequence alignment, such as, for example, NCBI Blast v2.0, using standard settings. Some other methods, computer algorithms and settings for determining the degree of sequence identity, for example, are described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768. Usually, with in order to determine the percentage of "sequence identity" between two nucleotide sequences according to the calculation method described above, the nucleotide sequence with the highest number of nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the "second" nucleotide sequence.

For purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between the first amino acid sequence and the second amino acid sequence (also referred to herein as "amino acid identity") can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], with 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 in one amino acid residue (position), i.e. as "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using a known computer algorithm, such as those mentioned above, to determine the degree of sequence identity for nucleotide sequences, again using standard settings. Generally, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method described above, the amino acid sequence with the most amino acid residues will be taken as the "first" amino acid sequence and the other amino acid sequence will be taken as the "second" amino acid sequence. subsequence.

In addition, when determining the degree of sequence identity between two amino acid sequences, one of skill in the art may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is changed to another amino acid residue with a similar chemical structure. , which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example 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 appropriate principles, for example from WO 04/037999 or, for example, WO 98/49185 and from the additional sources cited there.

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

Any amino acid substitutions applied to the polypeptides described herein can also be based on an analysis of amino acid variation frequencies between homologous proteins of different species, such as, for example, developed by Schulz et al. ("Principles of Protein Structure", Springer-Verlag, 1978), regarding structure-forming potential assays developed by, for example, Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45-149, 1978), and on the analysis of hydrophobicity patterns in proteins developed, for example, by Eisenberg et al. (Proc. Natl. Acad. 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 of which are incorporated herein by reference in their entirety. Information about the primary, secondary and tertiary structure of nanobodies is given in the description in this document and in the general prior art referred to above. In addition, for this purpose, the crystal structure of the VHH domain from llama, for example, is 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 on some of the amino acid residues that form the VH/VL interface in normal VH domains and potential camel substitutions at these positions can be found in the prior art referenced above.

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

When comparing two amino acid sequences, the term "difference in amino acid(s)" refers to the insertion, deletion or substitution of one amino acid residue at the position of the first sequence compared to the second sequence; it should be understood that two amino acid sequences may contain one, two or more such amino acid differences. More specifically, in the amino acid sequences and/or polypeptides of the present invention, the term "amino acid difference" refers to the insertion, deletion or substitution of one amino acid residue at the position of the CDR sequence indicated in b), d) or f) compared to the CDR sequence, respectively. a), c) or e); it should be understood that the CDR sequence b), d) and f) may contain one, two, three, four or a maximum of five such amino acid differences compared to the CDR sequence a), c) or e), respectively.

"Difference(s) in amino acid(s)" can be any one, two, three, four, or up to five substitutions, deletions or insertions, or any combination thereof, that enhances the properties of an aggrecan binding agent of the invention, such as a polypeptide of the invention. , or which at least do not detract too much from the desired properties or the balance or combination of desirable properties of an aggrecan binding agent of the invention, such as a polypeptide of the invention. In this regard, a resultant aggrecan binding agent of the invention, such as a polypeptide of the invention, should at least bind aggrecan with the same, about the same, or higher affinity as compared to a polypeptide containing one or more CDR sequences without one, two, three , four or a maximum of five substitutions, deletions or insertions, where said affinity is measured by surface plasmon resonance (SPR).

In this regard, the amino acid sequence of the CDR according to b), d) and/or f) may be the amino acid sequence derived from the amino acid sequence according to a), c) and/or e), respectively, by affinity maturation using one or more known affinity maturation technologies per se.

For example, and depending on the host organism used to express the polypeptide of the invention, such deletions and/or substitutions can be engineered such that one or more sites for post-translational modification (such as one or more glycosylation sites) are deleted, which will within the competence of a person skilled in the art.

"Nanobody family", "VHH family", or "family" as used herein refers to a group of nanobody and/or VHH sequences that are the same length (i.e., they have the same number of amino acids in their sequence), and from 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.

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

The portion of an antigen-binding molecule (such as an immunoglobulin, a conventional antibody, an ISV, and/or a polypeptide of the invention) that recognizes an epitope is referred to as a "paratope".

An amino acid sequence (such as an ISV, an antibody, a polypeptide of the invention, or generally an antigen-binding protein or polypeptide or fragment thereof) that can "bind" or "specifically bind", that "has affinity for" and/or that "has specificity for "to a particular epitope, antigen or protein (or at least one part, fragment or epitope thereof), is referred to as "against" or "directed against" the specified epitope, antigen or protein, or is a "binding" molecule with respect to such an epitope, an antigen or protein, or referred to as an "anti" epitope, "anti" antigen, or "anti" protein (eg, "anti" aggrecan).

Affinity refers to the strength or stability of a molecular interaction. Affinity is usually given as K _D , or dissociation constant, which has units of moles/liter (or M). Affinity can also be expressed as an association constant, K _A , which is equal to 1/K _D and has units of (mol/liter) ^-1 (or M ^-1 ). In the present description, the stability of the interaction between two molecules will be mainly expressed in terms of the K _D value of their interaction; one skilled in the art will appreciate that given the ratio K _A = 1/K _D , determining the strength of the molecular force from its K _D value can also be used to calculate the corresponding K _A value. The K _D value characterizes the strength of the molecular interaction also in a thermodynamic sense, since it is related to the change in the free energy (DG) of the binding using the well-known relationship DG=RT.ln(K _D ) (equivalent to DG=-RT.ln(K _A )) , where R is the gas constant, T is the absolute temperature, and ln is the natural logarithm.

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

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

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

The affinity of the molecular interaction between two molecules can be measured by various methods known per se, such as the well-known surface plasmon resonance biosensor method (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559), where one molecule is immobilized on the biosensor chip, and another molecule is passed over the immobilized molecule under flow conditions, giving measurements of k _on , K _off and hence K _D (or K _A ) values. This can be done, for example, using well known BIACORE® instruments (Pharmacia Biosensor AB, Uppsala, Sweden). The kinetic exclusion assay (KINEXA®) (Drake et al. 2004, Analytical Biochemistry 328: 35-43) measures binding events in solution without labeling binding partners and is based on the kinetic exclusion of complex dissociation. Solution affinity analysis can also be performed using the GYROLAB® Immunoassay System, which provides a platform for automated bioassay and rapid sample analysis (Fraley et al. 2013, Bioanalysis 5: 1765-74) or ELISA.

One skilled in the art will also appreciate that the measured K _D may correspond to the apparent K _D if the measurement process affects the intrinsic binding affinity of the implied molecules in any way, for example due to artifacts associated with single molecule coating on the biosensor. In addition, apparent K _D can be measured if one molecule contains more than one recognition site for another molecule. In such a situation, the measured affinity can be affected by the avidity of the interaction between the two molecules. In particular, accurate measurement of KD can be quite laborious, and as a result, often apparent KD values are determined to estimate the binding strength of two molecules. It should be noted that as long as all measurements are performed in a consistent manner (e.g., keeping the analysis conditions unchanged), apparent KD measurements can be used as an approximation to true K _D , and therefore, in this document, KD and apparent KD should be considered with equal importance or relevance.

The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an ISVD or a polypeptide of the invention) can bind. The specificity of an antigen binding protein can be determined based on affinity and/or avidity, for example, as described on pages 53-56 of WO 08/020079 (incorporated herein by reference), which also describes some preferred techniques for measuring binding between an antigen binding molecule (such as polypeptide or ISVD according to the invention) and the corresponding antigen. Typically, antigen-binding proteins (such as ISVDs and/or polypeptides of the invention) will bind their antigen with a dissociation constant (K _D ) of 10 ^-5 to 10 ^-12 mol/l or less and preferably 10 ^-7 to 10 ^-12 mol/liter or less, and more preferably 10 ^-8 to 10 ^-12 mol/liter (that is, with an association constant (K _A ) of 10 ⁵ to 10 ¹² L/mol or more, and preferably 10 ⁷ to 10 ¹² L /mol or more, and more preferably from 10 ⁸ to 10 ¹² l/mol). Generally, any K _D value above 10 ^-4 mol/liter (or any K _A value below 10 ⁴ L/mol) is considered to indicate non-specific binding. Preferably, the monovalent ISVD of the invention will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM, such as 10 to 5 pM or less. Also refer to point n) on pages 53-56 in WO 08/020079.

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

The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any suitable method known per se, including, for example, saturation binding assays and/or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and various thereof. options known in the art; and other methods mentioned in this document.

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

Finally, it should be noted that in many situations the experienced scientist may find it convenient to determine the binding affinity for some standard molecule. For example, to evaluate the strength of binding between molecules A and B, one can, for example, use a standard molecule C, which is known to bind to B and which is appropriately labeled with a fluorophore or chromophore group or other chemical moiety such as biotin for easy detection. in ELISA or FACS (fluorescence-activated cell sorting) or in another format (fluorophore for fluorescence, chromophore for light absorption, biotin for streptavidin-mediated ELISA). Typically, a standard molecule C is maintained at a fixed concentration and the concentration of A varies for a given concentration or amount of B. The result is an IC ₅₀ value corresponding to the concentration of A at which the signal for C is measured in the absence of A is halved. Provided that K _D ref, K _D of the standard molecule, is known, and the total concentration Cref of the standard molecule, the apparent K _D for the AB interaction can be obtained from the following formula: K _D =IC ₅₀ /(1+c _ref / K _Dref ) . Note that if c _ref << K _{D ref} , K _D ≈ IC ₅₀ . Provided that the measurement of IC ₅₀ is performed in a consistent manner (eg, keeping cref fixed) for the compared binders, the difference in strength or stability of the molecular interaction can be assessed by comparing the IC ₅₀ and this measurement is evaluated as equivalent to K _D or apparent K _D throughout text.

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

The term half maximum effective concentration (EC ₅₀ ) refers to the concentration of a compound that elicits a response halfway between baseline and maximum after a specified exposure time. In the present context, the term is used as a measure of the activity of a polypeptide, ISV (eg, nanobodies). The EC ₅₀ of the ramp curve is the concentration of a compound at which 50% of its maximum effect is observed. The concentration is preferably expressed in molar units.

In biological systems, small changes in ligand concentration usually result in rapid changes in response based on a sigmoidal function. The inflection point at which the increase in reaction with increasing ligand concentration begins to slow down is EC ₅₀ . It can be determined mathematically by deriving the most suitable line. Relying on a graph for evaluation is convenient in most cases. Where EC ₅₀ is indicated in the examples section, the experiments were designed to reflect K _D as accurately as possible. In other words, the EC ₅₀ values can then be considered as K _D values. The term "average K _D " refers to the average value of K _D obtained in at least 1, but preferably more than 1, for example, at least 2 experiments. The term "average" refers to the mathematical term "mean" (the sums of the data divided by the number of elements in the data).

This also applies to IC ₅₀ , which is a measure of the inhibition of a compound (50% inhibition). For competitive binding assays and functional antagonist assays, IC ₅₀ is the most common summary dose-response curve. For agonist/stimulator assays, the most common summary measure is the EC ₅₀ .

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

in which [L] is a fixed ligand concentration.

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

The terms "(cross) blocking", "(cross) blocking", "(cross) blocking", "competitive linking", "(cross)-competing", "(cross)-competing", and "(cross)-competing" are used interchangeably herein, means the ability of an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide, or other binding agent to interfere with the binding of other immunoglobulins, antibodies, immunoglobulin single variable domains, 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 a target, and therefore whether it can be said to be cross-blocked in accordance with the invention, can be determined using competitive assays that are well known in the art. this area. Particularly suitable quantitative cross-blocking assays include ELISA and fluorescence activated binding assay (FACS) with cell-expressed aggrecan. In the FACS settings, the degree of (cross) blocking can be measured by the (reduced) channel fluorescence.

Methods for determining whether an immunoglobulin, antibody, ISV, polypeptide or other binding agent is capable of (cross)-blocking, competitively binding or (cross)-competing as defined herein, for example, are described 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, for example, Example 2.3).

The amino acid sequence is said to be "cross-reactive" for two different antigens or antigenic determinants (such as, for example, aggrecan from different mammalian species, such as, for example, human aggrecan, dog aggrecan, bovine aggrecan, rat aggrecan, porcine aggrecan, mouse aggrecan, rabbit aggrecan, cynomolgus aggrecan and/or rhesus monkey aggrecan) if it is specific (as defined herein) for these various antigens or antigenic determinants.

In the context of the present invention, “modulating” or “modulating” generally means reducing or inhibiting the activity of a member of the serine protease family, cathepsins, matrix metalloproteinases (MMPs)/matrixins, or disintegrin and metalloproteinase with a thrombospondin motif (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20 , ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1), ADAMTS11 and/or pro-inflammatory cytokines such as, for example, interleukin-1α and -β, interleukin-6 and TNF-α by ISV, polypeptide or construct according to the invention measured using a suitable in vitro assay, cell assay, ex vivo assay, or in vivo assay (eg, those mentioned herein). In particular, "modulating" or "modulating" can mean either a reduction or inhibition of the activity of the aforementioned members, as measured using a suitable in vitro assay, cell assay, ex vivo assay, 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%, such as at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the activity of the aforementioned representatives in the same assay under the same conditions, but without the presence of an immunoglobulin or a polypeptide of the invention.

In the context of the present invention, "enhance" or "enhance" generally means an increase, potentiation, or stimulation of the activity of polypeptides or constructs of the invention, as measured using an appropriate in vitro assay, cell assay, ex vivo assay, or in vivo assay (such as those mentioned herein) . In particular, an increase or enhancement in the activity of a polypeptide or construct of the invention, as measured using a suitable in vitro assay, cell assay, ex vivo or in vivo assay (such as those mentioned herein) by at least 5%, preferably at least 10%, min 15%, min 20%, min 25%, min 30%, min 35%, min 40%, min 45%, min 50%, min 55%, min 60% at least 65% at least 70% at least 75% at least 80% at least 85% at least 90% at least 95% or more, e.g. 100%, compared to construct or polypeptide activity in the same assay under the same conditions , but without the presence of an aggrecan-binding agent, eg, aggrecan-binding ISV, of the invention.

A "synergistic effect" of two compounds is one in which the effect of the combination of two agents is greater than the sum of their individual effects, and preferably is statistically different from the standard and the individual drugs.

As used herein, the term "activity" of an ISV or polypeptide of the invention is a function of the amount of ISV or polypeptide of the invention required for its specific effect, such as, for example, cartilage penetration, specific binding to aggrecan, and/or cartilage retention. It can be measured simply by methods known to the person skilled in the art and, for example, those used in the Examples section.

In contrast, the "potency" of an ISV or polypeptide of the invention measures the maximum strength of the effect itself at saturation concentrations of the ISV or polypeptide. Efficacy indicates the maximum response achievable from an ISV or a polypeptide of the invention. This refers to the ability of an ISV or polypeptide to produce a desired (therapeutic) effect, such as, for example, binding to aggrecan or retaining aggrecan and/or inhibiting the activity of a member of the ADAMTS family or a member of the MMP family.

The "half-life" of a polypeptide or construct of the invention refers to the time it takes for the serum concentration of the construct or polypeptide to decrease by 50% in vivo, e.g., due to degradation of the construct or polypeptide and/or clearance or sequestration of the construct or polypeptide by natural mechanisms. , see, for example, paragraph o) on page 57 in WO 08/020079. The in vivo half-life of a construct or polypeptide of the invention can be determined by any known method, such as pharmacokinetic analysis. Suitable techniques will be understood by a person skilled in the art and may, for example, be generally 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, half-life can be expressed using parameters such as t1/2-alpha, t1/2-beta and area under the curve (AUC). Sources can be, for example, standard reference books such as Kenneth et al. (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, John Wiley & Sons Inc, 1986) and M Gibaldi and D Perron ("Pharmacokinetics", Marcel Dekker, 2nd ^Rev. Edition, 1982). The terms "increase in half-life" or "increased half-life" refers to an increase in t1/2-beta, either with or without an increase in t1/2-alpha and/or AUC, or both, for example, as described in paragraph o) on page 57 in WO 08/020079.

Unless otherwise indicated, the terms "immunoglobulin" and "immunoglobulin sequence"—whether used herein to refer to a heavy chain-only antibody or a conventional 4-chain antibody—are used as a generic term for inclusion as a full-length antibody, and its individual chains, as well as all parts, domains or fragments thereof (including, without limitation, antigen-binding domains or fragments, such as VHH domains or VH/VL domains, respectively).

As used herein, the term "domain" (of a polypeptide or protein) refers to the structure of a folded protein that has the ability to retain its tertiary structure independently of the rest of the protein. Typically, domains are responsible for discrete functional properties of proteins, and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and/or domain.

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

As used herein, the term "immunoglobulin variable domain" means an immunoglobulin domain essentially consisting of four "framework regions", which are referred to in this field and hereinafter as "framework region 1" or "FR1"; as "wireframe 2" or "FR2"; as "wireframe 3" or "FR3"; and as "frame region 4" or "FR4", respectively; wherein the framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in this area and below as "complementarity determination region 1" or "CDR1"; as "complementarity detection region 2" or "CDR2"; and as "complementarity detection region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be specified as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the variable domain (or domains) of an immunoglobulin which confers specificity of an antibody to an antigen by containing an antigen-binding site.

The term "immunoglobulin single variable domain" ("ISV" or "ISVD"), used interchangeably with "single variable domain", defines molecules in which an antigen binding site is present and constituted by a single immunoglobulin domain. This sets ISVs apart from "regular" immunoglobulins or fragments thereof, in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen binding site. In this case, complementarity determining regions (CDRs) of both VH and VL will contribute to the creation of an antigen binding site, ie. a total of 6 CDRs will be involved in creating the antigen binding site.

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 a Fab fragment, an F(ab')2 fragment, an Fv fragment, such as a disulfide-linked Fv either an scFv fragment or a diabody (all known in the art) derived from such a conventional 4 chain antibody are generally not considered an immunoglobulin single variable domain because, in these cases, binding to the corresponding antigen epitope is usually not a single (single) an immunoglobulin domain, but a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i. e. using a VH-VL pair of immunoglobulin domains that co-bind to an epitope of the corresponding antigen.

In contrast, ISVs are able to specifically bind to an antigen epitope without pairing with an additional immunoglobulin variable domain. The ISV binding site is formed by a single VH/VHH or VL domain. Therefore, an ISV antigen binding site is formed by no more than three CDRs.

Thus, a single variable domain may be a light chain variable region sequence (eg, VL sequence) or a suitable fragment thereof; or a heavy chain variable region sequence (eg, VH sequence or VHH sequence) or a suitable fragment thereof; since such a domain is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of a single variable domain such that one antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

In one embodiment of the invention, the ISVs are heavy chain variable domain sequences (eg, VH sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody, or heavy chain variable domain sequences that are derived from a heavy chain-only antibody.

For example, an immunoglobulin single variable domain can be a (single) domain antibody (or an amino acid suitable for use as a (single) domain antibody), a "dAb" or a dAb (or an amino acid suitable for use as a dAb), or a nanobody. (as defined herein and including, without limitation, VHH); other single variable domains, or any suitable fragment of any of them.

In particular, the immunoglobulin single variable domain may be Nanotel® (as defined herein) or a suitable fragment thereof. [Note: Nanobody® and Nanobody® are registered trademarks of Ablynx N.V.]. For a general description of nanobodies, reference herein may be made to the following description as well as to the prior art referred to herein, for example as described in WO 08/020079 (p. 16).

"VHH domains", also known as VHH, VHH domains, VHH antibody fragments, and VHH antibodies, were originally described as the antigen-binding immunoglobulin (variable) domain of "heavy chain-only antibodies" (i.e., "antibodies devoid of light chains", Hamers-Casterman et al. Nature 363: 446-448, 1993). The term "VHH domain" was chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4 chain antibodies (referred to herein as "V _H domains" or "VH domains") and from the light chain variable domains that are present in conventional 4 chain antibodies (referred to herein as "V _L domains" or "VL domains"). For a further description of VHH and nanobodies, see the review article Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as the following patent applications, which are cited as prior art: WO 94/04678, WO 95/04079 and WO 96/34103 from Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 from Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 from Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 from Algonomics NV and Ablynx NV; WO 01/90190 from the National Research Council of Canada; WO 03/025020 (= EP 1433793) from the Antibody Institute; and WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 086/1227 and WO 06/122825 from Ablynx NV and further published patent applications from Ablynx NV Reference may also be made to the additional prior art mentioned in these applications and in particular to the list of references mentioned on pages 41-43 of International Application WO 06/ 040153, the list and sources of which are incorporated herein by reference. As described in these references, ISVs, nanobodies (in particular, VHH sequences and partially humanized nanobodies), in particular, can be characterized by the presence of one or more "characteristic residues" in one or more framework sequences. Further description of ISVs, nanobodies, including humanization and/or overblending of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusion molecules", multivalent constructs (including some non-limiting examples of linker sequences), and various modifications to increase the period the elimination half-lives of ISVs, nanobodies and formulations thereof can be found, for example, in WO 08/101985 and WO 08/142164. For further general description of nanobodies, we hereby refer to the prior art cited herein, such as, for example, described in WO 08/020079 (p. 16).

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

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

Thus, within the meaning of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides that are derived from a non-human source, preferably a camel, preferably a heavy chain-only camel antibody. They can be humanized as described above. In addition, the term includes polypeptides derived from non-camelid sources, such as mice or humans, that have been "overtowed" as described, for example, in Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13 : 475-479, 1995; Prot. Eng. 9: 531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999).

The amino acid residues of the VHH domain are numbered according to the general numbering for VH domains given in Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), in relation to camelid VHH domains, as shown, for example, in figure 2 in Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999). Alternative methods for numbering amino acid residues of VH domains, which can also be applied in a similar manner to VHH domains, are known in the art. However, in the present specification, claims and figures, the Kabat numbering applicable to VHH domains as described above will be followed unless otherwise indicated.

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

The determination of the CDR regions may also be performed in accordance with various methods. In the Kabat definition of CDR, VHH FR1 includes amino acid residues at positions 1-30, VHH CDR1 includes amino acid residues at positions 31-35, VHH FR2 includes amino acids at positions 36-49, VHH CDR2 includes amino acid residues at positions 50- 65, VHH FR3 includes amino acid residues at positions 66-94, VHH CDR3 includes amino acid residues at positions 95-102, and VHH FR4 includes amino acid residues at positions 103-113.

(Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). Согласно этому способу FR1 включает аминокислотные остатки в положениях 1-25, CDR1 включает аминокислотные остатки в положениях 26-35, FR2 включает аминокислоты в положениях 36-49, CDR2 включает аминокислотные остатки в положениях 50-58, FR3 включает аминокислотные остатки в положениях 59-94, CDR3 включает аминокислотные остатки в положениях 95-102, а FR4 включает аминокислотные остатки в положениях 103-113 (по нумерации Kabat).However, in the present application, the CDR sequences have been determined according to Kontermann and

(Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). According to this method, FR1 includes amino acid residues at positions 1-25, CDR1 includes amino acid residues at positions 26-35, FR2 includes amino acids at positions 36-49, CDR2 includes amino acid residues at positions 50-58, FR3 includes amino acid residues at positions 59- 94, CDR3 includes amino acid residues at positions 95-102 and FR4 includes amino acid residues at positions 103-113 (Kabat numbering).

ISVs such as domain antibodies and nanobodies (including VHH domains) can be humanized. In particular, humanized immunoglobulin single variable domains such as nanobodies (including VHH domains) may be immunoglobulin single variable domains as generally defined in the preceding paragraphs, but in which at least one amino acid residue is present (and, in in particular at least one of the framework residues) that is and/or corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions can be identified by comparing the framework sequence of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, followed by one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into the specified VHH sequence (by any method known per se, as further described herein), and the resulting humanized VHH sequences can be tested for target affinity, stability, ease and level of expression, and/or other desired properties . Thus, with a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) may be determined by one of ordinary skill in the art based on the description disclosed herein. In addition, based on the above (framework regions), an immunoglobulin single variable domain such as Nanobody (including VHH domains) can be partially humanized or fully humanized.

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 changes in the amino acid sequence of one or more CDRs, which changes lead to an improvement in the affinity of the resulting immunoglobulin single variable domain to its corresponding antigen compared to the corresponding parent molecule. The affinity matured immunoglobulin single variable domain molecules of the invention can be prepared by methods known in the art, for example, as described by Marks et al. (Biotechnology 10: 779-783, 1992), Barbas et al. (Proc. Nat. Acad. Sci, USA 91: 3809-3813, 1994), Shier et al. (Gene 169: 147-155, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (J. MoI. Biol. 226: 889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996).

The process of constructing/selecting and/or producing a polypeptide starting from an ISV, such as a domain antibody or nanobody, is also referred to herein as "formatting" said ISV; and the ISV that is part of the polypeptide is said to be "formatted" or "in the format" of said polypeptide. Examples of ways in which an ISV may be formatted, and examples of such formats, will be understood by one of ordinary skill in the art based on the disclosures herein; and such a formatted ISV forms a further aspect of the invention.

For example, and without limitation, one or more immunoglobulin single variable domains can be used as a "binding unit", "binding domain", or "building block" (these terms are used interchangeably) to produce a polypeptide that may optionally contain one or more additional ISVs that can serve as a binding entity (ie, against the same or a different epitope on aggrecan and/or against one or more other antigens, proteins, or targets other than aggrecan).

The present invention provides aggrecan binding agents such as ISVs (also referred to herein as "ISVs of the invention") and/or polypeptides (also referred to herein as "polypeptides of the invention") that have specificity and/or bind aggrecan.

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

Aggrecan is a large multimodular molecule (2317 amino acids). Its main protein consists of three globular domains (G1, G2 and G3) and a large extended region (CS) between G2 and G3, to which are attached many N-linked oligosaccharides and chondroitin sulfate chains and keratan sulfate chains. Aggrecan is the main proteoglycan in articular cartilage. It plays an important role in the proper functioning of articular cartilage, providing a hydrated gel structure through its interaction with hyaluronan and binding proteins, which gives cartilage its load-bearing properties. The G1 domain interacts with hyaluronic acid and binds proteins, forming stable ternary complexes in the extracellular matrix (ECM). The G2 domain is homologous to G1 tandem repeats and binding proteins and is involved in product processing. G3 constitutes the carboxyl terminus of the main protein and enhances glycosaminoglycan modification and product secretion. In addition, the G3 domain binds proteoglycan aggregates to ECM proteins (fibulins and tenascins). The degradation of aggrecan seems to start at the C-terminus. The population of aggrecan molecules without the G3 domain increases with age. Aggrecan interacts with laminin, fibronectin, tenascin, and collagen, but it is also an enzymatic substrate of various disintegrins and thrombo-spondin-motif metalloproteases (ADAMTS), such as ADAMTS4, ADAMTS5, and ADAMTS11, and matrix metalloproteinases (MMPs), such as MMP8 , MMP13, MMP19 and MMP20.

In one aspect, the invention provides aggrecan binding agents such as ISVs and polypeptides that specifically bind aggrecan.

The aggrecan binding agents of the invention are ultimately intended for use as drugs in humans. Accordingly, in one aspect, the invention provides aggrecan binding agents such as ISVs and polypeptides that specifically bind human aggrecan (SEQ ID NO: 125).

The inventors have identified aggrecan binding agents with significantly improved cross-species cross-reactivity and exceptional selectivity properties.

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

The present inventors have surprisingly found that the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, have favorable characteristics compared to prior art molecules; they are stable in the joints, they persist in cartilage for a long time and are specific for cartilage, for example, they do not substantially bind to neurocan (O14594, SEQ ID NO: 134) and/or brevican (Q96GW7, SEQ ID NO: 135) ( see also Table B).

Accordingly, in one aspect, the invention provides an aggrecan binding agent, such as an ISV or a polypeptide, that does not substantially bind to neurocan (O14594, SEQ ID NO: 134) and/or brevican (Q96GW7, SEQ ID NO: 135), preferably where said aggrecan binds to neurocan and/or brevican with a K _D value greater than 10 ^-5 mol/liter, such as 10 ^-4 mol/liter.

In one aspect, the invention relates to an aggrecan binding agent, such as ISV, wherein it has greater than 10-fold, greater than 100-fold, preferably greater than 1000-fold selectivity over neurocan and/or brevican for binding to aggrecan.

Preferred aggrecan-binding agents of the invention include immunoglobulins (such as heavy chain-only antibodies, conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD or IgE molecules), Fab fragments, F(ab') fragments 2, Fv fragments, such as disulfide-linked Fv or scFv or diabody fragments derived from such a conventional 4-chain antibody, its individual chains, and all parts, domains, or fragments thereof (including, without limitation, antigen-binding domains or fragments , such as immunoglobulin single variable domains), monovalent polypeptides of the invention, or other binding agents).

The aggrecan binding agents of the invention were noted to have pIs greater than 8, with only one exception (see also Table 2.2). Without wishing to be bound by any theory, the inventors of the present invention hypothesized that the high positive charge of aggrecan may affect the retention and penetration into the cartilage of the whole group, i.e. even in combination with another building block, as in a multispecific polypeptide. Accordingly, the present invention relates to an aggrecan binding agent such as an ISV, a polypeptide or a construct of the invention, preferably an ISV of the invention, with a pI greater than 8, such as 8.1, 8.2, 8.3, 8.4, 8 .5, 8.6, 8.7, 8.8, 8.9, 9.0 or even more, such as 9.1, 9.2, 9.3, 9.4, 9.5, 9, 6, 9.7, 9.8 or even 9.8.

Binding of aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, to aggrecan can be measured in various binding assays commonly known in the art. Typical assays include (but are not limited to) fluorescent ligand binding assays, fluorescence activated cell sorting (FACS), radioligand binding assays, surface plasmon resonance (SPR), plasmon waveguide resonance (PWR), SPR imaging for affinity-based biosensors, microresonator whispering gallery mode (WGM), resonant waveguide grating (RWG), biolayer interferometry, biosensor (BIB), nuclear magnetic resonance (NMR), X-ray crystallography, thermal denaturation (TDA), isothermal titration calorimetry (ITC), ELISA, etc. .d. cell ligand binding assays such as surface acoustic wave (SAW) biosensor and RWG biosensor assay. A preferred assay for measuring the binding of aggrecan-binding agents of the invention, such as ISVs and/or polypeptides of the invention, to aggrecan is an SPR, such as, for example, the SPR described in the examples, in which the binding of aggrecan-binding agents of the invention, such as as ISV and/or polypeptides according to the invention, with aggrecan. Some preferred K _D values for binding agents of the invention that bind aggrecan, such as ISVs and/or polypeptides of the invention, to aggrecan will become clear from the further description and examples provided herein. Another particularly preferred assay is ELISA, as detailed in the examples (see also examples 1.2 and 2.4).

The binding of aggrecan binding agents of the invention to aggrecan can also be measured in binding assays that preferably retain the conformation of the aggrecan target. Exemplary assays include (without limitation) assays in which aggrecan is exposed to the cell surface (such as, for example, CHO cells).

In an embodiment of the invention, the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, have a rate constant (K _on ) for binding to said aggrecan selected from the group consisting of at least about 10 ² M ^-1 s ^{- 1} at least about 10 ³ M ^-1 s ^-1 at least about 10 ⁴ M ^-1 s ^-1 at least about 10 ⁵ M ^-1 s ^-1 at least about 10 ⁶ M ^-1 s ^-1 , 10 ⁷ M ^-1 s ^-1 at least about 10 ⁸ M ^-1 s ^-1 at least about 10 ⁹ M ^-1 s ^-1 and at least about 10 ¹⁰ M ^-1 s ^-1 , preferably according to with surface plasmon resonance measurement.

In an embodiment of the invention, the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, have a knock-off rate constant (K _off ) for binding to said aggrecan selected from the group consisting of a maximum of 10 ^-3 s ^-1 , a maximum of about 10 ^-4 s ^-1 , maximum about 10 ^-5 s ^-1 , maximum about 10 ^-6 s ^-1 , maximum about 10 ^-7 s ^-1 , maximum about 10 ^-8 s ^-1 , maximum about 10 ^-9 s ^-1 and at most about 10 ^-10 s ^-1 , preferably as measured by surface plasmon resonance.

In an embodiment of the invention, the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, bind to said aggrecan with an average KD of 100 nM to 10 pM, such as an average KD 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, K _D is determined by SPR, eg determined by Proteon.

Some preferred EC ₅₀ values for the binding of immunoglobulins and/or polypeptides of the invention to aggrecan will become clear from the further description and examples provided herein.

In an ELISA binding assay, aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or the G1-IGD-G2 domain, can have EC ₅₀ values for human aggrecan binding of 10 ^-8 M or less, more preferably 10 ^-9 M or less, or even 10 ^-10 M or less. For example, in such an ELISA binding assay, the immunoglobulins and/or polypeptides of the present invention may have EC ₅₀ values for human aggrecan binding between 10 ^-10 M and 10 ^-8 M, such as between 10 ^-9 M and 10 ^-8 M, or between 10 ^-10 M and 10 ^-9 M.

In such an ELISA binding assay, aggrecan-binding agents of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or the G1-IGD-G2 domain, can have EC ₅₀ values when binding cynomolgus monkey aggrecan (cyno) 10 ^-7 M or less, preferably 10 ^-8 M or less, more preferably 10 ^-9 M or less, or even 10 ^-10 M or less. For example, in such an ELISA binding assay, the polypeptides of the present invention may have EC ₅₀ values when binding cynomolgus monkey aggrecan from 10 ^-10 M to 10 ^-7 M, for example, from 10 ^-10 M to 10 ^-8 M, from 10 ^-10 M up to 10 ^-9 M.

In such an ELISA binding assay, aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or the G1-IGD-G2 domain, can have EC ₅₀ values with a rat aggrecan binding of 10 ^{- 6} M or less, preferably 10 ^-7 M or less, preferably 10 ^-8 M or less, more preferably 10 ^-9 M or less, or even 10 ^-10 M or less. For example, in such an ELISA binding assay, the polypeptides of the present invention may have rat aggrecan binding EC 50 values from 10 ^-10 M to 10 ^-6 M, for example, from 10 ^-10 M to 10 ^-7 M, from 10 ^-10 M to 10 ^-8 M, from 10 ^-10 M to 10 ^-9 M.

In such an ELISA binding assay, aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the present invention, preferably binding the G1 domain and/or the G1-IGD-G2 domain, may have an EC ₅₀ value for dog aggrecan binding of 10 ^{- 6} M or less, preferably 10 ^-7 M or less, preferably 10 ^-8 M or less, more preferably 10 ^-9 M or less, or even 10 ^-10 M or less. For example, in such an ELISA binding assay, the polypeptides of the present invention may have dog aggrecan binding EC ₅₀ values from 10 ^-10 M to 10 ^-6 M, for example, from 10 ^-10 M to 10 ^-7 M, from 10 ^-10 M to 10 ^-8 M, from 10 ^-10 M to 10 ^-9 M.

In such an ELISA binding assay, aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the present invention, may preferably bind the G1 domain and/or the G1-IGD-G2 domain, have EC ₅₀ values when binding bovine aggrecan equal to 10 ^-6 M or less, preferably 10 ^-7 M or less, preferably 10 ^-8 M or less, more preferably 10 ^-9 M or less, or even 10 ^-10 M or less. For example, in such an ELISA binding assay, the polypeptides of the present invention may have bovine aggrecan binding EC ₅₀ values from 10 ^-10 M to 10 ^-6 M, for example, from 10 ^-10 M to 10 ^-7 M, from 10 ^-10 M to 10 ^-8 M, from 10 ^-10 M to 10 ^-9 M.

As used herein, the term "cartilage tissue" refers to cartilage, including elastic cartilage, hyaline cartilage, and fibrocartilage, which is defined by the ratio of cells (chondrocytes) to intercellular space and the relative amounts of collagen and proteoglycan. "Articular cartilage" is the cartilage found on the articular surface of bones, and is basically hyaline cartilage. The menisci are made entirely of fibrocartilaginous tissue. Aggrecan is the main proteoglycan in the extracellular matrix (ECM) and makes up about 50% of the total protein content (the remaining approx. 50% is collagen II and some minor proteins, such as collagen IX).

The aggrecan-binding agents of the invention have shown preferential binding to cartilaginous tissues in the joint, such as cartilage and meniscus, as compared to non-cartilaginous tissue, such as the synovial membrane, tendon, and/or epimysium. Accordingly, the present invention relates to an aggrecan binding agent such as an ISV or a polypeptide, wherein said aggrecan binding agent preferably binds to cartilage tissue such as cartilage and/or meniscus, preferably at least 1.5 times, 2 times, 3 times, 4 times, 5 times or even more compared to non-cartilaginous tissue.

It should be taken into account that joints are areas where two or more bones meet. Most joints are movable, which allows the bones to move. Joints are made up of: cartilage, synovium, ligaments, tendons, bursa, and synovial fluid. Some joints also have a meniscus.

As shown in the examples, the aggrecan-binding agents of the invention have different cartilage retention characteristics, allowing for customization of joint retention according to specific needs (see also Example 2.2). Preferably, aggrecan binding agents are able to be retained in cartilage for longer periods of time after relatively short exposure of aggrecan binding agents to cartilage than would be expected from intra-articular injection. Cartilage retention can be measured using an ex vivo cartilage retention assay, as outlined in the examples section. The retention rate can be measured by visual inspection of a Western blot or by densitometric quantitation. The scale used to determine the degree of retention can be determined by a person skilled in the art, for example, a scale of 0 to 6 RU (Retention Units), where 0 is no retention and 6 is complete retention in this assay. If necessary, the scale can be quantified using the aggrecan binding agents of the invention, in which each aggrecan binding agent is given a score, eg, complete retention and no retention are recorded. Alternatively, the scale can be constructed with different intermediate scores that are assigned using the aggrecan binding agents of the invention, e.g., an aggrecan binding agent containing two 114F08 = 6 RU and a false aggrecan binding agent, e.g., ALB26-ALB26 = 0 RU; or an aggrecan binding agent containing two 114F08 = 6; aggrecan binding agent containing 608A05 = 5; aggrecan binding agent, 604G01 = 4; aggrecan binding agent containing two 601D02 = 3; aggrecan binding agent containing two 606A07 = 2; aggrecan binding agent, 112A01 = 1; and a decoy aggrecan binding agent, eg ALB26-ALB26 = 0 (see also Table 2.2). Accordingly, the present invention provides an aggrecan binding agent, such as an ISV and/or a polypeptide of the invention, wherein said aggrecan binding agent has a cartilage retention of at least 2, e.g., at least 3, 4, 5, or 6 RU in a retention assay in cartilage.

Aggrecan binding agents of the invention should preferably be stable. As a first premise, the biophysical properties of aggrecan binding agents were tested as detailed in Example 3, in which it was demonstrated that these aggrecan binding agents exhibited favorable stability characteristics, as evidenced by high melting points and no evidence of aggregation and multimerization. . The aggrecan-binding agents were then tested for their activity in the joints for extended periods by incubation in synovial fluid at 37° C. (see also Example 6). No degradation of any of the constructs was found, indicating that the constructs were stable under conditions mimicking the in vivo situation.

In one aspect, the invention relates to aggrecan binding agents, such as ISV, which have 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°C.

The present invention provides amino acid residue regions (SEQ ID NOs: 20-37 and 109, SEQ ID NOs: 38-55 and 110 and SEQ ID NOs: 56-74 and 111; Table A-2) that are particularly suitable for binding to aggrecan . In particular, the invention provides regions of amino acid residues that bind to human aggrecan, and in which the binding of said regions to said aggrecan remains present in cartilage (as described above). These amino acid residue regions may be present and/or may be included in a construct or polypeptide of the invention, in particular such that they form (part of) the antigen-binding site of the polypeptide of the invention. These amino acid residue regions were generated as heavy chain antibody CDR sequences or VHH sequences that were generated against aggrecan. These amino acid residue regions are also referred to herein as "inventive CDR sequence(s)" ("inventive CDR1 sequence(s)," "inventive CDR2 sequence(s)" and "inventive CDR3 sequence(s)," respectively. ).

However, it should be noted that the invention in its broadest sense is not limited to the specific structural role or function that these amino acid residue regions may have in the polypeptide of the invention, provided that these amino acid residue regions allow the polypeptide of the invention to bind to aggrecan with the desired affinity and potency. . Thus, in general, the invention provides, in its broadest sense, polypeptides (also referred to herein as "polypeptide(s) of the invention") that are capable of binding to aggrecan with a specific target affinity, avidity, potency, and/or effective concentration, and that contain one or more CDR sequences 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 additional amino acid sequences such that the entire polypeptide forms a binding domain and/ or a binding block capable of binding to aggrecan. However, it should also be noted that the presence of only one such CDR sequence in a polypeptide of the invention may by itself be sufficient to provide the polypeptide of the invention with the ability to bind to aggrecan; for example, reference can again be made to the so-called "contributory fragments" described in WO 03/050531.

In particular, but not in a limiting aspect, an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, may essentially consist of or contain at least one fragment of amino acid residues that is selected from the group consisting of :

i) CDR1 sequences:

a) SEQ ID NOs: 24, 32, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37 and 109; and

b) amino acid sequences that have a 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 24;

and/or

ii) CDR2 sequences:

c) SEQ ID NOs: 42, 50, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55 and 110; and

d) amino acid sequences that have a 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 42;

and/or

iii) CDR3 sequences:

e) SEQ ID NOs: 60, 68, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74 and 111; and

f) amino acid sequences that have a 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 60,

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, contains the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

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

Particularly preferably, an aggrecan binding agent of the invention, such as a polypeptide and/or ISV of the invention, may be an aggrecan binding agent that contains a single antigen binding site, wherein said antigen binding site contains at least one stretch of amino acid residues that is selected from the group consisting of CDR1 sequences, CDR2 sequences and CDR3 sequences as described above (or any suitable combination thereof). However, in a preferred aspect, preferably an aggrecan binding agent of the invention, such as a polypeptide and/or ISV of the invention, comprises more than one, for example two or more fragments of amino acid residues selected from the group consisting of CDR1 sequences of the invention, CDR2 sequences of the invention and/or CDR3 sequences of the invention. Preferably, an aggrecan binding agent of the invention, such as a polypeptide and/or ISV of the invention, comprises three stretches of amino acid residues selected from the group consisting of CDR1 sequences of the invention, CDR2 sequences of the invention, and CDR3 sequences of the invention, respectively. Combinations of CDRs that are mentioned herein as being preferred for an aggrecan binding agent of the invention, such as a polypeptide and/or ISV of the invention, are listed in Table A-2, i. preferably the combination of CDRs shown on one line in said table.

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

In a preferred embodiment, the present invention relates to an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, which contains 3 complementarity determining regions (CDR1-CDR3 respectively), where:

- CDR1 is selected from the group consisting of SEQ ID NOs: 24, 32, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37 and 109;

- CDR2 is selected from the group consisting of SEQ ID NOs: 42, 50, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55 and 110; and

- CDR3 is selected from the group consisting of SEQ ID NOs: 60, 68, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74 and 111

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In a preferred embodiment, the present invention relates to an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, which contains 3 complementarity determining regions (CDR1-CDR3 respectively), where:

- CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42 and CDR3 is SEQ ID NO: 60;

- CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50 and CDR3 is SEQ ID NO: 68;

- CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38 and CDR3 is SEQ ID NO: 56;

- CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39 and CDR3 is SEQ ID NO: 57;

- CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40 and CDR3 is SEQ ID NO: 58;

- CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41 and CDR3 is SEQ ID NO: 59;

- CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43 and CDR3 is SEQ ID NO: 61;

- CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 44 and CDR3 is SEQ ID NO: 62;

- CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45 and CDR3 is SEQ ID NO: 63;

- CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46 and CDR3 is SEQ ID NO: 64;

- CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47 and CDR3 is SEQ ID NO: 65;

- CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 48 and CDR3 is SEQ ID NO: 66;

- CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49 and CDR3 is SEQ ID NO: 67;

- CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 51 and CDR3 is SEQ ID NO: 69;

- CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 52 and CDR3 is SEQ ID NO: 70;

- CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50 and CDR3 is SEQ ID NO: 71;

- CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53 and CDR3 is SEQ ID NO: 72;

- CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54 and CDR3 is SEQ ID NO: 73;

- CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55 and CDR3 is SEQ ID NO: 74; or

- CDR1 is SEQ ID NO: 109, CDR2 is SEQ ID NO: 110, and CDR3 is SEQ ID NO: 111;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In a preferred embodiment, the present invention provides an aggrecan binding agent such as an ISV, wherein said ISV has been selected from the group consisting of SEQ ID NOs: 117, 5, 118, 13, 114-116, 1-4, 6-12 and 14-19.

In addition, it should be noted that the invention is not limited to the origin of the aggrecan binding agent of the invention, such as the ISV and/or the polypeptide of the invention (or the nucleic acid of the invention used to express it), nor as to how it is created or obtained ( or has been created or obtained), preferably an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, or a nucleic acid of the invention. Thus, preferably, an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, may be a naturally occurring ISV (from any suitable species) or synthetic or semi-synthetic ISVs and/or polypeptides.

In addition, one of skill in the art will also appreciate that one or more of the CDRs mentioned above can be "grafted" onto other "scaffolds", including, but not limited to, human or non-immunoglobulin scaffolds. Suitable scaffolds and methods for such CDR grafting will be understood by those skilled in the art and well known in the art, see, for example, US 7,180,370, WO 01/27160, EP 0605522, EP 0460167, US 7,054,297, Nicaise et al. (Protein Science 13: 1882-1891, 2004), Ewert et al. (Methods 34: 184-199, 2004), Kettleborough et al. (Protein Eng. 4: 773-783, 1991), O'Brien and Jones (Methods Mol. Biol. 207: 81-100, 2003), Skerra (J. Mol. Recognit. 13: 167-187, 2000) and Saerens et al. (J. Mol. Biol. 352: 597-607, 2005) and other sources cited therein. For example, methods known per se for grafting mouse or rat CDRs onto human scaffolds and supports can be used in a similar manner to produce 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 the presentation of amino acid sequences will be understood by one of skill in the art and, for example, include 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™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, ankyrin repeats, avimers and PDZ domains (Binz et al. Nat Biotech 23: 1257, 2005) and DNA-based binding fragments or RNA, including but not limited to DNA or RNA aptamers (Ulrich et al. Com Chem High Throughput Screen 9:619-32, 2006).

In an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, the CDRs may be linked to additional amino acid sequences and/or may be linked to each other via amino acid sequences, wherein said amino acid sequences are preferably frame sequences or are amino acid sequences that act as framework sequences, or together form a framework for CDR presentation.

According to a preferred embodiment, the aggrecan-binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, comprise at least three CDR sequences linked to at least two framework sequences, wherein preferably at least one of the three CDR sequences is is a CDR3 sequence, with the other two CDR sequences being CDR1 or CDR2 sequences, and preferably one CDR1 sequence and one CDR2 sequence. According to one particularly preferred but non-limiting embodiment, aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein CDR1, CDR2 and CDR3 are as defined herein for aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, and FR1, FR2, FR3 and FR4 are framework sequences. In such an aggrecan-binding agent of the invention, such as an ISV and/or a polypeptide of the invention, the framework sequences may be any suitable framework sequence, and examples of suitable framework sequences will be apparent to those skilled in the art, for example, based on standard reference books and the following disclosure and the prior art. techniques mentioned in this document.

Accordingly, preferably an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, contains 3 complementarity determining regions (CDR1-CDR3 respectively) in which:

(i) CDR1 is selected from the group consisting of:

(a) SEQ ID NOs: 24, 32, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37 and 109; and

(b) amino acid sequences that have a 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 24 or from any of SEQ ID NO: 20-23, 25-37 and 109; and/or

(ii) CDR2 is selected from the group consisting of:

(c) SEQ ID NOs: 42, 50, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55 and 110; and

(d) amino acid sequences that have a 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 42 or from any of SEQ ID NO: 38-41, 43-55 and 110; and/or

(iii) CDR3 is selected from the group consisting of:

(e) SEQ ID NOs: 60, 68, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74 and 111; and

(f) amino acid sequences that have a 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 60 or from any of SEQ ID NO: 56-59, 61-74 and 111

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

The aggrecan binding agents of the invention can be mapped to the G1 region, the G1-IGD-G2 region, or the G2 region of aggrecan.

Accordingly, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides that bind to the G2 domain of aggrecan. As indicated in the examples, these aggrecan binding agents of the invention, such as ISVs and/or polypeptides, have various preferred characteristics. Preferably, the aggrecan binding agents of the invention, such as ISVs and/or polypeptides, have a pI greater than 8 and/or have a K _off less than 2 x 10 ^-2 s ^-1 and/or have an EC _{50 of} less than 1 x 10 ^-6 M.

Comparison of the CDRs of the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, revealed a number of acceptable amino acid changes in the CDR while maintaining binding to the G2 domain of aggrecan. The sequence variability in the CDRs of all clones against the CDRs from 601D02, which were used as a standard, is presented in tables 1.5A, 1.5B and 1.5C.

In some embodiment, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides, wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOs: 28, 22, 26 and 33; and

b) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 28, where the amino acid difference is defined as follows:

- in position 1 G changed to R;

- in position 2 P changed to S or R;

- in position 3 T changed to I;

- in position 5 S changed to N;

- in position 6, R is changed to N, M or S;

- in position 7 Y changed to R or missing;

- in position 8 A changed to F or missing; and/or

- in position 10 G changed to Y;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOs: 46, 40, 44 and 52; and

d) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 46, where the amino acid difference is defined as follows:

- in position 1 A changed to S or Y;

- in position 4 W changed to L;

- in position 5 S changed to N;

- in position 6 S is absent;

- in position 7 G is absent;

- in position 8 G changed to A;

- in position 9 R changed to S, D or T; and/or

- in position 11 Y changed to N or R;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOs: 64, 58, 62 and 70; and

f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 64, where the amino acid difference is defined as follows:

- in position 1 A changed to R or F;

- in position 2 R changed to I or L;

- in position 3, I was changed to H or Q;

- in position 4 P changed to G or N;

- in position 5 V changed to S;

- in position 6 R changed to G, N or F;

- in position 7, T is changed to R, W or Y;

- in position 8, Y is changed to R or S or is missing;

- in position 9, T is changed to S or absent;

- in position 10 S changed to E, K or absent;

- in position 11 E changed to N, A or missing;

- in position 12 W changed to D or absent;

- in position 13 N changed to D or missing;

- in position 14 Y is absent; and/or

- D and N are added after position 14 in SEQ ID NO: 64;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents, wherein:

- CDR1 is selected from the group consisting of SEQ ID NO: 28, 22, 26 and 33;

- CDR2 is selected from the group consisting of SEQ ID NO: 46, 40, 44 and 52; and

- CDR3 is selected from the group consisting of SEQ ID NO: 64, 58, 62 and 70;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents, 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;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents selected from the group consisting of SEQ ID NOS: 9, 3, 7 and 15, and agents that bind aggrecan, which have more than 80%, for example, 90% or 95% sequence identity with any of SEQ ID NOS: 9, 3, 7 and 15.

In one aspect, the present invention relates to aggrecan-binding agents of the invention, such as ISVs and/or polypeptides that cross-block the binding 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 in as a single domain antibody, a dAb, an immunoglobulin which is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation for the G2 domain of aggrecan.

In one 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, an overblown VH sequence, or a VHH sequence that has been obtained by maturation of an affinity that binds to the G2 domain of aggrecan and that competes for binding to the G2 domain of aggrecan with aggrecan binding agents of the invention, such as ISV and/or polypeptides according to the invention, preferably presented in any of SEQ ID NOS: 9, 3, 7 and 15.

The present invention also relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides that bind to the G1-IGD-G2 domain of aggrecan. As indicated in the examples, these aggrecan binding agents of the invention, such as ISVs and/or polypeptides, have various preferred characteristics. Preferably, the aggrecan binding agents of the invention, such as ISVs and/or polypeptides, have a pI greater than 8 and/or have a K _off less than 2 x 10 ^-2 s ^-1 and/or have an EC _{50 of} less than 1 x 10 ^-6 M.

Comparison of the CDRs of the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, revealed a number of acceptable amino acid changes in the CDR while maintaining binding to the G1-IGD-G2 domain of aggrecan. The sequence variability in the CDR of all clones against the CDR from 604F02, which was used as a standard, is presented in tables 1.4A, 1.4B and 1.4C.

In an aspect, the present invention also provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides, wherein:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOs: 32, 30 and 23; and

b) amino acid sequences that have a 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 32, where the amino acid difference is defined as follows:

- in position 2 R changed to L;

- in position 6 S changed to T; and/or

- in position 8 T changed to A;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOs: 50, 41, 48 and 51; and

d) amino acid sequences that have a 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 36, where the amino acid difference is defined as follows:

- in position 7 G changed to S or R; and/or

- in position 8 R changed to T;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 68, 59, 66 and 69; and

f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 68, where the amino acid difference is defined as follows:

- in position 4 R changed to V or P;

- in position 6 A changed to Y;

- in position 7 S changed to T;

- in position 8 S is absent;

- in position 9 N changed to P;

- in position 10 R changed to T or L;

- in position 11 G changed to E; and/or

- in position 12 L changed to T or V;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides, wherein:

- CDR1 is selected from the group consisting of SEQ ID NO: 32, 30 and 23;

- CDR2 is selected from the group consisting of SEQ ID NO: 50, 41, 48 and 51; and

- CDR3 is selected from the group consisting of SEQ ID NO: 68, 59, 66 and 69;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents, 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;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group consisting of aggrecan binding agents of SEQ ID NOs: 118, 13, 4, 11 and 14, and agents that bind aggrecan, which have more than 80%, for example, 90% or 95% sequence identity with any of SEQ ID NOS: 118, 13, 4, 11 and 14.

In one aspect, the present invention relates to aggrecan-binding agents of the invention, such as ISVs and/or polypeptides that cross-block the binding 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 in as a single domain antibody, dAb, immunoglobulin which is suitable for use as a dAb, nanobody, VHH sequence, humanized VHH sequence, overblown VH sequence, or a VHH sequence that has been generated by affinity maturation for the G1-IGD-G2 domain of aggrecan.

In one 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, an overblown VH sequence, or a VHH sequence that has been obtained by maturation of an affinity that binds to the G1-IGD-G2 domain of aggrecan and that competes for binding to the Gg-IGD-G2 domain of aggrecan with a binding agent of the invention. aggrecan, such as an ISV and/or a polypeptide of the invention, preferably as shown in any of SEQ ID NOs: 118, 13, 4, 11 and 14.

In a particularly preferred embodiment, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, which bind to the G1 domain of aggrecan. As indicated in the examples, these aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, have various preferred characteristics. Preferably, the aggrecan binding agents of the invention, such as ISVs and/or polypeptides, have a pI greater than 8 and/or have a K _off less than 2 x 10 ^-2 s ^-1 and/or have an EC _{50 of} less than 1 x 10 ^-6 M.

Comparison of the CDRs of the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, revealed a number of acceptable amino acid changes in the CDR while maintaining binding to the G1 domain of aggrecan. The sequence variability in the CDR of all clones against the CDR from 114F08, which was used as a standard, is presented in tables 1.3A, 1.3B and 1.3C.

In a preferred aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, which contain 3 complementarity determining regions (CDR1-CDR3 respectively) in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36 and 37; and

b) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 24, where the amino acid difference is defined as follows:

- in position 2 S changed to R, F, I or T;

- in position 3 T changed to I;

- in position 5 I was changed to S;

- in position 6 I was changed to S, T or M;

- in position 7 N changed to Y or R;

- in position 8 V changed to A, Y, T or G;

- in position 9 V changed to M; and/or

- in position 10 R changed to G, K or A;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54 and 55; and

d) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 42, where the amino acid difference is defined as follows:

- in position 1, T is changed to A or G;

- S or N is inserted between position 3 and position 4 (position 2a, table 1.3B);

- in position 3 S changed to R, W, N or T;

- in position 4 S changed to T or G;

- in position 5 G changed to S;

- in position 6 G is changed to S or R;

- in position 7 N changed to S, T or R;

- in position 8 A changed to T; and/or

- in position 9, N is changed to D or Y;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73 and 74; and

f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 60, where the amino acid difference is defined as follows:

- in position 1 P changed to G, R, D or E or missing;

- in position 2, T is changed to R, L, P or V or absent;

- in position 3, T is changed to M, S or R or absent;

- in position 4 H changed to D, Y, G or T;

- in position 5 Y changed to F, V, T or G;

- in position 6 G is changed to L, D, S, Y or W;

- R, T, Y or V is inserted between position 6 and position 7 (position 6a, table 1.3C);

- in position 7 G is changed to P or S;

- in position 8 V is changed to G, T, H, R, L or Y;

- in position 9, Y is changed to R, A, S, D or G;

- in position 10 Y changed to N, E, G, W or S;

- W inserted between position 10 and position 11 (position 10a, table 1.3C);

- in position 11 G is changed to S, K or Y;

- in position 12 P is changed to E or D or missing; and/or

- in position 13 Y changed to L or missing;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

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

In a preferred aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents, 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;

- CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55 and CDR3 is SEQ ID NO: 74; and

- CDR1 is SEQ ID NO: 109, CDR2 is SEQ ID NO: 110, and CDR3 is SEQ ID NO: 111;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In the examples section, exemplary clone 114F08 has been shown to have particularly advantageous characteristics. Clone 114F08 is a family or set of clones further including clones 114A09 (SEQ ID NO: 114) and 114B04 (SEQ ID NO: 115) that have been grouped based on similarity in CDRs (see also Table A-2 and Tables 3.3A, 3.3B and 3.3C), resulting in similar functional characteristics. Therefore, in another particularly preferred aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides, which contain 3 complementarity determining regions (CDR1-CDR3 respectively) in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOs: 24 and 109; and

b) amino acid sequences that have a 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 24, where the amino acid difference is defined as follows:

- in position 7 N changed to S; and/or

- in position 9 V changed to M;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOS: 42 and 110; and

d) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 42, where the amino acid difference is defined as follows:

- in position 1, T is changed to A;

- in position 3 S changed to R;

- in position 4 S changed to T;

- in position 8 A changed to T; and/or

- in position 9 N changed to D;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 60 and 111; and

f) amino acid sequences that have a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 60, where the amino acid difference is defined as follows:

- in position 4 H changed to R; and/or

- in position 8 V changed to D;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents, wherein:

- CDR1 selected from the group consisting of SEQ ID NO: 24 and 109;

- CDR2 is selected from the group consisting of SEQ ID NO: 42 and 110; and

- CDR3 is selected from the group consisting of SEQ ID NOs: 60 and 111

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In addition, in the examples section, it was demonstrated that aggrecan binding agents that bind to the G1 region of aggrecan and belong to epitope group 1 or epitope group 4 are particularly effective in cartilage retention assays. In one aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides that belong to epitope group 1 or epitope group 4.

Comparison of the CDRs of aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention belonging to epitope group 1, revealed a number of acceptable amino acid changes in the CDR while maintaining binding to the G1 domain of aggrecan. The sequence variability in the CDRs of all clones against CDR 608A05, which was used as a standard, is presented in tables 2.3D, 2.3E and 2.3F.

In a preferred aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides, which contain 3 complementarity determining regions (CDR1-CDR3, respectively) in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOs: 36, 20 and 29; and

b) amino acid sequences that have a 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 36, where the amino acid difference is defined as follows:

- in position 3 T changed to S;

- in position 6 T changed to S;

- in position 8 T changed to A; and/or

- in position 9 M changed to V;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOs: 54, 38 and 37; and

d) amino acid sequences that have a 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 54, where the amino acid difference is defined as follows:

- in position 1 A changed to I;

- in position 4 W changed to R;

- in position 7 G changed to R; and/or

- in position 8 T changed to S;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 73, 56 and 65; and

f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 73, where the amino acid difference is defined as follows:

- in position 1 R changed to G;

- in position 2 P changed to R or L;

- in position 3, R is changed to L or S;

- in position 5 Y changed to R;

- in position 6 Y changed to S or A;

- in position 7 Y changed to T or missing;

- in position 8 S changed to P;

- in position 9 L changed to H or R;

- in position 10 Y changed to P or A;

- in position 11 S changed to A or Y;

- in position 12 Y changed to D;

- in position 13 D changed to F;

- in position 14 Y changed to G or missing; and/or

- S is inserted after position 14;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents, wherein:

- CDR1 is selected from the group consisting of SEQ ID NO: 20, 29 and 36;

- CDR2 is selected from the group consisting of SEQ ID NOs: 38, 47 and 54; and

- CDR3 is selected from the group consisting of SEQ ID NOs: 56, 65 and 73;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention relates to agents of the invention that bind aggrecan, such as ISVs and/or polypeptides belonging to epitope group 1, which cross-block the binding of a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, single domain antibody, immunoglobulin , which is suitable for use as a single domain antibody, a dAb, an immunoglobulin which is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been obtained by affinity maturation for the G1 domain of aggrecan.

In one 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 sequence VHH, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been generated by affinity maturation that binds to epitope group 1 of the aggrecan G1 domain and that competes for binding to the aggrecan G1 domain with aggrecan binding agents of the invention, such as ISVs and/or polypeptides that belong to epitope group 1, preferably such as, for example, for example, are presented in any of SEQ ID NOs: 1, 10 and 18.

Comparison of the CDRs of aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention belonging to epitope group 4, revealed a number of acceptable amino acid changes in the CDR while maintaining binding to the G1 domain of aggrecan. The sequence variability in the CDR of all clones against the CDR from 114F08, which was used as a standard, is presented in tables 2.3A, 2.3B and 2.3C.

In one aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides, which contain 3 complementarity determining regions (CDR1-CDR3 respectively) in which:

i) CDR1 is selected from the group consisting of:

a) SEQ ID NOs: 24, 25 and 27; and

b) amino acid sequences that have a 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 24, where the amino acid difference is defined as follows:

- in position 2 S changed to I or F;

- in position 5 I was changed to S;

- in position 6 I was changed to S or M;

- in position 7 N changed to R or Y;

- in position 8 V changed to A or Y;

- in position 9 V changed to M; and/or

- in position 10 R changed to K;

and/or

ii) CDR2 is selected from the group consisting of:

c) SEQ ID NOs: 42, 43 and 45; and

d) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 42, where the amino acid difference is defined as follows:

- in position 1, T is changed to A or G;

- N is inserted between position 2 and position 3 (position 2a, table 2.3B);

- in position 7 N changed to R;

- in position 8 A changed to T; and/or

- in position 9 N changed to D;

and/or

iii) CDR3 is selected from the group consisting of:

e) SEQ ID NOS: 60, 61 and 63; and

f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 60, where the amino acid difference is defined as follows:

- in position 1 P is absent;

- in position 2, T is changed to R or absent;

- in position 3, T is changed to M or absent;

- in position 4 H changed to D or Y;

- in position 5 Y changed to F or V;

- in position 6 G is changed to L or D;

- in position 8 V changed to G or T;

- in position 9 Y changed to R;

- in position 10 Y changed to N or E;

- in position 11 G changed to S or K;

- in position 12 P changed to E or missing; and/or

- in position 13 Y changed to L or missing;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention provides aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group of aggrecan binding agents, wherein:

- CDR1 is selected from the group consisting of SEQ ID NO: 24, 25 and 27;

- CDR2 is selected from the group consisting of SEQ ID NOs: 42, 43 and 45; and

- CDR3 is selected from the group consisting of SEQ ID NOs: 60, 61 and 63;

preferably, the aggrecan binding agent, such as an ISV and/or a polypeptide, comprises the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1, FR2, FR3 and FR4 are framework sequences.

In one aspect, the present invention relates to aggrecan-binding agents of the invention, such as ISVs and/or polypeptides belonging to epitope group 4, which cross-block the binding of a domain antibody, an immunoglobulin that is suitable for use as a domain antibody, single domain antibody, immunoglobulin , which is suitable for use as a single domain antibody, a dAb, an immunoglobulin which is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been obtained by affinity maturation for the G1 domain of aggrecan.

In one 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, an overblown VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to epitope group 4 of the aggrecan G1 domain and that competes for binding to the aggrecan G1 domain with aggrecan binding agents, and according to the invention , such as ISVs and/or polypeptides that belong to epitope group 4, such as, for example, those shown in any of SEQ ID NOs: 117, 114, 115, 116, 5, 6 and 8.

In one aspect, the present invention relates to aggrecan binding agents of the invention, such as ISVs and/or polypeptides selected from the group consisting of aggrecan binding agents shown in SEQ ID NOs: 117, 118, 116, 114, 115, 5 , 13, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19 and ISVs that have more than 80%, e.g. 90% or 95% or even more sequence identity with any of SEQ ID NO : 117, 118, 116, 114, 115, 5, 13, 1, 2, 6, 8, 10, 12, 16, 17, 18 and 19.

In a particular but non-limiting aspect, an aggrecan binding agent of the invention may be a region of amino acid residues that has a chain fold characteristic of immunoglobulins, or an aggrecan binding agent that, under suitable conditions (such as physiological conditions), is capable of forming a characteristic for immunoglobulin chain fold (i.e. folding). Among other things, the review of Halaby et al. (J. Protein Eng. 12: 563-71, 1999). Preferably, when properly folded to form the immunoglobulin-specific chain fold, amino acid residue regions may have the ability to correctly form an antigen-binding site for binding to aggrecan. Accordingly, in a preferred aspect, the aggrecan binding agent of the invention is an immunoglobulin, such as, for example, an immunoglobulin single variable domain.

Accordingly, framework sequences are preferably a (suitable combination) of immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (eg, by sequence optimization such as humanization or overblown). For example, framework sequences can be framework sequences derived from an immunoglobulin single variable domain, such as a light chain variable domain (eg, VL sequence) and/or from a heavy chain variable domain (eg, VH sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionally be partially or fully humanized) or are conventional VH sequences that have been overblown (as defined herein). document).

Framework sequences may preferably be such that the aggrecan binding agent 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); "dAb" (or an amino acid that is suitable for use as a dAb); Nanotelo®; VHH sequence; a humanized VHH sequence; overblown sequence VH; or a VHH sequence that was obtained by affinity maturation. Again, suitable framework sequences will be understood by those skilled in the art, for example, based on standard reference books and the further disclosure and prior art referred to herein.

Another particularly preferred class of ISVs of the invention includes an ISV with an amino acid sequence that matches the amino acid sequence of a naturally occurring VH domain, but which has been "overblue", i.e. obtained by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a normal A 4-chain antibody with one or more amino acid residues that are in the corresponding position(s) in the VHH domain of the heavy chain-only antibody. This can be done in a manner known to those skilled in the art, for example based on the description provided herein. Such "overlapping" substitutions are preferably introduced at amino acid positions which form and/or are present at the VH-VL interface and/or in the so-called characteristic Camelidae residues, which are well known to the person skilled in the art and which have been identified, for example, in WO 94/04678 and Davies and Riechmann (1994 and 1996). Preferably, the VH sequence that is used as a starting material or starting point for making or constructing overblown ISVs is preferably a mammalian VH sequence, more preferably a human VH sequence, such as a VH3 sequence. However, it should be noted that such overblown ISVs of the invention can be made by any suitable method known per se, and thus are not strictly limited to polypeptides that have been made using a polypeptide that contains a natural VH domain as a starting material.

For example, again, as further described herein, both "humanization" and "overlusion" can be performed by providing a nucleotide sequence that encodes a naturally occurring VHH domain or a VH domain, respectively, and then modifying, in a manner known per se, one or more codons in said nucleotide sequence, such that the new nucleotide sequence encodes a "humanized" or "overblown" ISV of the invention, respectively. This nucleic acid can then be expressed in a known manner to obtain the desired ISVs of the invention. Alternatively, based on the amino acid sequence of the naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or overblown ISVs of the invention, respectively, can be designed and then synthesized de novo using art-known peptide synthesis techniques. In addition, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or overblown ISVs of the invention, respectively, can be designed and then synthesized de novo using per se known methods for synthesis. nucleic acid, after which the nucleic acid thus obtained can be expressed in a manner known per se to provide the desired ISVs of the invention.

In particular, the framework sequences present in the aggrecan binding agents of the invention, such as the ISVs and/or the polypeptides of the invention, may contain one or more characteristic residues, for example, as defined in WO 08/020079 (Tables A-3 - A- 8), so that the aggrecan binding agent of the invention is a nanobody. Some preferred, but non-limiting examples (suitable combinations) of such framework sequences will become apparent from the further disclosure herein (see, for example, Table A-2). Typically, nanobodies (particularly VHH sequences and partially humanized nanobodies) can in particular be characterized by the presence of one or more "characteristic residues" in one or more framework sequences (as, for example, further described in WO 08/020079, page 61 , page 24 - page 98, line 3). As used herein, the term "represented" in the context of any SEQ ID NO is equivalent to "comprises or consists of" the specified SEQ ID NO, and is preferably equivalent to "consists of" the specified SEQ ID NO.

More specifically, the invention provides aggrecan binding agents comprising at least one ISV which is an amino acid sequence with the (general) structure

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

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

- 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 purposes of determining the degree of amino acid identity, the amino acid residues that form CDR sequences are not taken into account. In this regard, for example, Table A-2, which lists framework 1 sequences (SEQ ID NOs: 119, 120 and 75-84), framework 2 sequences (SEQ ID NOs: 121 and 85-93), framework sequences 3 (SEQ ID NOs: 123, 124, 122, 94-104 and 112-113) and framework sequences 4 (SEQ ID NOs: 105-108) immunoglobulin single variable domains SEQ ID NOs: 117, 118, 116, 115 , 114 and 1-19; or

- combinations of framework sequences shown in Table A-2;

and in which:

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

Accordingly, the present invention relates to an ISV and/or a polypeptide, wherein said ISV essentially consists of 4 framework regions (FR1-FR4, respectively) and said 3 complementarity-determining regions CDR1-CDR3, for example, an ISV that specifically binds aggrecan consists of 4 framework regions (FR1-FR4, respectively) and these three CDR1-CDR3 complementarity-determining regions, a therapeutic ISV, e.g., an ISV that binds to a member of the serine protease family, cathepsins, matrix metalloproteinases (MMPs)/matrixins, or disintegrin and a 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-FR4, respectively) and these three complementarity-determining regions CDR1-CDR3 ; The serum albumin binding ISV essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively).

Aggrecan binding agents of the invention, such as 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": US 61/ 994552 filed May 16, 2014; US 61/014015, filed June 18, 2014; U.S. Patent 62/040167, filed August 21, 2014; and US 62/047,560, filed September 8, 2014 (all assigned to Ablynx NV).

In particular, aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, may suitably contain (i) K or Q at position 112; or (ii) K or Q at position 110 in combination with V at position 11; or (iii) T at position 89; or (iv) L at position 89 with K or Q at position 110; or (v) V at position 11 and L at position 89; or any suitable combination of (i)-(v).

As also described in said co-pending U.S. Provisional Applications, when an aggrecan binding agent of the invention, such as an ISV and/or a polypeptide of the invention, contains mutations according to one of (i)-(v) above (or a suitable combinations):

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

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

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

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

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

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

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

As mentioned in these co-pending US Provisional Applications, these mutations are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to ISVs, polypeptides and constructs of the invention. For this purpose, aggrecan binding agents of the invention, such as 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 from 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4, or 5 (and preferably 1 or 2, such as 1), and each X is a (preferably naturally occurring) amino acid residue that is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)), for which we again refer to said US provisional applications and also to WO 12/175741. Particularly preferably, an aggrecan binding agent of the invention, such as an ISV and/or a 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 containing the same ( again, as further described, for example, in the referenced US Provisional Applications and also in WO 12/175741).

The aggrecan binding agent of the invention may be an immunoglobulin such as ISV obtained by any suitable method and from any suitable source, and may, for example, be naturally occurring VHH sequences (i.e. from a suitable camelid species) or synthetic or semi-synthetic amino acid sequences, including, but not limited to, “humanized” (as defined herein) nanobody or VHH sequences, “oversized” (as defined herein) immunoglobulin sequences (and in particular camelid heavy chain variable domain sequences ), as well as nanobodies that have been generated by methods such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using covering primers and similar methods for constructing an immunoglobulin sequence, well known to the person skilled in the art; or any suitable combination of any of the above, as further described herein. In addition, when an immunoglobulin contains a VHH sequence, said immunoglobulin may be suitably humanized, as further described herein, to provide one or more additional (partially or fully) humanized immunoglobulins of the invention. Similarly, when an immunoglobulin contains 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 to provide one or more additional (partially or completely) humanized immunoglobulins according to the invention.

In one aspect, the present invention relates to an aggrecan binding agent of the invention, such as an ISV, wherein said aggrecan binding agent is selected from the group consisting of SEQ ID NOS: 117, 118, 116, 115, 114, and 1-19.

ISVs can be used as a "building block" to produce a polypeptide, which may optionally contain one or more additional "building blocks", such as ISVs, against the same or a different epitope on aggrecan and/or against one or more other antigens, proteins, or targets other than aggrecan, eg, building blocks having a therapeutic mode of action, eg, therapeutic ISVs.

Generally, proteins or polypeptides or constructs that contain or essentially consist of a single building block, a single ISV, or a single nanobody will be referred to herein as "univalent" proteins or polypeptides or as "univalent constructs", respectively. Polypeptides or constructs that contain two or more structural elements or linking units (such as, for example, ISVs) will also be referred to herein as "multivalent" polypeptides or constructs, and structural elements/ISVs present in such polypeptides or constructs, will also be referred to herein as "multivalent format". For example, a "bivalent" polypeptide may contain two ISVs optionally linked via a linker sequence, while a "trivalent" polypeptide may contain three ISVs optionally linked via two linker sequences; while a "quadrivalent" polypeptide may contain four ISVs, optionally linked via three linker sequences, and so on.

In a polyvalent polypeptide or construct, two or more ISVs, such as nanobodies, may be the same or different and may be directed against the same antigen or antigenic determinant (e.g., against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or in any suitable combination thereof. Polypeptides or constructs that contain at least two structural elements (such as, for example, ISV), in which at least one structural element is directed against the first antigen (i.e., aggrecan), and at least one structural element is directed against second antigen (i.e., other than aggrecan, such as, for example, a therapeutic target) will also be referred to as "multispecific" polypeptides or multispecific constructs, respectively, and structural elements (such as, for example, ISV) present in such polypeptides or constructs will also be referred to herein as "multi-specific format". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide that contains at least one ISV directed against a first antigen (i.e., aggrecan) and at least one additional ISV directed against a second antigen (i.e., aggrecan). e. different from aggrecan, such as, for example, a therapeutic target), while a "trispecific" polypeptide of the invention is a polypeptide that contains at least one ISV directed against the first antigen (i.e., aggrecan) at least one additional An ISV directed against a second antigen (i.e., other than aggrecan, such as, for example, a therapeutic target) and at least one other ISV directed against a third antigen (i.e., different from both aggrecan and the second antigen ); etc.

"Multiparatopic" polypeptides and "multiparatopic" constructs, such as, for example, "biparatopic" polypeptides or constructs and "triparatopic" polypeptides or constructs, contain or essentially consist of two or more structural elements, each of which has its own separate paratope.

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

One or more ISVs of the invention can be used as a binding unit or in such a polypeptide or construct to provide a monovalent, multivalent or multiparatopic polypeptide or construct of the invention, respectively, all as described herein. Thus, the present invention also relates to a polypeptide which is a monovalent construct comprising or essentially consisting of a single monovalent polypeptide or ISV of the invention. Thus, the present invention also relates to a polypeptide or construct which is a multivalent polypeptide or a multivalent construct, respectively, such as, for example, a divalent or trivalent polypeptide or a construct comprising or essentially consisting of two or more ISVs of the invention (for multivalent and polyspecific polypeptides containing one or more VHH domains and their preparation are also referred to, for example, Conrath et al. /34103, WO 99/23221 and WO 2010/115998.

The invention also relates to a polyvalent polypeptide (also referred to herein as "multivalent(s) polypeptide(s) according to the invention"), which includes or (essentially) consists of at least one ISV, such as one or two ISV (or their suitable fragments) directed against aggrecan, preferably human aggrecan, and another additional ISV.

In one aspect, in its simplest form, a multivalent polypeptide or construct of the invention is a divalent 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 ISV, such as a nanobody, may optionally be linked via a linker sequence (as defined herein). In another form, a polyvalent polypeptide or construct of the invention may be a trivalent polypeptide or construct of the invention comprising a first ISV, such as a nanobody directed against aggrecan, identical to a second ISV, such as a nanobody directed against aggrecan, and a third ISV, such as a nanobody , is directed against an antigen other than aggrecan, such as, for example, a therapeutic target, in which said first, second and third ISVs, such as nanobodies, may optionally be linked via one or more, in particular two, linker sequences.

In another aspect, a polyvalent 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, for example, a therapeutic a target in which said first and second ISVs, such as nanobodies, may optionally be linked via a linker sequence (as defined herein); while the multivalent polypeptide or construct of the 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, for example, a therapeutic target, and a third ISV, such as a nanobody directed against a third antigen, such as, for example, also a therapeutic target, but different from said second antigen, in which said first, second and third ISV, such as nanobodies, may optionally be linked through one or more, and in particular two, linker sequences.

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, can 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 nanobodies directed against another antigen, such as, for example, a therapeutic target, in which said first, second and third ISVs, such as nanobodies, may optionally be linked via one or more, in particular two, linker sequences.

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, for example, a therapeutic target, where said first, second and third ISVs, such as nanobodies, may optionally be linked via one or more, in particular two, linker sequences.

Particularly preferred trivalent, bispecific polypeptides or constructs according to the invention are those shown in the examples described herein and in Tables E-1 and E-2.

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 can 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 a different antigen, such as, for example, a therapeutic target in which said first and second ISVs, such as nanobodies, may optionally be linked via a linker sequence.

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

The two or more ISVs present in a polyvalent polypeptide or construct of the invention may consist of a light chain variable domain sequence (eg, a VL sequence) or a heavy chain variable domain sequence (eg, a VH sequence); they may consist of a heavy chain variable domain sequence that is derived from a conventional four-chain antibody, or a heavy chain variable domain sequence that is derived from a heavy chain-only antibody. In a preferred aspect, they consist of a domain antibody (or an amino acid that is suitable for use as a domain antibody), a single domain antibody (or an amino acid that is suitable for use as a single domain antibody), a "dAb" (or an amino acid that is suitable for use in as a dAb), Nanotela® (including but not limited to VHH), humanized VHH sequence, overblown VH sequence; or a VHH sequence that was 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.

In one 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 containing two or more ISVs, where each ISV belongs to a different family. In one aspect, the first ISV of the multiparatopic (preferably biparatopic) polypeptide or construct of the invention does not block cross-linking to aggrecan of the second ISV of the multi-paratopic (preferably biparatopic) polypeptide or construct of the invention and/or the first ISV does not cross-block the second ISV from aggrecan. In another aspect, the first ISV of the multiparatopic (preferably biparatopic) polypeptide or construct of the invention cross-blocks the binding to aggrecan of the second ISV of the multiparatopic (preferably biparatopic) polypeptide or construct of the invention and/or the first ISV cross-blocks the binding to aggrecan of the second ISV.

In a preferred aspect, the polypeptide or construct of the invention contains 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 contains or essentially consists of three or more ISVs, of which at least two ISVs are directed against aggrecan. It is understood that the at least two ISVs directed against aggrecan may be the same or different, may be directed against the same epitope or different aggrecan epitopes, may belong to the same epitope group or different epitope groups, and/or may bind to one or different aggrecan domains.

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

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 any other of SEQ ID NOs: 117, 118, 114, 115, 116 and 1-19.

The final format of the molecule for clinical use is expected to include one or two building blocks, such as ISVs that bind aggrecan, and one or more building blocks, such as ISVs, with a therapeutic mode of action, and possibly additional fragments. In the examples section, it is shown that such formats retain aggrecan binding and retention properties, as well as therapeutic effect, such as enzymatic and/or inhibitory functions. One or more building blocks, such as an ISV 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, e.g., arthritic disease, osteoarthritis , spondyloepimetaphyseal dysplasia, lumbar disc degeneration disease, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, aggrecanopathy and/or in which aggrecan is used to guide, anchor and/or maintain other, for example, therapeutic structural elements at a desired site, such as joint. Thus, the present invention relates to a polypeptide or construct according to the invention, where one or more additional structural elements, for example, additional ISVs, retained activity.

The present invention relates to a polypeptide or construct that contains or essentially consists of at least one ISV of the invention, such as one or more ISVs of the invention (or suitable fragments thereof) that binds aggrecan and at least one additional ISV, in particular , a therapeutic ISV, wherein said at least one additional ISV preferably binds to a therapeutic target, e.g., binds to a member of the serine protease family, cathepsins, matrix metalloproteinases (MMPs)/matrixins, or disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and/or ADAMTS11.

In one aspect, the present invention relates to a polypeptide or construct of the invention essentially consisting of or comprising at least one aggrecan-binding ISV and at least one additional ISV that exerts a therapeutic effect, e.g., a therapeutic building block. A therapeutic effect can be any desired effect that improves, treats, or prevents a disease, as will be further detailed below. Preferably, the additional ISV, e.g., the therapeutic ISV inhibits or reduces the activity of a protease, e.g., inhibits or reduces the activity of a therapeutic target, i.e. a member of the serine protease family, cathepsins, matrix metalloproteinases (MMPs)/matrixins, or disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and/or ADAMTS11. Inhibition or reduction of activity can be achieved by binding to the active site or by altering the structure of the protease or proteinase, thereby preventing and/or reducing hydrolysis of the target protein by the protease or proteinase.

In one aspect, the present invention provides a polypeptide or construct of the invention selected from the polypeptides and constructs of Table E-1 and Table E-2.

In one aspect, the present invention provides 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°C.

In one aspect, the present invention provides an ISV, polypeptide or construct of the invention having a cartilage retention of at least 2, eg at least 3, 4, 5 or 6 RU in a cartilage retention assay.

In one aspect, the present invention provides an ISV, polypeptide or construct of the invention that penetrates cartilage at least 5 µm, such as at least 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, or even more.

The stability of a polypeptide, construct, or ISV of the invention can be measured using routine assays known to those skilled in the art. Exemplary assays include, but are not limited to, assays that measure the activity of a designated polypeptide, construct, or ISV, followed by incubation in synovial fluid for a desired period of time, after which the activity is measured again, for example, as detailed in the examples section (see See also Example 6).

The desired activity of a therapeutic building block in a multivalent polypeptide or construct of the invention can be measured using routine assays known to one of skill in the art. Typical assays include assays that analyze GAG release as detailed in the examples section (see Example 8).

The relative affinity may depend on the location of the ISVD in the polypeptide. It is understood that the order of the ISVDs in the 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 contains a linker, is a matter of design choice. Certain orientations, with or without linkers, may provide preferred binding characteristics over other orientations. For example, the order of the first ISV (eg, ISV 1) and second ISV (eg, ISV 2) in a polypeptide of the invention may be (N-terminus to C-terminus): (i) ISV 1 (eg, Nanobody 1) - [ linker] - ISV 2 (eg Nanobody 2) - [C-terminal extension]; or (ii) ISV 2 (eg Nanobody 2) - [linker] - ISV 1 (eg Nanobody 1) - [C-terminal extension]; (where the fragments between square brackets, i.e. linker and C-terminal extension, are optional). All orientations are covered by the invention. Polypeptides that contain an ISV orientation that provides the desired binding characteristics can be easily identified by routine screening, for example, as shown in the examples section. The preferred order is from N-terminus to C-terminus: therapeutic ISV - [linker] - aggrecan binding ISV - [C-terminal extension], where fragments between square brackets are optional. Another preferred order is from N-terminus to C-terminus: therapeutic ISV - [linker] - ISV, aggrecan binding - [linker] - ISV, aggrecan binding - [C-terminal extension], where the fragments between square brackets are optional.

The aggrecan binding agents of the invention, such as the polypeptides and/or ISVs of the invention, may or may not additionally contain one or more other groups, residues (e.g., amino acid residues), fragments, or binding units (these aggrecan binding agents, such as polypeptides and/or ISVs (with or without additional groups, residues, fragments or linking units) are all referred to as "compound(s) of the invention", "construct(s) of the invention" and/or "polypeptide(s) of the invention" ). If present, such additional groups, residues, fragments, or linking units may or may not provide additional functionality to the aggrecan binding agent, such as a polypeptide and/or ISV, and may or may not change the properties of the aggrecan binding agent, such as a polypeptide and /or or ISV.

For example, such additional groups, residues, fragments or linking units may be one or more additional amino acid sequences such that the resulting polypeptide is a (hybrid) polypeptide. In a preferred, but non-limiting aspect, said one or more other groups, residues, fragments or binding units are immunoglobulins. Even more preferably, said one or more other groups, residues, fragments or binding units are ISVs selected 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 dAb, amino acids suitable for use as dAbs, nanobodies (such as, for example, VHH, humanized VHH or overblown VH sequences).

As described above, additional binding units, such as ISVs, having different antigenic specificities, can be associated with the formation of polyspecific polypeptides. By combining ISVs of two or more specificities, bispecific, trispecific, and so on can be obtained. polypeptides or constructs. For example, a polypeptide of the invention may contain one, two or more ISVs directed against aggrecan and at least one ISV domain against another target. Such constructs and modifications thereof, which one of ordinary skill in the art can readily foresee, are encompassed by the term "compound of the invention, construct of the invention, and/or polypeptide of the invention" as used herein.

In the compounds, constructs and/or polypeptides described above, one, two, three or more ISVs and one or more groups, residues, or fragments may be linked directly to each other and/or through one or more suitable linkers or spacers. For example, when one or more groups, residues, or fragments are amino acid sequences, the linkers can also be amino acid sequences such that the resulting polypeptide is a hybrid (protein) or hybrid (polypeptide).

One or more additional groups, residues, fragments or linking units may be any suitable and/or desired amino acid sequences. Additional amino acid sequences may or may not change, transform, or otherwise affect the (biological) properties of the polypeptide of the invention, and may or may not add additional functionality to the polypeptide of the invention. Preferably, the additional amino acid sequence is such that it imparts one or more desired properties or functionality to the polypeptide of the invention.

Examples of such amino acid sequences will be understood by those skilled in the art and may generally include all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including, but not limited to, ScFv antibodies and single domain antibodies). Sources include, for example, the review by Holliger and Hudson (Nature Biotechnology 23: 1126-1136, 2005).

For example, such an amino acid sequence may be an amino acid sequence that increases half-life, solubility, or absorption, reduces immunogenicity or toxicity, eliminates or reduces unwanted side effects, and/or confers other beneficial properties and/or reduces undesirable properties of a compound, construct, and/or polypeptide according to the invention compared to the polypeptide according to the invention as such. Some non-limiting examples of such amino acid sequences are serum proteins such as human serum albumin (see e.g. WO 00/27435) or hapten molecules (e.g. haptens that are recognized by circulating antibodies, see e.g. WO 98/22141).

In a particular aspect of the invention, a construct or polypeptide of the invention may have a fragment that provides an increased half-life compared to the corresponding construct or polypeptide of the invention without said fragment. Some preferred, but non-limiting, examples of such constructs and polypeptides of the invention will become apparent to those skilled in the art based on further disclosure herein, and include, for example, ISVs or polypeptides of the invention that have been chemically modified to increase half-life (e.g., with PEG- ylation); aggrecan binding agents of the invention, such as ISVs and/or polypeptides of the invention, which contain at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that contain at least one amino acid sequence of the invention that is linked to at least one fragment (and in particular at least one amino acid sequence) that increases the half-life of the amino acid sequence of the invention. Examples of the constructs of the invention, such as the polypeptides of the invention, which contain such half-life increasing moieties or ISVs, will become apparent to those skilled in the art based on the further disclosure herein; and, for example, include, but are not limited to, polypeptides wherein 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 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 (e.g. human serum albumin), serum immunoglobulins such as IgG, or transferrin; we hereby refer to the additional description and references cited herein) ; polypeptides in which the amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable portion or fragment thereof; or polypeptides in which one or more ISVs of the invention are suitable for binding to one or more small proteins or peptides that can bind to serum proteins, such as, for example, the proteins and peptides described in WO 91/01743, WO 01/45746 , WO 02/076489, WO2008/068280, WO2009/127691 and PCT/EP2011/051559.

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

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

In general, constructs or polypeptides of the invention with an extended 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, such as at least 10 times, or more than 20 times the half-life of the respective constructs or polypeptides of the invention as such, ie without the half-life extension portion. For example, constructs or polypeptides of the invention with an extended half-life may have a half-life, e.g. in humans, that is increased by over 1 hour, preferably over 2 hours, more preferably over 6 hours, e.g. over 12 hours. or even more than 24, 48 or 72 hours compared to the corresponding constructs or polypeptides of the invention as such, ie without the moiety conferring an extended half-life.

In a preferred but non-limiting aspect of the invention, the constructs of the invention, such as the polypeptides of the invention, have a serum half-life, e.g., in humans this is increased by more than 1 hour, preferably by more than 2 hours, more preferably by more than 6 hours , for example, more than 12 hours, or even more than 24, 48 or 72 hours, compared with the corresponding constructs or polypeptides according to the invention per se, i. without a residue giving an increase in the half-life.

In another preferred, but non-limiting, aspect of the invention, such constructs of the invention, such as the polypeptides of the invention, exhibit a human serum half-life of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, yet 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 (e.g., about 5 to 10 days), preferably at least 9 days (e.g., about 9 to 14 days), more preferably at least about 10 days (e.g., about 10 to 15 days) or at least about 11 days (e.g., about 11 to 16 days), more preferably at least about 12 days (e.g., about 12 to 18 days or more) or more than 14 days (for example, about 14-19 days).

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, in addition to one or more aggrecan-binding building blocks and optionally one or more therapeutic building blocks, at least one building block, a serum albumin-binding ISV such as a serum albumin-binding ISV such as human serum albumin described herein, wherein said serum albumin-binding ISV contains or essentially consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity determining regions (CDR1-CDR3, respectively), in which CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR. Preferably, said human serum albumin binding ISV is selected from the group consisting of Alb8, Alb23, Alb129, Alb132, Alb135, Alb11, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82- G, Alb82-GG, Alb82-GGG, Alb92 or Alb223 (see also Table C).

In an embodiment, the present invention provides a construct of the invention, such as a polypeptide comprising a serum protein-binding fragment, wherein said serum protein-binding fragment is a non-antibody-based polypeptide.

In one aspect, the present invention relates to a compound or construct described herein, containing one or more other groups, residues, fragments or binding units, preferably selected from the group consisting of a polyethylene glycol molecule, whey proteins or their fragments, binding units, which can bind to serum proteins, part of the Fc and small proteins or peptides that can bind to serum proteins.

In one embodiment, the present invention relates to a construct of the invention, such as a polypeptide containing a fragment that provides an increase in half-life, where the specified fragment is a PEG. Therefore, the present invention relates to a construct or polypeptide according to the invention containing PEG.

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

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

- can form a signal sequence or leader sequence that directs the secretion of the polypeptide from the host cell upon synthesis (for example, to provide a pre-, pro-, or pre-pro form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention). Suitable secretory leader peptides will be understood by those skilled in the art and may be as described below. Typically, such a leader sequence will be linked to the N-terminus of the polypeptide, although the invention is not limited to this in its broadest sense;

- can form a "tag", for example, an amino acid sequence or residue, which allows or facilitates the purification of the polypeptide, for example, using affinity methods directed against the indicated sequence or residue. Thereafter, said sequence or residue may be removed (eg, by chemical or enzymatic cleavage) to produce a polypeptide (for this purpose, the label may optionally be linked to an 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;

- can be one or more amino acid residues that have been functionalized and/or that can serve as a site for attaching functional groups. Suitable amino acid residues and functional groups will be understood by those skilled in the art and include, without limitation, the amino acid residues and functional groups mentioned herein for derivatives of the polypeptides of the invention.

In the constructs of the invention, such as the polypeptides of the invention, two or more building blocks, such as, for example, ISVs and optionally one or more other groups, drugs, agents, residues, fragments, or linkers, 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. The skilled artisan will recognize which spacers or linkers for use in multivalent and polyspecific polypeptides are suitable, and they can 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.

For example, a polypeptide of the invention may be, for example, a trivalent trispecific polypeptide containing one structural element, such as ISV, binding aggrecan, one therapeutic structural element, such as ISV, and one structural element, such as ISV, binding (human) serum albumin, in which said first, second and third building blocks, such as ISV, may optionally be linked through one or more, and in particular two, linker sequences. Furthermore, the present invention relates to a construct or polypeptide of the invention comprising a first aggrecan-binding ISV and/or a second ISV and/or possibly a third ISV and/or possibly a serum albumin-binding ISV, wherein said first ISV and /or said second ISV and/or possibly said third ISV and/or possibly said serum albumin-binding ISV are linked through a linker.

Some particularly preferred spacers include spacers and linkers which are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general art cited above, as well as, for example, linkers that are used in the art to construct diabodies or ScFv fragments (however, it should be noted in this regard that although the linker used in diabodies and ScFv fragments the sequence must have a length, degree of flexibility, and other properties that allow suitable VH and VL domains to combine to form a complete antigen-binding site, there are no particular restrictions on the length or flexibility of the linker used in the polypeptide of the invention, since each ISV, such as a nanobody, is itself form a complete antigen-binding site).

For example, the linker may be a suitable amino acid sequence, and in particular amino acid sequences of 1 to 50, preferably 1 to 30, eg 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, e.g. of the (glyxsery)z type, such as (e.g. (gly4ser)3 or (gly3ser2)3 as described in WO 99/42077 and GS30, GS15, GS9 linkers and GS7 as described in the Ablynx applications referenced herein (see, for example, WO 06/040153 and WO 06/122825), as well as hinge-like regions such as natural antibody heavy chain hinges or similar sequences (such as, for example, described in WO 94/04678) Preferred linkers are shown in Table D (SEQ ID NOS: 154-170).

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

It is within the scope of the invention that the length, degree of flexibility and/or other properties of the linker(s) used (although not critical, as is usually the case for linkers used in ScFv fragments), may have some influence on the properties of the final constructs of the invention, such as a polypeptide of the invention, including, but not limited to, affinity, specificity, or avidity for a chemokine or one or more other antigens. Based on the disclosure herein, one skilled in the art 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 experimentation.

For example, in polyvalent polypeptides of the invention that contain building blocks, ISVs or nanobodies directed against aggrecan and another target, the length and flexibility of the linker is preferably such that it allows each building block, such as ISV, of the present invention to be present in the polypeptide for binding to its related target, for example, the antigenic determinant on each of the targets. Again, based on the disclosure herein, one skilled in the art will be able to determine the optimal linker(s) for use in a specific construct of the invention, such as a polypeptide of the invention, optionally after some limited routine experimentation.

It is also within the scope of the invention that the linker(s) used impart one or more other beneficial properties or functionality to the constructs of the invention, such as the polypeptides of the invention, and/or provide one or more sites for derivatization and/ or to attach functional groups (eg, as described herein for the ISV derivatives of the invention). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, while linkers that form or contain small epitopes or tags can be used for detection, identification and/or purification purposes. Again, based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linkers for use in a specific polypeptide of the invention, perhaps after some limited routine experimentation.

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

Typically, for 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 to this. For example, when a construct of the invention, such as a polypeptide of the invention, contains three or more building blocks, ISVs or nanobodies, they can be linked using a linker with three or more "arms", where each of the "arms" is associated with a building block, ISV or nanobody to provide a "star" design. It is also possible, although usually less preferred, to use ring structures.

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 one another or linked through a linker.

Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein a first ISV and/or a second ISV and/or optionally a serum albumin binding ISV are linked through a linker.

Accordingly, the present invention provides a construct of the invention, such as a polypeptide of the invention, wherein said linker is selected from the group consisting of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, 35GS, poly-A, 8GS, 40GS, G1 hinge, 9GS-G1 hinge, lama top long hinge and G3 hinge.

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

Also included in the present invention are compounds, constructs and/or polypeptides that contain an ISV or a polypeptide of the invention and further contain labels or other functional moieties, eg, toxins, labels, radiochemicals, etc.

Other groups, residues, fragments or linking units may, for example, be chemical groups, residues, fragments, which themselves may be biologically and/or pharmacologically active or not be biologically and/or pharmacologically active. For example, and without limitation, such groups can be linked to one or more ISVs or polypeptides of the invention to provide a "derivative" of the polypeptide of the invention.

Accordingly, the invention in its broadest sense also includes 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 (eg enzymatic) modification of the polypeptides of the invention and/or one or more amino acid residues that form the polypeptide of the invention.

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

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

For example, such modification may include the introduction (for example, by covalent bonding or any other suitable method) of one or more functional groups that increase the half-life, solubility and/or absorption of the polypeptide of the invention, reduce the immunogenicity and/or toxicity of the polypeptide of the invention, eliminate or reduce any undesirable side effects of the polypeptide of the invention and/or impart other beneficial properties and/or reduce undesirable properties of the polypeptide of the invention, or any combination of two or more of the above. Examples of such functional groups and methods of introducing them will be understood by the person skilled in the art and, as a rule, can include all the functional groups and methods mentioned in the general prior art cited above, as well as functional groups and methods known per se for the modification of pharmaceutical proteins. and, in particular, for the modification of antibodies or antibody fragments (including ScFv and single domain antibodies), in this connection, for example, reference is made to Remington (Pharmaceutical Sciences, 16 ^th ed., Mack Publishing Co., Easton, PA, 1980). Such functional groups may, for example, be linked directly (eg, covalently) to a polypeptide of the invention, or optionally via a suitable linker or spacer, as again will be appreciated by one of skill in the art.

One specific example is a derivative polypeptide of the invention, wherein the polypeptide of the invention has been chemically modified to increase its half-life (eg, by PEGylation). This is one of the most widely used methods for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins and involves the attachment of a suitable pharmacologically acceptable polymer such as poly(ethylene glycol) (PEG) or derivatives thereof (such as methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments such as, for example, (single) domain antibodies and ScFvs; the source can be, 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.

Preferably, site-directed PEGylation is used, in particular through a cysteine residue (see, for example, Yang et al. (Protein Engineering 16: 761-770, 2003). For example, for this purpose, PEG can be attached to a cysteine residue, which naturally occurs in a polypeptide of the invention, the polypeptide of the invention may be modified to suitably introduce one or more cysteine residues for PEG attachment, or an amino acid sequence containing one or more cysteine residues for PEG attachment may be combined with N - and/or the C-terminus of a polypeptide of the invention, all using protein engineering techniques known per se to a person skilled in the art.

Preferably, for the polypeptides of the invention, PEGs with a molecular weight greater than 5,000, such as greater than 10,000 and less than 200,000, such as less than 100,000, are used; for example, in the range from 20000 to 80000.

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

Another modification may include the introduction of one or more detectable labels or other signal-generating groups or fragments, depending on the intended use of the labeled polypeptide of the invention. Suitable labels and methods for attaching, using and detecting them will be understood by those skilled in the art and include, for example, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde). and fluorescent and fluorescent metals such as 152Eu or other metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, acridinium aromatic ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogues ), radioisotopes (such as ³ H, ¹²⁵ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe and ⁷⁵ Se), metals, metal chelates or metal cations (for example, metal cations such as ⁹⁹ mTc, ¹²³ I, ¹¹¹ In, ¹³¹ I, ⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga and ⁶⁸ Ga or other metals or metal cations which are particularly suitable for in vivo, in vitro or in situ diagnostic and imaging , such as ( ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr and ⁵⁶ Fe)), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphate fatase, asparaginase, glucose oxidase, β-galactose, β-galactose glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels will be understood by one of skill in the art and include, for example, fragments that can be detected by NMR or ESR spectroscopy.

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

As will be appreciated by one of skill in the art, another modification may include the introduction of a chelate group, for example to chelate one of the metals or metal cations mentioned above. Suitable chelating groups, for example, include, without limitation, diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

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

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

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 _D value (actual or apparent), a K _A value (actual or apparent) of the k _on and/or K _off rates or, alternatively, IC ₅₀ values, as further described herein) as defined herein (ie, as defined for the polypeptides of the invention).

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

In one aspect, the present invention relates to a construct of the invention that includes or essentially consists of an ISV of the invention or a polypeptide of the invention, and which further comprises one or more other groups, residues, fragments, or linking units, optionally linked through one or more peptide linkers.

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

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

The polyvalent polypeptides of the invention can generally be prepared by a method that includes at least the step of suitably linking an ISV and/or a monovalent polypeptide of the invention to one or more additional ISVs, optionally via one or more suitable linkers, so as to provide a polyvalent polypeptide at invention. The polypeptides of the invention can also be produced by a method that typically includes at least the steps of obtaining a nucleic acid encoding a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and isolating the expressed polypeptide of the invention. Such methods can be performed in a manner known to those skilled in the art, for example, based on the methods and techniques further described herein.

The process for producing polyvalent polypeptides of the invention may include 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 linked by any method known in the art and described hereinafter. Preferred methods include linking nucleic acid sequences that encode the ISVs of the invention (and linkers) to obtain a genetic construct that expresses a multivalent polypeptide. Methods for linking amino acids or nucleic acids will be understood by those skilled in the art, and again reference may be made to standard guidelines such as Sambrook et al. and Ausubel et al., mentioned above, as well as the examples below.

Accordingly, the present invention also relates to the use of the ISV of the invention for the production of a polyvalent polypeptide of the invention. A method for producing a polyvalent polypeptide will include linking an ISV of the invention to at least one additional ISV of the invention, optionally via one or more linkers. The ISV of the invention is then used as a binding domain or to provide and/or obtain a polyvalent polypeptide comprising 2 (for example, in a divalent polypeptide), 3 (for example, in a trivalent polypeptide), 4 (for example, in a tetravalent) or more (for example, in a polyvalent polypeptide) structural elements. In this regard, an ISV of the invention can be used as a binding domain or binding unit in providing and/or in obtaining a multivalent, eg, divalent, trivalent or tetravalent polypeptide of the invention containing 2, 3, 4 or more building blocks.

Accordingly, the present invention also relates to the use of an ISV polypeptide of the invention (as described herein) in the preparation of a polyvalent polypeptide. A method for producing a polyvalent polypeptide will include linking an ISV of the invention to at least one additional ISV of the invention, optionally via one or more linkers.

The polypeptides and nucleic acids of the invention may be prepared in a manner known per se, as will be apparent to those skilled in the art from the following description. For example, the polypeptides of the invention can be obtained by any known method for the production of antibodies and, in particular, for the production of antibody fragments (including, without limitation, (single)domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for producing polypeptides and nucleic acids include the methods and techniques described herein.

The method for producing a polypeptide according to the invention may include the following steps:

- 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 for a nucleic acid that encodes said polypeptide of the invention (also referred to herein as "nucleic acid of the invention "),

which is optionally followed by:

- isolation and/or purification of the polypeptide according to the invention thus obtained.

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

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").

The nucleic acid of the invention may 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 substantially isolated 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, for example an expression vector such as, for example, a plasmid, cosmid, or YAC, which again may be in substantially isolated form. Accordingly, the present invention also relates to an expression vector containing the nucleic acid or nucleotide sequence of the invention.

Nucleic acids of the invention can be prepared or obtained by a method that is itself known based on the information about the polypeptides of the invention given in this document, and/or can be isolated from a suitable natural source. Also, as will be understood by one of skill in the art, multiple nucleotide sequences can also be linked to obtain a nucleic acid of the invention, such as at least two nucleic acids encoding an ISV of the invention, and, for example, nucleic acids encoding one or more linkers. may be linked together in an appropriate manner. Methods for obtaining nucleic acids according to the invention will be understood by a person skilled in the art and may, for example, include, but are not limited to, automatic DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introducing mutations that result in the expression of a truncated expression product; introduction of one or more restriction sites (for example, to create cassettes and/or regions that can be easily cut and/or ligated using suitable restriction enzymes), and/or introduction of mutations via a PCR reaction using one or more "mismatched" primers ". These and other techniques will be understood by those skilled in the art, and again standard guidelines such as Sambrook et al. and Ausubel et al. mentioned above as well as examples below.

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

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

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

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

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

The genetic constructs of the invention can generally be prepared by appropriately linking the nucleotide sequence(s) of the invention to one or more of the additional elements described above, for example, using the techniques described in general guidelines such as Sambrook et al. . and Ausubel et al. mentioned above.

The nucleic acids of the invention and/or the genetic constructs of the invention can be used to transform a host cell or host organism, ie to express and/or produce a polypeptide of the invention. Suitable hosts or host cells will be understood by those skilled in the art 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 hosts. (non-human) known per se to express and produce antibodies and antibody fragments (including, but not limited to (single) domain antibodies and ScFv fragments), as will be apparent to those skilled in the art. The general prior art cited above can also serve as a source, as well as, for example, WO 94/29457; W096/34103; W099/42077; Franken et al. (Res Immunol. 149: 589-99, 1998); Riechmann and Muyldermans (1999), supra; van der Linden (J. Biotechnol. 80: 261-70, 2000); Joosten et al. (Microb. Cell Fact. 2: 1, 2003); Joosten et al. (Appl. Microbiol. Biotechnol. 66: 384-92, 2005); additional sources cited in this document. In addition, the polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be apparent to those skilled in the art. Suitable methods for transforming a host cell or host cell of the invention will be apparent to those skilled in the art and may depend on the intended host cell/host organism and the genetic construct used. The guides and patent applications mentioned above can again serve as a source. A transformed host cell (which may be in the form of a stable cell line or strain) 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 host cells or host organisms containing a nucleic acid of the invention or an expression vector of the invention. Preferably, these host cells or host organisms are such that they express or (at least) are capable of expressing (e.g., under suitable conditions) a polypeptide of the invention (and in the case of a host organism, in at least one cell, part, tissue, or organ). The invention also includes the next generations, progeny and/or descendants of the host cell or host organism of the invention, which can be obtained, for example, by cell division or by sexual or asexual reproduction.

In order to produce/express the polypeptides of the invention, the transformed host cell or transformed host organism can generally be stored, maintained and/or cultured under conditions under which the (desired) polypeptide of the invention is expressed/produced. Suitable conditions will be understood by one of skill in the art and will generally depend on the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference can be made to the manuals and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

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

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

In an aspect, the invention relates to a composition containing a construct, polypeptide, ISV, or nucleic acid of the invention.

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

Thus, in a further aspect, the invention relates to a pharmaceutical composition that comprises 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 at least one suitable carrier, diluent or excipient (ie suitable for pharmaceutical use) and optionally one or more other active substances. In a particular aspect, the invention relates to a pharmaceutical composition that contains 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 (t i.e. suitable for pharmaceutical use), and optionally one or more other active substances.

In general, the constructs, polypeptides and/or ISVs of the invention may be formulated and administered by any suitable method known per se. The source may be, for example, the general prior art mentioned above (and in particular WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079), as well as standard guidelines such as Remington's Pharmaceutical Sciences, 18th ^Ed ., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21st Edition, ^Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see, for example, pages 252-255).

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

The constructs, polypeptides and/or ISVs of the invention may be formulated and administered by any method known per se for conventional antibodies and antibody fragments (including ScFvs and diantibodies) and other pharmaceutically active proteins. Such formulations and methods for their preparation will be understood by those skilled in the art and, for example, include formulations suitable for parenteral administration (eg, intravenous, intra-abdominal, subcutaneous, intramuscular, intraluminal, intra-arterial, or intrathecal administration) or topical (eg, intra-articular, transdermal, or intradermal administration). ) introductions.

Formulations for parenteral administration may, for example, be sterile solutions, suspensions, dispersions or emulsions which are suitable for infusion or injection. Suitable carriers or diluents for such formulations, for example, include those mentioned on page 143 of WO 08/020079. Generally, aqueous solutions or suspensions will be preferred.

The constructs, polypeptides and/or ISVs of the invention can also be administered using delivery methods known from gene therapy, see, for example, US Pat. No. 5,399,346, which is incorporated by reference in connection with its delivery methods for gene therapy. Using a gene therapy delivery method, primary cells transfected with a gene encoding a construct, polypeptide and/or ISV of the invention can be further transfected with tissue-specific promoters to target specific organs, tissues, grafts, tumors, joints, or cells, and can be further transfected with signaling and stabilizing sequences for expression localized within the cell.

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

Useful dosages of the constructs, polypeptides and/or ISVs of the invention can be determined by comparing their in vitro activity with in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art, see, for example, US 4,938,949.

The number of constructs, polypeptides and/or ISVs of the invention required for use in treatment will vary not only depending on the specific ISV, polypeptide, compound and/or construct chosen, but also on the route of administration, the nature of the condition being treated and age and the patient's condition, and will ultimately be determined by the attending 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.

The desired dose may conveniently be presented as a single dose or as divided doses administered at appropriate intervals, such as two, three, four or more sub-doses per day. The sub-dose itself may be further subdivided into, for example, several separate, freely distributed administrations. Preferably, the dose is administered once a week or even less frequently, such as once every two weeks, once every three weeks, once a month, or even once every two months.

The mode of administration may include long-term treatment. By "long term" is meant at least two weeks, and preferably several weeks, months or years. Necessary modifications within this dosage range can be determined by one of ordinary skill in the art using only the routine experiments provided herein. See, for example, Remington's Pharmaceutical Sciences (Martin, EW, ed. 4th), ^Mack Publishing Co., Easton, PA. The dosage may also be adjusted by the individual physician in case of any complications.

There is a need in the art for more effective treatments for disorders affecting cartilage in the joints, such as osteoarthritis. Even with intra-articular administration, the residence time of most drugs in the treatment of affected cartilage is not enough. The present inventors have hypothesized that the effectiveness of a therapeutic drug can be significantly improved by combining the therapeutic drug with a component that "anchors" the drug in the joint and therefore increases drug retention, but this should not interfere with the effectiveness of said therapeutic drug. drug (also referred to as "cartilage anchoring protein" or "CAP"). This concept of anchoring not only improves drug efficacy, but also functional specificity for the diseased joint by reducing toxicity and side effects, thus increasing the number of possible beneficial drugs. The present inventors also hypothesized that aggrecan binding agents could potentially function as such an anchor, although aggrecan is highly glycosylated and degraded in various disorders affecting cartilage in the joints. In addition, due to the cost and extensive testing in various animal models required for a drug to enter the clinic, such aggrecan binding agents should preferably have broad cross-reactivity, e.g., aggrecan binding agents should bind to aggrecan. various types. Using various original methods of immunization, screening and characterization, the present inventors were able to identify various aggrecan binding agents with excellent properties of selectivity, stability and specificity, which allowed long-term retention and activity in the joint.

In one aspect, the present invention relates to a composition of the invention, an ISV of the invention, a polypeptide of the invention, and/or a construct of the invention for use as a medicament.

In one aspect, the present invention relates to a method for reducing and/or inhibiting efflux of a composition, polypeptide, or construct from a joint, said method comprising administering a pharmaceutically active amount of at least one polypeptide of the invention, construct of the invention, or composition of the invention to a person in need.

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

It is contemplated that the aggrecan-binding agents of the invention can be used in various diseases affecting cartilage such as arthropathies and chondrodystrophies, gouty diseases such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costochondritis , spondyloepimetaphyseal dysplasia, spinal herniation, lumbar disc degeneration, degenerative joint disease, and relapsing polychondritis (commonly referred to herein as "aggrecan-associated diseases").

In one aspect, the present invention relates to a composition, ISV, polypeptide and/or construct of the invention for use in the prevention or treatment of an aggrecan-associated disease such as, for example, arthropathy and chondrodystrophy, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or avulsion, achondroplasia, costochondritis, spondyloepimetaphyseal dysplasia, spinal disc herniation, degenerative lumbar disc disease, degenerative joint disease, and relapsing polychondritis.

In one aspect, the present invention relates to a method for the prevention or treatment of arthropathies and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costochondritis, spondyloepimetaphyseal dysplasia, spinal herniation, lumbar disc degeneration, degenerative joint diseases; 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 of the invention to a person in need thereof.

In one aspect, the present invention relates to the use of an ISV, polypeptide, composition or construct of the invention, in the preparation of a pharmaceutical composition for the treatment or prevention of arthropathy and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gout arthritis, psoriatic arthritis, traumatic laceration or detachment , achondroplasia, costochondritis, spondyloepimetaphyseal dysplasia, spinal disc herniation, degenerative lumbar disc disease, degenerative joint disease, and relapsing polychondritis.

By binding to aggrecan, the aggrecan binding agents of the invention are expected to reduce or inhibit the activity of a member of the serine protease family, cathepsins, matrix metalloproteinases (MMP)/matrixins or disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19 , MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 (aggrecanase-1) and/or ADAMTS11, when aggrecan is degraded.

Accordingly, in one aspect, the invention relates to a method of reducing or inhibiting the activity of a member of the family of serine proteases, cathepsins, matrix metalloproteinases (MMP)/matrixins or disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase -2), ADAMTS4 (aggrecanase-1) and/or ADAMTS11, upon degradation of 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.

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

The object to be treated may be any warm-blooded animal, but in particular it is a mammal and in particular a human. As will be appreciated by one of skill in the art, the subject to be treated will in particular be a human suffering from or at risk of the diseases, disorders and conditions mentioned herein.

Typically, the treatment regimen includes the introduction of one or more ISV, polypeptides, compounds and/or constructs according to the invention or one or more compositions containing them, in one or more pharmaceutically effective amounts or doses. The specific amount or doses to be administered may be determined by the physician, again based on the factors noted above.

Generally, depending on the specific disease, disorder, or condition being treated, the activity 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 the appropriate daily dose.

Typically, the above method will use an ISV, polypeptide, compound and/or construct of the invention. However, it is within the scope of the invention to use two or more ISVs, polypeptides and/or constructs of the invention in combination.

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

Again, the clinician will be able to select such additional compounds or active principles, as well as an appropriate combination treatment regimen, based on the above factors and their expert judgment.

In particular, the ISVs, polypeptides and/or constructs of the invention may be used in combination with other pharmaceutically active compounds or active principles that are or may be used in the prevention and/or treatment of the diseases, disorders and conditions described herein, in as a result, a synergistic effect may or may not be obtained. Examples of such compounds and active principles, as well as ways, methods and pharmaceutical compositions or compositions for their introduction will be clear to the doctor.

Where two or more substances or active principles are to be used as part of a combination treatment regimen, they may be administered by the same route of administration or by different routes of administration at substantially the same time or at different points in time (e.g., substantially simultaneously, sequentially or according to the alternation mode). When the substances or active principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions, or as part of combined pharmaceutical formulations or compositions, as will be appreciated by those skilled in the art.

In addition, when two or more active substances or active principles are to be used as part of a combined treatment regimen, each of the substances or active principles may be administered in the same amount and according to the same regimen as used when the compound is used by itself or active principle, and such combined use may or may not lead to a synergistic effect. However, when the combined use of two or more active substances or active principles results in a synergistic effect, it may also be possible to reduce the amount of one, several or all of the substances or active principles to be administered while still achieving the desired therapeutic effect. This may, for example, be useful in preventing, limiting or reducing any unwanted side effects that are associated with the use of one or more substances or active principles, when used in their usual amounts, while obtaining the desired pharmaceutical or therapeutic effect.

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

Typically, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or until the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

In another aspect, the invention relates to the use of an ISV, a polypeptide, a compound and/or a construct of the invention in the preparation of a pharmaceutical composition for the 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 in this document.

The invention also relates to the use of an ISV, a polypeptide, a compound and/or a construct according to 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, for example by inhibiting the degradation of aggrecan .

The invention also relates to the use of an ISV, a polypeptide, a compound and/or a construct according to 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, a polypeptide, compounds and/or constructs of the invention to a patient.

The invention further relates to an ISV, a polypeptide, a compound and/or a construct of the invention, or a pharmaceutical composition containing the same, for use in the prevention and/or treatment of at least one aggrecan-associated disease.

The object to be treated may be any warm-blooded animal, but in particular it is a mammal and in particular a human. In veterinary applications, the subject to be treated includes any animal raised commercially or kept as a pet. As will be appreciated by one of skill in the art, the subject to be treated will in particular be a human suffering from or at risk of the diseases, disorders and conditions mentioned herein.

Again, in such a pharmaceutical composition, one or more ISVs, polypeptides, compounds and/or constructs of the invention, or a nucleotide encoding them and/or a pharmaceutical composition containing them, can also be appropriately combined with one or more other active principles, such as mentioned in this document.

The invention also relates to a composition (such as, but not limited to, a pharmaceutical composition or formulation as further described herein) for use either in vitro (for example, in an in vitro assay or cellular assay) or in vivo (for example, in a separate cell or multicellular organism, and in particular in a mammal, and more specifically in a human, such as a human who is at risk of or suffering from the disease, disorder or condition of the present invention).

When referring to treatment, it should be understood that this includes both treatment of established symptoms and prophylactic treatment, unless expressly stated otherwise.

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

The invention will now be further described using the following non-limiting preferred aspects, examples and figures.

The entire contents of all sources (including references to the literature, issued patents, published patent applications, and patent applications contemplated in this application) cited throughout this application are expressly incorporated herein by reference, in particular in connection with the manuals to which There are links above.

EXAMPLES

Example 1 Immunization of llamas with aggrecan, cloning of repertoires of heavy chain-only antibody fragments and phage production

The present inventors are aware that the goal of animal models of OA is to controllably reproduce the extent and progression of joint damage, so that opportunities to detect and modulate symptoms and disease progression can be identified and novel treatments can be developed. An ideal animal model is relatively low cost and exhibits reproducible disease progression with an effect scale large enough to detect differences within a short period of time. If the model progresses too rapidly to the end stage of degeneration, intermediate time points that are representative of the pathophysiology of OA may not be available, and in the absence of this information, the subtle effects of potential interventions may be missed. Recognizing that OA is an end-stage phenotype resulting from the interaction of mechanical and biochemical processes, animal models allow these factors to be studied in a controlled environment (see also Teeple et al. 2013 AAPS J. 15: 438-446).

The ultimate goal of animal models is to replicate human disease (see also Cohen-Solal et al. 2013 Bonekey Rep. 2: 422). Given the heterogeneity of profiles in human OA, multiple models are needed. They are either spontaneous or induced. Most of them focus on one factor that contributes to the development of OA, such as aging, mechanical stress (surgery), chemical defect (enzyme), or genetic factors. All of them differ in severity, localization of lesions and pathogenesis. However, no animal model addresses all aspects of the development of OA.

Thus, to be useful in various animal models, and ultimately in the patient, the CAP binding agent preferably has broad cross-reactivity, eg, binds to more than one species of aggrecan. Preferably, the aggrecan binding agent binds to human aggrecan, as well as one or more of dog aggrecan, bovine aggrecan, rat aggrecan, porcine aggrecan, mouse aggrecan, rabbit aggrecan, cynomolgus monkey aggrecan and/or rhesus monkey aggrecan.

In addition, the authors of the present invention realized that the degradation of aggrecan, apparently, is initiated in the C-terminal region. The population of aggrecan molecules without the G3 domain also increases with age. The main feature of arthritis-related cartilage degeneration is the loss of aggrecan due to proteolytic cleavage in the interglobular region between the G1 and G2 domains. Therefore, it is preferable if the aggrecan binding agent binds to the N-terminal region of the aggrecan, i.e. to a region other than the CS or G3 domain, such as the G1-IGD-G2 region, or to the G1, IGD or G2 domain. Most preferably, the aggrecan binding agent will bind to the G1 domain that remains in chondrocytes and in the ECM.

1.1 Immunizations

Five llamas were immunized with recombinant (rec) human aggrecan (domains G1-IGD-G2, R&D Systems # 1220-PG) (see Example 1.2). Serum samples were taken after antigen challenge and titers were determined by ELISA against human recombinant aggrecan G1-IGD-G2. All llamas gave certain serum titers.

1.2 Primary screening

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

Phage display selections were performed on five immune libraries and two ISV synthetic libraries. Libraries were subjected to two to three rounds of enrichment against various combinations of recombinant human aggrecan G1-IGD-G2 domain and biotinylated rat aggrecan, extracted full-length bovine aggrecan, or intact bovine cartilage. Individual clones from the selection products were screened for binding in an ELISA (using periplasmic extracts from E. coli cells expressing ISV) 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 individual human G1, IGD and G2 domains. Only a few ISVs showed similar levels of binding to recombinant G1-IGD-G2 human, rat, dog and bovine aggrecan. Limited species cross-reactivity was particularly evident for G1 domain binding agents, for which binding to bovine and dog aggrecans was particularly poor. To identify more species of cross-reactive ISVs binding to the G1 domain, phage display was performed against the bovine G1-IGD-G2, canine G1-IGD-G2 and human G1 domains. Of 1245 clones screened in ELISA for binding to human, cynomolgus, rat, dog and bovine G1-IGD-G2, only 15 new cross-reactive ISV species were identified, nine of which could be mapped to the G1 domain.

A total of 19 unique clones were chosen as the "master panel" for further characterization. An overview of domain mapping data and species cross-reactivity for this master panel is shown in Table 1.2.

Table 1.2. Overview of periplasmic extract screening data for a master panel. Nd: not determined.

Periplasmic extract after ELISA. OD 450 nm mapping Clone Chel G1-IGD-G2 JAV G1-IGD-G2 Rat G1-IGD-G2 Dog G1-IGD-G2 Bull G1-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 the control cAbLys3 0.05 0.06 0.06 0.06 0.06 the control cAbLys3 0.05 0.05 0.06 0.06 0.05

1.3 G1 binding agents

CDR sequence variability of G1 binding agents was determined relative to clone 114F08. The amino acid sequences of the CDRs of clone 114F08 were used as the standard against which the CDRs of all other clones (G1 binding agents) were compared and are presented in Tables 1.3A, 1.3B and 1.3C below (CDR1 starts at Kabat Position 26, CDR2 starts at Kabat position 50 and CDR3 starts at Kabat position 95).

Table 1.3A

G1 CDR1* based numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence G S T F I I N V V R 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 1.3B

G1 CDR2* based numbering one 2 2a 3 4 5 6 7 eight nine wild-type sequence T I - S S G G N A N 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 1.3C

G1 CDR3* based numbering one 2 3 4 5 6 6a 7 eight nine ten 10a eleven 12 thirteen wild-type sequence P T T H Y G - G V Y Y - G P Y 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 binding agents

CDR sequence variability of G1-IGD-G2 (GIG) binding agents was determined relative to clone 604F02. The amino acid sequences of the CDRs of clone 604F02 were used as a standard against which the CDRs of all other clones (binding GIGs) were compared and are presented in Tables 1.4A, 1.4B and 1.4C below (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50 and CDR3 starts at Kabat position 95).

Table 1.4A

gig CDR1* main numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence G R T F S S Y T M G mutation L T A

*up to 2 CDR1 mutations in one clone.

Table 1.4B

gig CDR2* main numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence A I S W S G G R T Y mutations S T R

*up to 2 CDR2 mutations in one clone.

Table 1.4C

gig CDR3* main numbering one 2 3 4 5 6 7 eight nine ten eleven 12 thirteen fourteen fifteen wild-type sequence Y R R R R A S S N R G L W D Y mutations V Y T - P T E T P L V

*up to 5 CDR3 mutations in one clone.

1.5 G2 binding agents

CDR sequence variability of G2 binding agents was determined relative to clone 601D02. The amino acid sequences of the CDRs of clone 601D02 were used as a standard against which the CDRs of all other clones (G2 binding agents) were compared and are shown in Tables 1.5A, 1.5B and 1.5C below (CDR1 starts at position 26 in Kabat, CDR2 starts at Kabat position 50 and CDR3 starts at Kabat position 95).

Table 1.5A

G2 CDR1* main numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence G P T F S R Y A M G mutation R S I N N R F Y mutation R M - - S

*up to 5 CDR3 mutations in one clone.

Table 1.5B

G2 CDR2* main numbering one 2 3 4 5 6 7 eight nine ten eleven wild-type sequence A I T W S S G G R T Y mutations S L N - - A S N Y D R T

*up to 5 CDR2 mutations in one clone.

Table 1.5C

G2 CDR3* main numbering one 2 3 4 5 6 7 eight nine ten eleven 12 thirteen fourteen fifteen sixteen wild-type sequence A R I P V R T Y T S E W N Y - - 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 ISV sequence optimization

Various ISVs were subjected to a sequence optimization process. Sequence optimization is a process in which the parent ISV sequence is mutated. This process covers humanization (i) of ISVs and knockout post-translational modifications (ii) as well as epitopes for potential pre-existing antibodies (iii).

(i) For humanization purposes, the parental ISV sequence is mutated to produce an ISV sequence that 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 characteristic residues) that differ between ISV and the human IGHV3-IGHJ germline consensus are altered for the human counterpart such that protein structure, activity and stability are kept intact. Several characteristic residues are known to be critical to ISV stability, activity and affinity and therefore have not been mutated.

(ii) Those amino acids that are present in the CDR and for which there is experimental evidence that they are sensitive to post-translational modifications (PTM) are changed in such a way that the PTM site is inactivated, while the structure, activity and stability of the protein remain unchanged.

(iii) The ISV sequence is optimized without affecting the structure, activity and stability of the protein to minimize the binding of any naturally occurring pre-existing antibodies and to reduce the possibility of causing an immunogenic response that occurs during treatment - a sudden immunogenic response.

To generate sequence-optimized formatted ISVs, engineered ISVs were produced in Pichia pastoris as unlabeled proteins and purified by protein A affinity chromatography followed by desalting, all according to standard protocols.

Various sequence-optimized formatted ISVs are shown in Tables A-1 and A-2.

Example 2 Characterization of Leading Panel (Purified ISVs) - Aggrecan

After primary screening, initial evaluation of binding by ELISA, determination of dissociation rate and species cross-reactivity, the ISVs of the master panel were subjected to further characterization.

2.1 Formatting the leading panel against aggrecan with ALB26 (n = 19)

It is expected that the final format of the molecule for clinical use will include one or two aggrecan-binding ISVs ("anchors"), as well as one, two or more ISVs or other fragments with a therapeutic mode of action. Therefore, 19 selected clones were fused in a monovalent or divalent format with ALB26 (CAP-ALB26 or ALB26-CAP-CAP) and expressed in P. pastoris. ALB26 is a variant of ALB11 (an albumin-binding ISV) with two mutations in CDR1 that completely abolish albumin binding from different species. Fusion with ALB26 was performed to mimic the size of the final polypeptide format containing the aggrecan binding agent. Without wishing to be bound by any theory, the authors hypothesized that pI may affect penetration and retention in cartilage. Divalent ALB26 (C01010030) was used as a negative control or blank.

2.2 Retention in ex vivo bovine cartilage

Because there is no established pattern assay to assess cartilage retention, the inventors developed a reliable and reproducible ex vivo cartilage retention assay using bovine cartilage.

Cow bones were usually collected from the local slaughterhouse. Cartilage was cut from the bones in strips ~1 mm thick and then cut into round discs 3 mm in diameter using biopsy cutters. Cartilage discs were predominantly taken from fresh cartilage.

The ability of ISV to be retained in the cartilage for a long period of time after a relatively short exposure of the nanobody to the cartilage (which would be expected with intra-articular injection) was determined. The assay consisted of ex vivo incubation of cartilage, typically 3 mm bovine discs (~10 mg ww) with 10 μg/mL nanobody (100 μL), followed by washing for up to 5 days (PBS/0.1% BSA/ 0.1%). NaN ₃ /100 mM NaCl). The bound (retained) nanobody was then released from cartilage into SDS-containing SDS-PAGE sample buffer (LDS Invitrogen sample buffer) and analyzed by Western blotting (WB). The analysis was typically performed with 4 cartilage discs per nanobody sample; 2 disks were analyzed immediately after nanobody incubation (t0) to determine the initial amount of bound nanobody; 2 disks were analyzed after washing (t1-5 days). The retention rate was defined as the ratio of the amount of nanobody detected on days t1-5 and t0. To increase the throughput of the assay, this ratio was determined by visual inspection of a Western blot with a score of 0-6, where 0 is no retention and 6 is complete retention.

A summary of the results is shown in Table 2.2.

Target epitope group C01010# Design pi Retention in cartilage* G1 4 118 ALB26-114F08-114F08 9.09 6.00 G1 one 131 ALB26-601E08-601E08 9.00 6.00 G1-IGD-G2 eight 106 ALB26-604F02-604F02 9.61 6.00 G1-IGD-G2 eight 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 one 97 608A05-ALB26 9.09 5.00 G1 one 109 ALB26-608A05-608A05 8.95 5.00 G2 7 115 ALB26-117D05-117D05 8.73 5.00 G1-IGD-G2 eight 47 601E09-ALB26 9.13 4.83 G2 6 108 ALB26-604G09-604G09 9.13 4.00 G1-IGD-G2 eight 95 604G01-ALB26 6.96 4.00 G1-IGD-G2 eight 116 ALB26-113A01-113A01 8.73 4.00 G1-IGD-G2 eight 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 ALB26-601D02-601D02 9.24 3.00 G1 one 46 601E08-ALB26 8.96 2.60 G1 5 60 606A07-ALB26 9.09 2.25 G1 5 113 ALB26-606A07-606A07 8.62 2.00 G1 4 119 ALB26-115B08-115B08 9.49 2.00 G2 6 117 ALB26-112A03-112A03 9.12 2.00 G2 6 62 112A03-ALB26 9.21 1.66 G1 4 104 115B08-ALB26 8.66 1.50 G1 one 40 102G11-ALB26 9.20 1.33 G1 2 53 112A01-ALB26 9.17 1.00 G1 2 111 ALB26-112A01-112A01 8.64 1.00 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 ALB26-609C09-609C09 8.13 0.00 G1 3 112 ALB26-604B05-604B05 9.06 0.00 G1 3 114 ALB26-606A05-606A05 9.03 0.00 False thirty ALB26-ALB26 8.75 0.00

Table 2.2. Epitope binding and retention in cartilage of the host panel against aggrecan in a format using ALB26. *Table shows mean scores from (n) independent ex vivo bovine cartilage retention assays on a scale of 0 to 6 with 0 being no retention and 6 being complete retention.

It was found that 9 structures were held very well (5-6 points) in the cartilage. These "top 9" included both univalent and divalent aggrecan binding moiety constructs that bind to all G1, G2 or G1-IGD-G2 recombinant domains. In this analysis, 14 constructs showed moderate retention (scores between <5 and 2) and 5 constructs showed low, albeit detectable retention (scores between <2 and 1). Notably, all but one of the anti-aggrecan constructs had pI values ranging from 8 to 9.

2.3 Sorting of antibodies depending on the binding epitope

To sort antibodies depending on the epitope to be bound, the purified ALB26-fused nanobody constructs were screened against the same set of FLAG-tagged nanobodies in a competitive ELISA.

Briefly, the analysis was as follows. Monoclonal phage ELISAs were incubated at half-saturated phage dilution with or without 1 μM purified nanobody (or 5 μg/ml mAb). The ratio between absorbance at 450 nm in the presence and absence of the purified nanobody (or mAb) was used to determine whether the nanobody recognizes overlapping or non-overlapping epitopes.

The resulting epitope groups are shown in Table 2.2 (above). The constructs in epitope groups 2 and 3 (at the G1 domain) had low cartilage retention scores (0-1) in an ex vivo bovine cartilage retention assay. However, there does not seem to be a direct correlation between binding to bovine aggrecan G1-IGD-G2 as measured by ELISA and retention in bovine cartilage. Without being limited by any theory, the authors suggested that these epitopes may be difficult to access in native cartilage tissue.

The sequence variability of the CDR clones belonging to the group is shown below and above (ie group 8 with 604F02 as reference compound; tables 1.4A-C).

Sequence variability of G1 binding agents from epitope group 4 against 114F08 is shown in Tables 2.3A, 2.3B and 2.3C below. The amino acid sequences of the CDRs of clone 114F08 were used as a reference against which the CDRs of all other clones (binding group 4 agents) were compared (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95 ).

Table 2.3A (114F08)

G1-group 4 CDR1* main numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence G S T F I I N V V R mutations I S S R Y M K F M Y A

*up to 2 CDR1 mutations in one clone.

Table 2.3B (114F08)

G1-group 4 CDR2* main numbering one 2 2a 3 4 5 6 7 eight nine wild-type sequence T I - S S G G N A N mutations A N R T D G

*up to 2 CDR2 mutations in one clone.

Table 2.3C (114F08)

G1-group 4 CDR3* main numbering one 2 3 4 5 6 7 eight nine ten eleven 12 thirteen wild-type sequence P T T H Y G G V Y Y G P Y 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.

Sequence variability of G1 binding agents from epitope group 1 against 608A05 is shown in Tables 2.3D, 2.3E and 2.3F below. The amino acid sequences of the CDRs of clone 608A05 were used as a reference against which the CDRs of all other clones (epitope group 1 binders) were compared (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95 ).

Table 2.3D (608A05)

G1-group 1 CDR1* main numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence G R T F S T Y T M G mutation S S A V

*up to 2 CDR1 mutations in one clone.

Table 2.3E (608A05)

G1-group 1 CDR2* main numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence A I S W S G G T T Y mutations I R R S

*up to 2 CDR2 mutations in one clone.

Table 2.3F (608A05)

G1-group 1 CDR3* main numbering one 2 3 4 5 6 7 eight nine ten eleven 12 thirteen fourteen fifteen wild-type sequence R P R Y Y Y Y S L Y S Y D Y - 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

Based on ex vivo retention data in bovine cartilage and data on sorting of antibodies depending on the binding epitope, some exemplary constructs from different epitope groups were selected for further characterization. G2 domain binding agents were excluded from further characterization at this stage for the reasons outlined previously.

Selected constructs were characterized by ELISA on the recombinant G1-IGD-G2 region of human, cynomolgus, rat, dog and bovine aggrecan to determine species cross-reactivity, and on recombinant human neurocan and brevican to determine selectivity. The determined EC ₅₀ values are given in Table 2.4A.

SPR experiments (ProteOn) were carried out on "monovalent" aggrecan-ALB26 formats to determine dissociation rates. The interaction of nanobodies with the aggrecan surface turned out to be inhomogeneous. Heterogeneity may be due to rebinding events, a heterogeneous population of immobilized aggrecan, and/or heterogeneous glycosylation patterns. As a consequence, the calculated deviations are indicative only. Overall, it appears that the dissociation kinetics were fast for nanobodies including aggrecan (Table 2.4B).

Table 2.4A. Characterization of the master panel against aggrecan in ALB26 format by ELISA

G1-IGC -G2 (kd 1/s) Target C01010
# Design Man Javanese macaque Rat Dog Cow 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 GMGD-G2 94 604F02-ALB26 1.7E-01 1.5E-01 2.6E-01 1.2E-01 2.6E-01

Table 2.4B: Characterization of the "univalent" master panel against aggrecan in ALB26 format (n = 5) by SPR (rate of dissociation). Dissociation rates are indicative only due to heterogeneous binding patterns.

Example 3 Biophysical Characterization of Monovalent Lead Constructs - Aggrecan

Because all selected designs exhibited various favorable characteristics, whether they were in combination or not, ISV 114F08 and 604F02 and their respective ALB26 formats (C010100054, -118 and 094) were used as exemplary designs representing the master panel for further characterization.

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

For biophysical characterization, 114F08 and 604F02 monovalent nanobodies were expressed with FLAG3-His6 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 pI, Tm and analytical SEC for 114F08 and 604F02

For thermal shift analysis (TSA), 5 µl of purified monovalent nanobody (800 µg/ml) was incubated with 5 µl of Sypro Orange fluorescent probe (Invitrogen, S6551) (final concentration 10x) in 10 µl of buffer (100 mM phosphate, 100 mM borate, 100 mM citrates, 115 mM NaCl, buffered at various pH values ranging from 3.5 to 9). The samples were heated in a LightCycler 480II (Roche) at a rate of 37-99°C at a rate of 4.4°C/s, after which they were cooled to 37°C at a rate of 0.03°C/s. Thermal unfolding exposes hydrophobic regions of proteins to which Sypro Orange binds, which leads to 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 point (Tm), essentially according to Ericsson et al. 2006 (Anals of Biochemistry, 357: 289-298).

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

The isoelectric points of the two ISVs against aggrecan are relatively basic. The sequences are shown in Table A-1). The melting point was determined to be 61.0°C for 114F08 and 70.0°C for 604F02. None of the clones showed signs of aggregation or multimerization as determined by analytical SEC.

Accordingly, along with positive functional properties, ISVs exhibit favorable biophysical properties.

3.3 Members of the 114F08 family

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

Table 3.3A

114F08 CDR1* Numbering by Kabat 26 27 28 29 thirty 31 32 33 34 35 main numbering one 2 3 4 5 6 7 eight nine ten wild-type sequence G S T F I I N V V R mutations S M

*up to 2 CDR1 mutations in one clone.

Table 3.3B

114F08 CDR2* Numbering by Kabat fifty 51 52 53 54 55 56 57 58 main numbering one 2 3 4 5 6 7 eight nine wild-type sequence T I S S G G N A N mutations A R T T D

*up to 5 CDR2 mutations in one clone.

Table 3.3C

Example 4 Ex vivo Binding to Various Types of Cartilage

Exemplary CAP-containing polypeptides (also referred to herein as "CAP-containing constructs" or "constructs") have been shown to bind recombinant/extracted human proteins and bovine cartilage in an ex vivo bovine cartilage retention assay. To demonstrate that these exemplary CAP-containing constructs also bind to other cartilage species, the experiments described above with bovine cartilage were essentially repeated with human cartilage and rat cartilage.

4.1 Ex vivo binding to human cartilage

To confirm that exemplary constructs containing CAP also bind to human cartilage, selected constructs were tested in an ex vivo cartilage binding assay using frozen human cartilage chips. Binding was determined after a 30 minute wash using Western blot.

The results are summarized in Table 4.1.

Target C010100
# Design _t0 G1 054 114F08-ALB26

G1 118 ALB26-114F08-114F08

G1-IGD-G2 094 604F02-ALB26

False 030 ALB26-ALB26

Table 4.1. Binding to human cartilage ex vivo. The amount of construct bound to cartilage after a 30 minute wash (T0) was analyzed by Western blotting.

All constructs were found to bind better to human cartilage than the false construct.

4.2 Binding to ex vivo rat cartilage

To facilitate testing of the constructs in an in vivo rat model, binding to rat cartilage was evaluated. Therefore, an analysis was performed using a femur from a rat with intact cartilage. Representative constructs C010100054, -118 and -094 were incubated with rat cartilage overnight followed by a 30 minute wash, releasing bound constructs followed by Western blot analysis.

The results are shown in Table 4.2.

All constructs tested were found to bind well to rat cartilage.

Target C010100
# Design Intact surface G1 054 114F08-ALB26

G1 118 ALB26-114F08-114F08

G1-IGD-G2 094 60F02-ALB26

False 030 ALB26-ALB26

Table 4.2: Binding of rat cartilage. The constructs were incubated with femoral heads. After incubation of the nanobody construct, the unbound construct was washed away and the bound construct was analyzed by Western blotting.

Example 5 Tissue Specificity

It has been demonstrated above that the constructs of the invention bind specifically to aggrecan both in vitro and ex vivo. In addition, these constructs should also preferentially bind to the cartilage of the joint, while should not bind or bind less to other tissues of the joint.

Binding of exemplary CAP-containing constructs to the synovial membrane, tendon, epimysium and meniscus was assessed using the same scheme as for the ex vivo cartilage binding assay. Construct release and Western blotting analysis was performed after a short tissue wash (30 min) after incubation with the constructs.

The results are summarized in Table 5.

The results show that CAP binders show preferential binding to cartilage tissues, including the meniscus, compared to other tissues found in the joint.

Target C010100
# Design Cartilage synovial membrane Tendon Epimysium Meniscus G1 054 114F08-ALB26 +++++ +/- + +/- nd G1 118 ALB26-114F08-114F08 +++++ + + + nd G1-IGD-G2 094 604F02-ALB26 +++++ + + +/- nd G1 046 601E08-ALB26 ++++ +/- + nd +++ False 030 ALB26-ALB26 +/- +/- +/- - -

Table 5. Tissue specificity. Binding of the leading panel in ALB26 format (n = 10) to articular cartilage, synovial membrane, tendon, epimysium and meniscus.

Example 6 Stability of nanobodies in bovine synovial fluid

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

Accordingly, the stability of exemplary ALB26-fused CAP constructs in synovial fluid (SF) was assessed by incubating the constructs in non-arthritic bovine SF for up to 7 days at 37°C.

The results are summarized in Table 6.

Target C010100# Design Stability in bovine SF, 37°C G1 054 114F08-ALB26 > 7 days G1 118 ALB26-114F08-114F08 > 7 days G1-IGD-G2 094 604F02-ALB26 > 7 days False +030 ALB26-ALB26 > 7 days

Table 6 Stability of the master panel format using ALB26 in bovine synovial fluid (SF).

No degradation of any of the structures was found.

Example 7 Retention in IL-1α Stimulated Cartilage Explant

Up to this point, all experiments regarding cartilage binding and retention of CAP containing nanobodies have been performed in healthy (non-arthritic) cartilage ex vivo. Arthritis cartilage is characterized by degraded collagen and aggrecan. Therefore, it is also important to evaluate the binding and retention of aggrecan-inducing agents in cartilage where degradation of these proteins has occurred. To this end, exemplary CAP constructs fused to ALB26 were tested in a cartilage explantation assay in which degradation induction was stimulated in cartilage.

Briefly, typical, CAP-containing constructs were incubated overnight (ON) with bovine cartilage explants cultured with or without IL-1α or oncostatin M, followed by a 5 day culture with daily media change (wash). IL-1α and oncostatin M mainly cause the degradation of aggrecan during the 6 days of the experiment. The cartilage explants were analyzed for binding and retention of the construct with WB. Two independent experiments were carried out (Exp A and Exp B).

Western blot results are presented in Table 7.1.

Target G1 G1 G1-IGD-G2 G2 False Design #54 #118 #94 #45 #thirty Stimulation - + - + - + - + - + - + - + - + - + - + Flushing (days) 0 5 0 5 0 5 0 5 0 5 Arr A

arr B

Table 7.1. Preservation of the master panel in ALB26 format in stimulated bovine cartilage explants. Two independent experiments were carried out: A and B.

The retention results of the CAP-containing construct in stimulated cartilage explants are summarized in Table 7.2.

Target C010100
# Design Binding stimulated versus unstimulated Retention Day 5 G1 054 114F08-ALB26 Reduced Partial G1 118 ALB26-114F08-114F08 Equal Complete G1-IGD-G2 094 604F02-ALB26 Reduced Partial G1 045 601D02-ALB26 Reduced Partial False 030 ALB26-ALB26 No binding No binding

Table 7.2. Summary of CAP binding and retention in stimulated bovine cartilage explant assay

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

In addition, it appears that binding to the G2 domain of aggrecan (example C01010045) is reduced to a greater extent than binding to other domains, consistent with the hypothesis that aggrecan degradation occurs from the C-terminus.

Example 8 ADAMTS5-CAP GAG Release Assay

To account for the possible effect of CAP, a cartilage anchoring moiety, on cartilage protease inhibitory nanobody activity, exemplary CAP constructs were combined with ADAMTS5 (ATS5)-blocking ISV and tested in a GAG (glycosaminoglycan) release assay in cartilage explant.

Prior to testing the constructs in a cartilage explant GAG release assay, cartilage binding and ADAMTS5 inhibitory properties were confirmed in vitro. For the latter, an enzymatic peptide analysis was carried out, which showed that the blocking function of the ISAM ADAMTS5 enzyme was not impaired in any of the in vitro CAP hybrid constructs.

In the GAG release assay, bovine cartilage explants were cultured for 5 days in the presence of IL-1α and oncostatin M (to induce ADAMTS5) and a range of construct doses, followed by quantification of released GAG in the culture supernatant.

The tested constructs and the results of the GAG release assay are shown in Table 8.

IC50(nM) Target ID Design Peptide analysis GAG release ADAMTS-5-G1 C010100270 ATS5-114F08 0.11 4.17 ADAMTS-5-G1 C010100276 ATS5-114F08-114F08 0.06 19.15 ADAMTS-5-G1-IGD-G2 C010100271 ATS5-604F02 0.19 2.15 ADAMTS-5-G1 C011400510 ATS5 (no label) 0.12 0.87

Table 8 Summary of ADAMTS5-CAP GAG Release Assay

The results show that the addition of an anchor arm (CAP-ISV construct) to the ADAMTS5 inhibitor still effectively inhibited GAG release.

Example 9 Biological imaging of CAP constructs in vivo

In parallel with the in vitro and ex vivo characterization of exemplary anti-aggrecan CAP constructs, in vivo biodistribution was determined for several ALB26 hybrid constructs to confirm retention properties.

9.1 Biodistribution studies of ALB26-CAP constructs

The nanobodies were labeled with ¹²⁵ I (via ¹²⁵ I-SIB lysine attachment). The constructs were injected into the knee joints of healthy rats. Autoradiographic images of the joints were obtained at various time points up to 4 weeks after injection. These images made it possible to evaluate the retention and tissue (cartilaginous) specificity of the constructs in vivo.

Representative images are shown in Figure 1.

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

9.2 MARG designs ALB26-CAP

The biodistribution study described above (Example 9.1) demonstrated specific cartilage retention of the ALB26-CAP constructs. However, the resolution of the images did not allow us to investigate the depth of penetration into the cartilage. MARG (microautoradiography) was used to increase the image resolution and thus assess cartilage penetration.

Exemplary constructs that were included in the study are listed in Table 9.2A. For this study, nanobodies were labeled with ³ H (by attaching lysine to ³ H-NSP (N-succinimidyl propionate)) and injected into healthy and osteoarthritic (surgically induced by cutting the anterior cruciate ligament joints) rat joints; 8 rats per group. 7-14 days after injection, rats were sacrificed and injected healthy and OA-induced joints were subjected to MARG.

Typical MARG images are shown in figure 2.

Target C010100# Design Agrekan #54 114F08-ALB26 Agrekan #626 ALB26-114F08-114F08 SO Agrekan #94 604F02-ALB26 False #thirty ALB26-ALB26

Table 9.2A. Tested Exemplary Nanobody Designs

All aggrecan-binding agents generally showed penetration into healthy cartilage. The 626 design sometimes also showed more intense staining on the surface. Varying degrees of staining and cartilage penetration were found in the operated knee: no staining was observed with the 054 monovalent construct; there was no or little staining with the monovalent construct 094, while the bivalent construct 626 resulted in slightly more permanent staining, albeit with varying penetration depths (see Table 9.2B).

Design* healthy knee joint operated knee joint Silver Grain Grade Penetration depth Silver Grain Grade Penetration depth +030 0% stained samples missing 0% stained samples missing 054 100% samples with minimal staining With 0% stained samples missing 094 83% of samples with predominantly soft staining With 60% samples mostly with soft staining With 626 100% samples with mostly soft staining B-C 100% samples with minimal or mild staining A-B-C

Table 9.2B. Summary of MARG staining results. *Global results for 8 animals based on silver grain evaluation are shown. Distribution score: A = cartilage surface without intrinsically deeper staining, B = cartilage surface with deeper staining, C = staining in deeper cartilage layers without accumulation on the surface.

Example 10 In Vivo MMT DMOAD in Rats Demonstrated a Statistically Significant Effect

To further demonstrate the in vivo efficacy of CAP binders of the invention, a surgically induced medial meniscal tear (MMT) rat model was used. Briefly, the CAP binding agents of the invention were coupled to an anti-MMP13 ISV (designated "0754" or "C010100754") or an anti-ADAMTS5 ISV (designated "0954" or "C010100954"). Rats were operated on in one knee to induce OA-like symptoms. Treatment began 3 days after surgery using intra-component injection. Histopathology was performed 42 days after surgery. Intermediate and final serum samples were collected for exploratory biomarker analysis. The medial and overall significant width of cartilage degeneration was determined, as well as the percentage reduction in cartilage degeneration. The group used 20 animals.

Inhibition of cartilage degradation by nanobodies in the middle tibia is shown in figure 3.

The results show that cartilage width was significantly reduced by the ADAMTS5-CAP construct and by the MMP13-CAP construct at 42 days compared to vehicle. These results suggest that CAP fragment (a) does not adversely affect the activity of anti-MMP13 ISV (0754) or anti-ADAMTS5 ISV (0954); and (b) allows these constructs to be retained in the joints for an extended period of time.

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

In a cartilage retention study in healthy rats, it was demonstrated that the polypeptides of the invention can be detected in measurable amounts in cartilage up to 112 days after intra-articular (I.A.) injection (data not shown). Because the cartilage composition can affect cartilage binding and systemic absorption, the pharmacokinetics of the polypeptides of the invention were compared in vivo in osteoarthritis patients and healthy rats by monitoring serum polypeptide levels as a function of time.

In particular, a surgically induced medial meniscus tear (MMT) model in rats was used as described in Example 10, but with some modifications. Briefly, the polypeptides of the invention were linked to anti-MMP13 ISV and anti-ADAMTS5 ISV, resulting in the MMP13-ADAMTS5-CAP-CAP construct (designated "0949" or "C010100949" nanobodies). Rats were operated on in one knee to induce OA-like symptoms (OA group). Each treatment group (healthy and OA) consisted of 15 animals and received one injection of I.A. 400 µg/30 µl Nanobody on day 7 (healthy) or 7 days after surgery (MMT). Serum samples were taken from anesthetized rats on day 0, day 7 (at 0 hours = pre-dose sample), day 8 (various times post-treatment up to 24 hours), day 9 (48 hours post-treatment), d10 ( 3 days) post-treatment), d14 (7 days post-treatment), d21 (14 days post-treatment), and d42 (35 days post-treatment). The collected serum samples were used to determine the concentrations of the polypeptide in the format of a general PK analysis based on electrochemiluminescence (ECL), followed by non-compartmental analysis.

The retention of polypeptides in the blood serum of healthy and OA rats is shown in Figure 4.

The results demonstrate that there are no obvious differences in serum concentrations of polypeptides between healthy rats and rats with OA. These results suggest that cartilage degradation does not affect the pharmacokinetics of the polypeptides of the invention.

Table A-1: Amino acid sequences of monovalent aggrecan-inducing agents ("ID" refers to SEQ ID NO, as used herein)

Name ID Amino acid sequence 102G11 one EVQLVESGGGLVQAGGSLRLSCAASGRSFSSYAMGWFRQAPGKEREFVSIISWSGGSTVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAGRLYRATPRPADFGSWGQGTQVTVSS 112A01 2 EVQLVESGGGLVQTGGSLRLSCVASGRAFSNYIMGWFRQAPGKERDFVAAINWNGVTTHYTDSVKGRFTISRDNAKSTSYLQMDSLKPDDTAVYFCAARGTVYSRTYGVSEEGYMYWGQGTQVTVSS 112A03 3 EVQLVESGGGLVQPGGSLRLSCAASGSIFSNRFMYWYRQAPGKQRELVASITLSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTFLQNSFYWGQGTQVTVSS 113A01 4 EVQLVESGGGLVQPGGSLRLSCSASGFTFSGSWMFWVRQAPGKDYEWVASINSSGGRTYYDDSVKGRFTISRDSAKNTLYLEMNNLKPEDTALYFCARSPRVGSWGQGTQVTVSS 114F08 5 EVQLVESGGGLVQAGGSLRLSCAASGSTFIINVVRWYRRTPGKQRELVATISSGGNANYVDSVRGRFSISRDGAKNAVDLQMNGLKPEDTAVYYCNVPTTHYGGVYYGPYWGQGTQVTVSS 115B08 6 KVQLVESGGGLVQPGGSLRLSCAASGFTFSMYAMKWVRQAPGKGLEWVSGINSSGGRTNYAGSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATDFLGGRNSRGQGTQVTVSS 117D05 7 KVQLVESGGGLVQAGGSLRLSCAASRRTFNMMGWFRQAPGKEREFVAYITWNGGDTRYAESVKGRFTVSRDDVKNTMALQMNRLDPLDTAVYYCGVRIHGSNWSTKADDYDNWGQGTQVTVSS 117G09 eight EVQLVESGGGSALPGGSLRLSCAASGITFSSRYMRWYRQAPGRQRELVAAISSGGRTDYVDSVRGRFTLSINNAKNTVYLQMNDLKPEDTAVYYCYRPRMYVDGTYEKELWGQGTLVTVSS 601D02 nine DVQLVESGGGLVQPGGSLRLSCAASGPTFSRYAMGWFRQAPGKEREFVAAITWSSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAARIPVRTYTSEWNYWGQGTLVTVSS 601E08 ten DVQLVESGGGLVQPGGSLRLSCTASGRTFSSYAVGWFRQAPGKEREFVAAISRSGRSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLSYYSPHAYYDYWGQGTLVTVSS 601E09 eleven DVQLVESGGGLVQPGGSLRLSCAASGLTFSTYAMGWFRQAPGKEREFVAAISWSGSRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAYRRPRYSPTGTWDYWGQGTLVTVSS 604B05 12 DVQLVESGGGLVQPGGSLRLSCVASGRTFSIYTMAWFRQAPGKEREFVAAISWSSGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTAYTGPRSGYDYWGQGTLVTVSS 604F02 thirteen DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYTMGWFRQAPGKEREFVAAISWSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAYRRRASSNRGLWDYWGQGTLVTVSS 604G01 fourteen DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYTMGWFRQAPGKEREFVAAISWSGRTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAYRRVRYTNLEVWDYWGQGTLVTVSS 604G09 fifteen DVQLVESGGGLVQPGGSLRLSCVASGRTFSSYAMGWFRQAPGKEREFVAAITWSSATTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAARIPVGRRSENWDYWGQGTLVTVSS 606A05 sixteen DVQLVESGGGLVQPGGSLRLSCVASGRTFSIYTMGWFRQAPGKEREFVAAISWSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCTAYTGRSYGSYDYWGQGTLVTVSS 606A07 17 DVQLVESGGGLVQPGGSLRLSCVASGRTFSIYGMGWFRQAPGKEREFVAAINGGSRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRSGYGTSLDWWYDYWGQGTLVTVSS 608A05 eighteen DVQLVESGGGLVQPGGSLRLSCAASGRTFSTYTMGWFRQAPGKEREFVAAISWSGGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAARPRYYYYSLYSYDYWGQGTLVTVSS 609C09 nineteen DVQLVESGGGLVQPGGSLRLSCAASGTIFSINVMGWYRQAPGKEREFVAAITTGGRTNYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNAVTTGWVGYSWYDYWGQGTMVTVSS 114A09 114 evqlvesggglvqaggslrlscaasGSTFIISVMRwyrqapgkqrelvaAIRTGGNTDyagpvrgrfsisrdgaknavdlqmnglkpedtavyycnvPTTRYGGDYYGPYwgqgtqvtvss 114B04 115 evqlvesggglvqaggslrlscaasGSTFIISVMRwyrqapgkqrelvaAIRTGGNTDyagpvrgrfsisrdgakdavdlqmnglkpedtavyycnvPTTRYGGDYYGPYwgqgtqvtvss 00269 SO114F08 116 EVQLVESGGGLVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNVPTTHYGGVYYGPYWGQGTLVTVSS 00745 PEA114F08 117 EVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 00747 PEA604F02 118 EVQLVESGGGVVQPGGSLRLSCAASGRTFSSYTMGWFRQAPGKEREFVAAISWSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAYRRRRASSNRGLWDYWGQGTLVTVSSA

Table B: Sequences of aggrecan and others from various species ("ID" refers to SEQ ID NO as used herein)

Name ID Amino acid sequence Human aggrecan 125 MTTLLWVFVTLRVITAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEVQSLRSNDSGVYRCEVMHGIEDSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGHVYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYVHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAPEIGATAFAEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGPTRYSLTFEEAQQACPGTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSTETYDVYCFVDRLEGEVFFATRLEQFTFQEALEFCESHNATATTGQLYAAWSRGLDKCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGISAVPSPGEEEGGTPTSPSGVEEWIVTQVVPGVAAVPVEEETTAVPSGETTAILEFTTEPENQTEWEPAYTPVGTSPLPGILPTWPPTGAETEESTEGPSATEVPSASEEPSPSEVPFPSEEPSPSEEPFPSVRPFPSVELFPSEEPFPSKEPSPSEEPSASEEPYTPSPPEPSWTELPSSGEESGAPDVSGDFTGSGDVSGHLDFSGQLSGDRASGLPSGDLDSSGLTSTVGSGLTVESGLPSGDEERIEWPSTPTVGELPSGAEILEGSASGVGDLSGLPSGEVLETSASGVGDLSGLPSGEVLETTAPGVEDISGLPSGEVL ETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETTAPGVEEISGLPSGEVLETTAPGVDEISGLPSGEVLETTAPGVEEISGLPSGEVLETSTSAVGDLSGLPSGGEVLEISVSGVEDISGLPSGEVVETSASGIEDVSELPSGEGLETSASGVEDLSRLPSGEEVLEISASGFGDLSGVPSGGEGLETSASEVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDLVGSASGDLDLGKLPSGTLGSGQAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTIGISGAGEISGLPSSELDISGRASGLPSGTELSGQASGSPDVSGEIPGLFGVSGQPSGFPDTSGETSGVTELSGLSSGQPGVSGEASGVLYGTSQPFGITDLSGETSGVPDLSGQPSGLPGFSGATSGVPDLVSGTTSGSGESSGITFVDTSLVEVAPTTFKEEEGLGSVELSGLPSGEADLSGKSGMVDVSGQFSGTVDSSGFTSQTPEFSGLPSGIAEVSGESSRAEIGSSLPSGAYYGSGTPSSFPTVSLVDRTLVESVTQAPTAQEAGEGPSGILELSGAHSGAPDMSGEHSGFLDLSGLQSGLIEPSGEPPGTPYFSGDFASTTNVSGESSVAMGTSGEASGLPEVTLITSEFVEGVTEPTISQELGQRPPVTHTPQLFESSGKVSTAGDISGATPVLPGSGVEVSSVPESSSETSAYPEAGFGASA APEASREDSGSPDLSETTSAFHEANLERSSGLGVSGSTLTFQEGEASAAPEVSGESTTTSDVGTEAPGLPSATPTASGDRTEISGDLSGHTSQLGVVISTSIPESEWTQQTQRPAETHLEIESSSLLYSGEETHTVETATSPTDASIPASPEWKRESESTAAAPARSCAEEPCGAGTCKETEGHVICLCPPGYTGEHCNIDQEVCEEGWNKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGEPPVVEHARTFGQKKDRYEINSLVRYQCTEGFVQRHMPTIRCQPSGHWEEPRITCTDATTYKRRLQKRSSRHPRRSRPSTAH Aggrekan dogs 126 MTTLLWVFVTLRVITAASSEETSDHDNSLSVSIPEPSPMRVLLGSSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRITKEKEVVLLVATEGQVRINSAYQDKVSLPNYPAIPSDATLEIQNLRSNDSGIYRCEVMHGIEDSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVLYATSPEKFTFQEAANECRRLGARLATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITIQTVTWPDVELPLPRNITEGEARGNVILTVKPIFDLSPTAPEPEEPFTFVPEPEKPFTFATDVGVTAFPEAENRTGEATRPWGVPEESTPGPAFTAFTSEDHVVQVTAVPGAAEVPGQPRLPGGVVFHYRPGSARYSLTFEEAQQACLRTGAVIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPPSETYDVYCYVDKLEGEVFFITRLEQFTFQEALAFCESHNATLASTGQLYAAWRQGLDKCYAGWLSDGSLRYPIVTPRPSCGGDKPGVRTVYLYPNQTGLPDPLSRHHVFCFRGVSGVPSPGEEEGGTPTPSVVEDWIPTQVGPVVPSVPMGEETTAILDFTIEPENQTEWEPAYSPAGTSPLPGIPPTWPPTSTATEESTEGPSGTEVPSVSEEPSPSEEPFPWEELSTLSPPGPSGTELPGSGEASGVPEVSGDFTGSGEVSGHPDSSGQLSGESASGLPSEDLDSSGLTSAVGSGLASGDEDRITLSSIPKVEGEGLETSASGVEDLSGLPSGREGLETSTSGVGDLSGLPSGEGLEVSASGVEDLSGLPSGEGPETSTSGVGDLSRLPSGEGPEVSASGVGDLSGLPSGREGLETS TSGVEDLSGLPSGEGPEASTSGVGDLSRLPSGEGPEVSASGVEDLSGLPSGEGLEASASGVGDLSGLPSGEGPEASASGVGDLSRLPSGEGPEVSASGVEDLSGLSSGESPEASASGVGDLSGLPSGREGLETSASGVGDLSGLPSGEGQEASASGVEDLSRLPSGEGPEASASGVGELSGLPSGREGLETSASGVGDLSGLPSGEGPEAFASGVEDLSILPSGEGPEASASGVGDLSGLPSGREGLETSTSGVGDLSGLPSGREGLETSTSGVGDLSGLPSGEGPEASASGIGDISGLPSGREGLETSSSGVEDHPETSASGVEDLSGLPSGVEGHPETSASGVEDLSDLSSGGEGLETSASGAEDLSGFPSGKEDLIGSASGALDFGRIPSGTLGSGQAPEASSLPSGFSGEYSGVDFGSGPISGLPDFSGLPSGFPTISLVDTTLVEVITTTSASELEGRGTIGISGAGETSGLPVSELDISGAVSGLPSGAELSGQASGSPDMSGETSGFFGVSGQPSGFPDISGGTSGLFEVSGQPSGFSGETSGVTELSGLYSGQPDVSGEASGVPSGSGQPFGMTDLSGETSGVPDISGQPSGLPEFSGTTSGIPDLVSSTMSGSGESSGITFVDTSLVEVTPTTFKEKKRLGSVELSGLPSGEVDLSGASGTMDISGQSSGATDSSGLTSHLPKFSGLPSGAAEVSGESSGAEVGSSLPSGTYEGSGNFHPAFPTVFLVDRTLVESVTQAPTAQEAGEGPSGILELSGAHSGAPDVSGDHSGSLDLSGMQSGLVEPSGEPSSTPYFSGDFSGTMDVTGEPSTAMSASGEASGLLEVTLITSEFVEGVTEPTVSQELAQRPPVTHTPQLFESSGEASASGEISGATPAFPGSGLEASSVPESSSETSDFPERAVGVSAAPEASGGASGAPDVSEATSTFPEADVEGASGLGVSGGTSAFPEAPREGSATPEVQEEPTTSYDVGREALGWPSATPTASGDRIEV SGDLSGHTSGLDVVISTSVPESEWIQQTQRPAEAHLEIEASSPLHSGEETQTAETATSPTDDASIPTSPSGTDESAPAIPDIDECLSSPCLNGATCVDAIDSFTCLCLPSYRGDLCEIDQELCEEGWTKFQGHCYRYFPDRESWVDAESRCRAQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHSLQFENWRPNQPDNFFVSGEDCVVMIWHEKGEWNDVPCNYYLPFTCKKGTVACGDPPVVEHARTFGQKKDRYEINSLVRYQCTEGFVQRHVPTIRCQPSGHWEKPRITCTDPSTYKRRLQKRSSRAPRRSRPSTAH Aggrekan cows 127 MTTLLLVFVTLRVITAAISVEVSEPDNSLSVSIPEPSPLRVLLGSSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRISKEKEVVLLVATEGRVRVNSAYQDKVTLPNYPAIPSDATLEIQNMRSNDSGILRCEVMHGIEDSQATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPESFFGVGGEEDITIQTVTWPDVELPLPRNITEGEARGSVILTAKPDFEVSPTAPEPEEPFTFVPEVRATAFPEVENRTEEATRPWAFPRESTPGLGAPTAFTSEDLVVQVTLAPGAAEVPGQPRLPGGVVFHYRPGSSRYSLTFEEAKQACLRTGAIIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPPSETYDVYCYVDRLEGEVFFATRLEQFTFWEAQEFCESQNATLATTGQLYAAWSRGLDKCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLLDPLSRHHAFCFRGVSAAPSPEEEEGSAPTAGPDVEEWMVTQVGPGVAAVPIGEETTAIPGFTVEPENKTEWELAYTPAGTLPLPGIPPTWPPTGEATEEHTEGPSATEVPSASEKPFPSEEPFPPEEPFPSEKPFPPEELFPSEKPFPSEKPFPSEEPFPSEKPFPPEELFPSEKPIPSEEPFPSEEPFPSEKPFPPEEPFPSEKPIPSEEPFPSEKPFPSEEPFPSEEPSTLSAPVPSRTELPSSGEVSGVPEISGDFTGSGEISGHLDFSGQPSGESASGLPSEDLDSSGLTSTVGSGLPVESGLPSGEEERITWTSAPKVDRLPS GGEGPEVSGVEDISGLPSGGEVHLEISASGVEDISGLPSGGEVHLEISASGVEDLSRIPSGEGPEISASGVEDISGLPSGEEGHLEISASGVEDLSGIPSGEGPEVSASGVEDLIGLPSGEGPEVSASGVEDLSRLPSGEGPEVSASGVEDLSGLPSGEGPEVSVSGVEDLSRLPSGEGPEVSASGVEDLSRLPSGEGPEISVSGVEDISILPSGEGPEVSASGVEDLSVLPSGEGHLEISTSGVEDLSVLPSGEGHLETSSGVEDISRLPSGEGPEVSASGVEDLSVLPSGEDHLEISASGVEDLGVLPSGEDHLEISASGVEDISRLPSGEGPEVSASGVEDLSVLPSGEGHLEISASGVEDLSRLPSGGEDHLETSASGVGDLSGLPSGREGLEISASGAGDLSGLTSGKEDLTGSASGALDLGRIPSVTLGSGQAPEASGLPSGFSGEYSGVDLESGPSSGLPDFSGLPSGFPTVSLVDTTLVEVVTATTAGELEGRGTIDISGAGETSGLPFSELDISGGASGLSSGAELSGQASGSPDISGETSGLFGVSGQPSGFPDISGETSGLLEVSGQPSGFYGEISGVTELSGLASGQPEISGEASGILSGLGPPFGITDLSGEAPGIPDLSGQPSGLPEFSGTASGIPDLVSSAVSGSGESSGITFVDTSLVEVTPTTFKEEEGLGSVELSGLPSGELGVSGTSGLADVSGLSSGAIDSSGFTSQPPEFSGLPSGVTEVSGEASGAESGSSLPSGAYDSSGLPSGFPTVSFVDRTLVESVTQAPTAQEAGEGPSGILELSGAPSGAPDMSGDHLGSLDQSGLQSGLVEPSGEPASTPYFSGDFSGTTDVSGESSAATSTSGEASGLPEVTLITSELVEGVTEPTVSQELGQRPPVTYTPQLFESSGEASASGDVPRFPGSGVEVSSVPESSGETSAYPEAEVGASAAPEASGGASGSPNLSETTSTFHEADLEGTSGLGVSGSPSAFPEGPTEGLATP EVSGESTTAFDVSVEASGSPSATPLASGDRTDTSGDLSGHTSGLDIVISTTIPESEWTQQTQRPAEARLEIESSSPVHSGEESQTADTATSPTDASIPASAGGTDDSEATTTDIDECLSSPCLNGATCVDAIDSFTCLCLPSYQGDVCEIQKLCEEGWTKFQGHCYRHFPDRATWVDAESQCRKQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDKTIEGDFRWSDGHSLQFENWRPNQPDNFFATGEDCVVMIWHEKGEWNDVPCNYQLPFTCKKGTVACGEPPVVEHARIFGQKKDRYEINALVRYQCTEGFIQGHVPTIRCQPSGHWEEPRITCTDPATYKRRLQKRSSRPLRRSHPSTAH Rat aggrecan 128 MTTLLLVFVTLRVIAAVISEEVPDHDNSLSVSIPQPSPLKALLGTSLTIPCYFIDPMHPVTTAPSTAPLTPRIKWSRVSKEKEVVLLVATEGQVRVNSIYQDKVSLPNYPAIPSDATLEIQNLRSNDSGIYRCEVMHGIEDSEATLEVIVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRTVGARLATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITIQTVTWPDLELPLPRNITEGEARGNVILTAKPIFDMSPTVSEPGEALTLAPEVGTTVFPEAGERTEKTTRPWGFPEEATRGPDSATAFASEDLVVRVTISPGAVEVPGQPRLPGGVVFHYRPGSTRYSLTFEEAQQACIRTGAAIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSSETYDVYCYVDKLEGEVFFATQMEQFTFQEAQAFCAAQNATLASTGQLYAAWSQGLDKCYAGWLADGTLRYPIVNPRPACGGDKPGVRTVYLYPNQTGLPDPLSKHHAFCFRGVSVVPSPGGTPTSPSDIEDWIVTRVEPGVDAVPLEPETTEVPYFTTEPEKQTEWEPAYTPVGTSPLPGIPPTWLPTVPAAEEHTESPSASQEPSASQVPSTSEEPYTPSLAVPSGTELPSSGDTSGAPDLSGDFTGSTDTSGRLDSSGEPSGGSESGLPSGDLDSSGLGPTVSSGLPVESGSASGDGEIPWSSTPTVDRLPSGGESLEGSASASGTGDLSGLPSGGEITETSASGTEEISGLPSGGDDLETSTSGIDGASVLPTGRGGLETSASGVEDLSGLPSGEEGSETSTSGIEDISVLPTGESPETSASGV GDLSGLPSGGESLETSASGVEDVTQLPTERGGLETSASGIEDITVLPTGRENLETSASGVEDVSGLPSGKEGLETSASGIEDISVFPTEAEGLETSASGGYVSGIPSGEDGTETSTSGVEGVSGLPSGGEGLETSASGVEDLGLPTRDSLETSASGVDVTGYPSGREDTETSVPGVGDDLSGLPSGQEGLETSASGAEDLGGLPSGKEDLVGSASGALDFGKLPSGTLGSGQTPEASGLPSGFSGEYSGVDIGSGPSSGLPDFSGLPSGFPTVSLVDSTLVEVITATTASELEGRGTISVSGSGEESGPPLSELDSSADISGLPSGTELSGQTSGSLDVSGETSGFFDVSGQPFGSSGTGEGTSGIPEVSGQAVRSPDTTEISELSGLSSGQPDVSGEGSGILFGSGQSSGITSVSGETSGISDLSGQPSGFPVLSGTTPGTPDLASGAMSGSGDSSGITFVDTSLIEVTPTTFREEEGLGSVELSGLPSGETDLSGTSGMVDVSGQSSGAIDSSGLISPTPEFSGLPSGVAEVSGEVSGVETGSSLSSGAFDGSGLVSGFPTVSLVDRTLVESITLAPTAQEAGEGPSSILEFSGAHSGTPDISGDLSGSLDQSTWQPGWTEASTEPPSSPYFSGDFSSTTDASGESITAPTGSGETSGLPEVTLITSELVEGVTEPTVSQELGHGPSMTYTPRLFEASGEASASGDLGGPVTIFPGSGVEASVPEGSSDPSAYPEAGVGVSAAPEASSQLSEFPDLHGITSASRETDLEMTTPGTEVSSNPWTFQEGTREGSAAPEVSGESSTTSDIDAGTSGVPFATPMTSGDRTEISGEWSDHTSEVNVTVSTTVPESRWAQSTQHPTETLQEIGSPNPSYSGEETQTAETAKSLTDTPTLASPEGSGETESTAADQEQCEEGWTKFQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEFVNKNAQDYQWIGLNDRTIEGDFRWSDGHSLQFEKWRPNQP DNFFATGEDCVVMIWHERGEWNDVPCNYQLPFTCKKGTVACGEPPAVEHARTLGQKKDRYEISSLVRYQCTEGFVQRHVPTIRCQPSADWEEPRITCTDPNTYKHRLQKRTMRPTRRSRPSMAH Aggrecan pig (cow) 129 AISVEVSEPDNSLSVSIPQPSPLRVLLGGSLTIPCYFIDPMHPVXTAPXTAPLAPRIKWSRVSKEKEVVLLVATEGQVRVNSAYQDRVTLPNYPAIPSDATLEIQNLRSNDSGIYRCEVMHGIEDSEATLEVVVKGIVFHYRAISXRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVITYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGQLYLAWRGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITIQTVTWPDVELPLPRNITEGEARGTVILTVKPVFEFSPTAPEPEEPFTFAPGTGATAFPEAENRTGEATRPWAFPEESTPGLGAPTAFTSEDLVVQVTSAATEEGTEGPSATEAPSTSEEPFPSEKPFPSEEPFPSEEPFPSEKPSASEEPFPSEQPSTLSAPVPSRTELPGSGEVSGAPEV mouse aggrecan 130 MTTLLLVFVTLRVIAAVISEEVPDHDNSLSVSIPQPSPLKVLLGSSLTIPCYFIDPMHPVTTAPSTAPLTPRIKWSRVSKEKEVVLLVATEGQVRVNSIYQDKVSLPNYPAIPSDATLEIQNLRSNDSGIYRCEVMHGIEDSEATLEVIVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGQLYLAWQGGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEDDITIQTVTWPDLELPLPRNVTEGEALGSVILTAKPIFDLSPTISEPGEALTLAPEVGSTAFPEAEERTGEATRPWGFPAEVTRGPDSATAFASEDLVVRVTISPGAAEVPGQPRLPGGVVFHYRPGSTRYSLTFEEAQQACMHTGAVIASPEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSSETYDVYCYVDKLEGEVFFATRLEQFTFQEARAFCAAQNATLASTGQLYAAWSQGLDKCYAGWLADGTLRYPIITPRPACGGDKPGVRTVYLYPNQTGLPDPLSKHHAFCFRGVSVAPSPGEEGGSTPTSPSDIEDWIVTQVGPGVDAVPLEPKTTEVPYFTTEPRKQTEWEPAYTPVGTSPQPGIPPTWLPTLPAAEEHTESPSASEEPSASAVPSTSEEPYTSSFAVPSMTELPGSGEASGAPDLSGDFTGSGDASGRLDSSGQPSGGIESGLPSGDLDSSGLSPTVSSGLPVESGSASGDGEVPWSHTPTVGRLPSGGESPEGSASASGTGDLSGLPSGGEITETSTSGAEETSGLPSGGDGLETSTSGVDDVSGIPTGREGLETSASGVEDLSGLPSGEEGSETSTSGIEDISVLPTGGESLET SASGVGDLSGLPSGGESLETSASGAEDVTQLPTERGGLETSASGVEDITVLPTGRESLETSASGVEDVSGLPSGREGLETSASGIEDISVFPTEAEGLDTSASGGYVSGIPSGGDGTETSASGVEDVSGLPSGGEGLETSASGVEDLGPSTRDSLETSASGVDVTGFPSGRGDPETSVSGVGDDFSGLPSGKEGLETSASGAEDLSGLPSGKEDLVGSASGALDFGKLPPGTLGSGQTPEVNGFPSGFSGEYSGADIGSGPSSGLPDFSGLPSGFPTVSLVDSTLVEVITATTSSELEGRGTIGISGSGEVSGLPLGELDSSADISGLPSGTELSGQASGSPDSSGETSGFFDVSGQPFGSSGVSEETSGIPEISGQPSGTPDTTATSGVTELNELSSGQPDVSGDGSGILFGSGQSSGITSVSGETSGISDLSGQPSGFPVFSGTATRTPDLASGTISGSGESSGITFVDTSFVEVTPTTFREEEGLGSVELSGFPSGETELSGTSGTVDVSEQSSGAIDSSGLTSPTPEFSGLPSGVAEVSGEFSGVETGSSLPSGAFDGSGLVSGFPTVSLVDRTLVESITQAPTAQEAGEGPSGILEFSGAHSGTPDISGELSGSLDLSTLQSGQMETSTETPSSPYFSGDFSSTTDVSGESIAATTGSGESSGLPEVTLNTSELVEGVTEPTVSQELGHGPSMTYTPRLFEASGDASASGDLGGAVTNFPGSGIEASVPEASSDLSAYPEAGVGVSAAPEASSKLSEFPDLHGITSAFHETDLEMTTPSTEVNSNPWTFQEGTREGSAAPEVSGESSTTSDIDTGTSGVPSATPMASGDRTEISGEWSDHTSEVNVAISSTITESEWAQPTRYPTETLQEIESPNPSYSGEETQTAETTMSLTDAPTLSSSEGSGETESTVADQEQCEEGWTKFQGHCYRHFHDRETWVDAERRCREQQSHLSSIVTPEEQEFVNKNAQDYQWIGLNDRTIEGDFRWSDGHSLQF EKWRPNQPDNFFATGEDCVVMIWHERGEWNDVPCNYQLPFTCKKGTVACGDPPVVEHARTLGQKKDRYEISSLVRYQCTEGFVQRHVPTIRCQPSGHWEEPRITCTDPNTYKHRLQKRSMRPTRRSRPSMAH Rabbit aggrecan 131 MTTLLLVLVALRVIAAAISGDVSDLDNALSVSIPQPSPVRALLGTSLTIPCYFIDPVHPVTTAPSTAPLTPRIKWSRISKDKEVVLLVANEGRVRINSAYQDKVSLPNYPAIPSDATLEIQSLRSNDSGIYRCEVMHGLEDSEATLEVVVKGVVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAASECRRLGARLATTGQLYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYVHANQTGYPDPSSRYDAICYTGEDFMDIPENFFGVGGEEDITVQTVTWPDVELPVPRNITEGEARGSVVLTAKPVLDVSPTAPQPEETFAPGVGATAFPGVENGTEEATRPRGFADEATLGPSSATAFTSADLVVQVTAAPGVAEVPGQPRLPGGVVFHYRPGPTRYSLTFEEAQQACLRTGAAMASAEQLQAAYEAGYEQCDAGWLQDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPPSETYDVYCYVDRLEGEVFFATRLEQFTFQEALEFCESHNATLASTGQLYAAWSRGLDRCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGTSEAPSPGPEEGGTATPASGLEDWIVTQVGPGVAATPRAEERTAVPSFATEPGNQTGWEAASSPVGTSLLPGIPPTWPPTGTAAEGTTEGLSTAAMPSASEGPYTPSSLVARETELPGLGVTSVPPDISGDLTSSGEASGLFGPTGQPLGGSASGLPSGELDSGSLTPTVGSGLPIGSGLASGDEDRIQWSSSTEVGGVTSGAEIPETSASGVGTDLSGLPSGAEIPETFASGVGTDLSGLPSGAEIPETFASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASG VGTDLSGLPSGAEIPETFASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEIPETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGAEILETSASGVGTDLSGLPSGGEIPETFASGVGDLSGLPPGREDLETLTSGVGDLSGLSSGKDGLVGSASGALDFGGTLGSGQIPETSGLPSGYSGEYSEVDLGSGPSSGLPDFSGLPSGFPTVSLVDTPLVEVVTATTARELEGRGTIGISGAGEISGLPSSELDVSGGTSGADISGEADVGGEASGLIVRGQPSGFPDTSGEAFGVTEVSGLSSGQPDLSGEASGVLFGSGPPFGITDLSGEPSGQPSGLPEFSGTTHRIPDLVSGATSGSGESSGIAFVDTSVVEVTPTTLREEEGLGSVEFSGFPSGETGLSGTPETIDVSGQSSGTIDSSGFTSLAPEVSGSPSGVAEVSGEASGTEITSGLPSGVFDSSGLPSGFPTVSLVDRTLVESVTQAPTAQEAEGPSDILELSGVHSGLPDVSGAHSGFLDPSGLQSGLVEPSGEPPRTPYFSGDFPSTPDVSGEASAATSSSGDISGLPEVTLVTSEFMEGVTRPTVSQELGQGPPMTHVPKLFESSGEALASGDTSGAAPAFPGSGLEASSVPESHGETSAYAEPGTKAAAAPDASGEASGSPDSGEITSVFREAAGEGASGLEVSSSSLASQQGPREGSASPEVSGESTTSYEIGTETSGLPLATPAASEDRAEVSGDLSGRTPVPVDVVTNVPEAEWIQHSQRPAEMWPETKSSSPSYSGEDTAGTAASPASADTPGEPGPTTAAPRSCAEEPCGPGTCQETEGRVTCLCPPGHTGEYCDIDIDECLSSPCVNGATCVDASDSFTCLCLPSYGGDLCETDQEVCEEGWTKFQGHCYRHFPDRETWVDAEGRCREQQSHLSSIVTPEE QEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPLQFENWRPNQPDNFFATGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGDPPVVEHARTFGQKKDRYEINSLVRYQCAEGFTQRHVPTIRCQPSGHWEEPRITCTHPTTYKRRVQKRSSRTLQRSQASSAP Aggrecan cynomolgus macaque 132 MTTLLWVFVTLRVIAAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEIQSLRSNDSGVYRCEVMHGIEDSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGQLYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFDVSPSPLEPEEPFTFAPEIGATAFPEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGSTRYSLTFEEAQQACLRTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSTETYDVYCYVDRLEGEVFFATRLEQFTFQEALEFCESHNATLATTGQLYAAWSRGLDKCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGVSAVPSPGEEEGGTPTSPSGVEDWIATQVVPGVAAVPVEEETTAVPLGETTAILEFTTEPENQTEWEPAYTPMGTSPLPGILPTWPPTGTATEESTEGPSATEVLTASKEPSPPEVPFPSEEPSPSEEPFPSVRPFPSVEPSPSEEPFPSVEPSPSEEPSASEEPYTPSPPVPSWTELPGSGEESGAPDVSGDFIGSGDVSGHLDFSGQLSGDRISGLPSGDLDSSGLTSTVGSGLPVDSGLASGDEERIEWSSTPTVGELPSGAEILEGSASEVGDLSGLPSGDVLETSASGVGDLSGLPSGEVLETSASGVGDLSGLPSGEV LETSTSGVGDLSGLPSGEVLETSTSGVGDLSGLPSAGEVLETTASGVEDISGLPSGEVLETTASGVEDISGFPSGEVLETTASGVEDISGLPSGEVLETTASGVEDISGLPSGEVLETTASGVGDLGGLPSGEVLETSTSGVGDLSGLPSGEVVETSTSGVEDLSGLPSGGEVLETSTSGVEDISGLPSGEVLETTASGIEDVSELPSGEGLETSASGVEDLSRLPSGEVLETSASGVGDISGLPSGGEVLEISASGVGDLSGLPSGGEGLETSASGVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDLVGPASGDLDLGKLPSGTLRSGQAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTIGISGAGEISGLPSSELDISGEASGLPSGTELSGQASGSPDVSRETPGLFDVSGQPSGFPDISGGTSGISEVSGQPSGFPDTSGETSGVTELSGLPSGQPGVSGEASGVPYGSSQPFGITDLSGETSGVPDLSGQPSGLPGFSGATSGVPDLVSGATSGSGESSGITFVDTSLVEVTPTTFKEEEGLGSVELSGLPSGEADLSGRSGMVDVSGQFSGTVDSSGFTSQTPEFSGLPIGIAEVSGESSGAETGSSLPSGAYYGSGLPSGFPTVSLVDRTLVESVTQAPTAQEAGEGPPGILELSGTHSGAPDMSGDHSGFLDVSGLQFGLVEPSGEPPSTPYFSGDFASTTDVSGESSAAMGTSGEASGLPGVTLITSEFMEGVTEPTVSQELGQRPPVTHTPQLFESSGEASAAGDISGATPVLPGSGVEVSSVPESSSETSAYPEAGVGASAAPETSGEDSGSPDLSETTSAFHEADLERSSGLGVSGSTLTFQEGEPSASPEVSGESTTTGDVGTEAPGLPSATPTASGDRTEISGDLSGHTSGLGVVISTSIPESEWTQQTQRPAEAHLETESSSLLYSGEETHTAETATSPTDASIPA SPEWTGESESTVADIDECLSSPCLNGATCVDAIDSFTCLCLPSYGGDLCEIDQEVCEEGWTKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGEPPMVQHARTFGQKKDRYEINSLVRYQCTEGFVQRHVPTIRCQPSGHWEEPRITCTDATAYKRRLQKRSSRHPRRSRPSTAH Rhesus monkey aggrecan XM_002804944.1 133 MTTLLWVFVTLRVIAAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEIQSLRSNDSGVYRCEVMHGIEDSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGQLYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYLHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFDVSPSPLEPEEPFTFAPEIGATAFPEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGSTRYSLTFEEAQQACLRTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSTETYDVYCYVDRLEGEVFFATRLEQFTFQEALEFCESHNATLATTGQLYAAWSRGLDKCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGVSAVPSPGEEEGGTPTSPSGVEDWIATQVVPGVAAVPVEEETTAVPLGETTAILEFTTEPENQTEWEPAYTPMGTSPLPGILPTWPPTGTATEESTEGPSATEVLTASKEPSPPEVPFPSEEPSPSEEPFPSVRPFPSVEPSPSEEPFPSVEPSPSEEPSASEEPYTPSPPVPSWTELPGSGEESGAPDVSGDFIGSGDVSGHLDFSGQLSGDRISGLPSGDLDSSGLTSTVGSGLPVDSGLASGDEERIEWSSTPTVGELPSGAEILEGSASEVGDLSGLPSGDVLETSASGVGDLSGLPSGEVLETSVSGVGDLSGLPSGEV LETSTSGVGDLSGLPSGEVLETSTSGVGDLSGLPSAGEVLETTASGVEDISGLPSGEVLETTASGVEDISGFPSGEVLETTASGVEDISGLPSGEVLETTASGVEDISGLPSGEVLETTASGVGDLGGLPSGEVLETSTSGVGDLSGLPSGEVVETSTSGVEDLSGLPSGGEVLETSTSGVEDISGLPSGEVLETTASGIEDVSELPSGEGLETSASGVEDLSRLPSGEVLETSASGVGDISGLPSGGEVLEISASGVGDLSGLPSGGEGLETSASGVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDLVGPASGDLDLGKLPSGTLGSGQAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTIGISGAGEISGLPSSELDISGEASGLPSGTELSGQASGSPDVSRETSGLFDVSGQPSGFPDTSGETSGVTELSGLPSGQPGVSGEASGVPYGSSQPFGITDLSGETSGVPDLSGQPSGLPGFSGATSGVPDLVSGATSGSGESSDITFVDTSLVEVTPTTFKEEEGLGSVELSGLPSGEADLSGRSGMVDVSGQFSGTVDSSGFTSQTPEFSGLPIGIAEVSGESSGAETGSSLPSGAYYGSELPSGFPTVSLVDRTLVESVTQAPTAQEAGEGPPGILELSGTHSGAPDMSGDHSGFLDVSGLQFGLVEPSGEPPSTPYFSGDFASTTDVSGESSAAMGTNGEASGLPEVTLITSEFMEGVTEPTVSQELGQRPPVTHTPQLFESSGEASAAGDISGATPVLPGSGVEVSSVPESSSETSAYPEAGVGASAAPETSGEDSGSPDLSETTSAFHEADLERSSGLGVSGSTLTFQEGEPSASPEVSGESTTTGDVGTEAPGLPSATPTASGXXXXXXPTRSCAEEPCGAGTCKETEGHVICLCPPGYTGEHCNIDQEVCEEGWTKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEE QEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGEPPMVQHARTFGQKKDRYEINSLVRYQCTEGFVQRHVPTIRCQPSGHWEEPRITCTDATAYKRRLQKRSSRHPRRSRPSTAH human neurocan 134 MGAPFVWALGLLMLQMLLFVAGEQGTQDITDASERGLHMQKLGSGSVQAALAELVALPCLFTLQPRPSAARDAPRIKWTKVRTASGQRQDLPILVAKDNVVRVAKSWQGRVSLPSYPRRRANATLLLGPLRASDSGLYRCQVVRGIEDEQDLVPLEVTGVVFHYRSARDRYALTFAEAQEACRLSSAIIAAPRHLQAAFEDGFDNCDAGWLSDRTVRYPITQSRPGCYGDRSSLPGVRSYGRRNPQELYDVYCFARELGGEVFYVGPARRLTLAGARAQCRRQGAALASVGQLHLAWHEGLDQCDPGWLADGSVRYPIQTPRRRCGGPAPGVRTVYRFANRTGFPSPAERFDAYCFRAHHPTSQHGDLETPSSGDEGEILSAEGPPVRELEPTLEEEEVVTPDFQEPLVSSGEEETLILEEKQESQQTLSPTPGDPMLASWPTGEVWLSTVAPSPSDMGAGTAASSHTEVAPTDPMPRRRGRFKGLNGRYFQQQEPEPGLQGGMEASAQPPTSEAAVNQMEPPLAMAVTEMLGSGQSRSPWADLTNEVDMPGAGSAGGKSSPEPWLWPPTMVPPSISGHSRAPVLELEKAEGPSARPATPDLFWSPLEATVSAPSPAPWEAFPVATSPDLPMMAMLRGPKEWMLPHPTPISTEANRVEAHGEATATAPPSPAAETKVYSLPLSLTPTGQGGEAMPTTPESPRADFRETGETSPAQVNKAEHSSSSPWPSVNRNVAVGFVPTETATEPTGLRGIPGSESGVFDTAESPTSGLQATVDEVQDPWPSVYSKGLDASSPSAPLGSPGVFLVPKVTPNLEPWVATDEGPTVNPMDSTVTPAPSDASGIWEPGSQVFEEAESTTLSPQVALDTSIVTPLTTLEQGDKVGVPAMSTLGSSSSQPHPEPEDQVETQGTSGASVPPHQSSPLGKPAVPPGTPTAASVGESASVSSGEPTVPWDPSSTLLPVTLGIEDFELEVLAGSPGVESFWEEVASGEEPALPGTPM NAGAEEVHSDPCENNPCLHGGTCNANGTMYGCSCDQGFAGENCEIDIDDCLCSPCENGGTCIDEVNGFVCLCLPSYGGSFCEKDTEGCDRGWHKFQGHCYRYFAHRRAWEDAEKDCRRRSGHLTSVHSPEEHSFINSFGHENTWIGLNDRIVERDFQWTDNTGLQFENWRENQPDNFFAGGEDCVVMVAHESGRWNDVPCNYNLPYVCKKGTVLCGPPPAVENASLIGARKAKYNVHATVRYQCNEGFAQHHVATIRCRSNGKWDRPQIVCTKPRRSHRMRRHHHHHQHHHQHHHHKSRKERRKHKKHPTEDWEKDEGNFC Brevican of man 135 MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLQGVLGGALTIPCHVHYLRPPPSRRAVLGSPRVKWTFLSRGREAEVLVARGVRVKVNEAYRFRVALPAYPASLTDVSLALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGVVFLYREGSARYAFSFSGAQEACARIGAHIATPEQLYAAYLGGYEQCDAGWLSDQTVRYPIQTPREACYGDMDGFPGVRNYGVVDPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQERGAEIATTGQLYAAWDGGLDHCSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPSAIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSSTPEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYEDEEEKEEEEEEEEVEDEALWAWPSELSSPGPEASLPTEPAAQEESLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPTETLPTPRERNLASPSPSTLVEAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRAPEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAVVPASGDCVPSPCHNGGTCLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSWEEAETQCRMYGAHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPDSYFLSGENCVVMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVLRYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKALLIPPSSPMPGP

Table C: Serum albumin-binding ISV sequences ("ID" refers to SEQ ID NOs used herein)

Name ID Amino acid sequence Alb8 136 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 137 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 138 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 139 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 140 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11(S112K)-A 141 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 142 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 143 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 144 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 145 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 146 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSSG Alb82-GG 147 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 148 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG Alb92 149 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 150 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA ALB-CDR1 151 SFGMS ALB-CDR2 152 SISGSGSDTLYADSVKG ALB-CDR3 153 GGSLSSR Alb135 171 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVKSA

Table D: Linker sequences ("ID" refers to SEQ ID NOs used in this document)

Name ID Amino acid sequence 3A linker (Poly-A) 154 AAA Linker 5GS 155 GGGGS Linker 7GS 156 sggsggs Linker 8GS 157 Gggggggs Linker 9GS 158 GGGGSGGGS Linker 10GS 159 GGGGSGGGGS Linker 15GS 160 GGGGSGGGGSGGGGS Linker 18GS 161 GGGGSGGGGSGGGGGGGS Linker 20GS 162 GGGGSGGGGSGGGGSGGGGS Linker 25GS 163 GGGGSGGGGSGGGGSGGGGSGGGGS Linker 30GS 164 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Linker 35GS 165 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Linker 40GS 166 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1-hinge 167 EPKSCDKTHTCPPCP 9GS-G1-hinge 168 GGGGSGGGSEPKSCDKTHTCPPCP upper long hinge area of the llama 169 epktpkpqpaaa G3 hinge 170 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

Table E-1: Polypeptides/Constructs Containing Specified Therapeutic ISV and Specified Aggrecan Binding ISV

Target
(binding ISV) CAP (ISV) Target
(binding ISV) CAP (ISV) Target
(binding ISV) CAP (ISV) Cathepsin A 604F02 ADAMTS1 604F02 MMP1 604F02 Cathepsin B 604F02 ADAMTS2 604F02 MMP2 604F02 Cathepsin C 604F02 ADAMTS3 604F02 MMP3 604F02 Cathepsin D 604F02 ADAMTS4 604F02 MMP7 604F02 Cathepsin E 604F02 ADAMTS5 604F02 MMP8 604F02 Cathepsin F 604F02 ADAMTS6 604F02 MMP9 604F02 Cathepsin G 604F02 ADAMTS7 604F02 MMP10 604F02 Cathepsin N 604F02 ADAMTS8 604F02 MMP11 604F02 Cathepsin K 604F02 ADAMTS9 604F02 MMP12 604F02 Cathepsin L1 604F02 ADAMTS10 604F02 MMP13 604F02 Cathepsin L2 (or V) 604F02 ADAMTS11 604F02 MMP14 604F02 Cathepsin O 604F02 ADAMTS12 604F02 MMP15 604F02 Cathepsin C 604F02 ADAMTS13 604F02 MMP16 604F02 Cathepsin W 604F02 ADAMTS14 604F02 MMP17 604F02 Cathepsin Z (or X) 604F02 ADAMTS15 604F02 MMP18 604F02 ADAMTS16 604F02 MMP19 604F02 ADAMTS17 604F02 MMP20 604F02 ADAMTS18 604F02 MMP21 604F02 ADAMTS19 604F02 MMP23A 604F02 ADAMTS20 604F02 MMP23B 604F02 MMP24 604F02 MMP25 604F02 MMP26 604F02 MMP27 604F02 MMP28 604F02

Table E-2. Polypeptides/constructs containing the specified therapeutic ISV and the two specified aggrecan-binding ISVs

Target
(associated
ISV) CAP (ISV) Target
(associated
ISV) CAP (ISV) Target
(associated
ISV) CAP (ISV) Cathepsin A 114F08-114F08 ADAMTS1 114F08-114F08 MMP1 114F08-114F08 Cathepsin B 114F08-114F08 ADAMTS2 114F08-114F08 MMP2 114F08-114F08 Cathepsin C 114F08-114F08 ADAMTS3 114F08-114F08 MMP3 114F08-114F08 Cathepsin D 114F08-114F08 ADAMTS4 114F08-114F08 MMP7 114F08-114F08 Cathepsin E 114F08-114F08 ADAMTS5 114F08-114F08 MMP8 114F08-114F08 Cathepsin F 114F08-114F08 ADAMTS6 114F08-114F08 MMP9 114F08-114F08 Cathepsin G 114F08-114F08 ADAMTS7 114F08-114F08 MMP10 114F08-114F08 Cathepsin N 114F08-114F08 ADAMTS8 114F08-114F08 MMP11 114F08-114F08 Cathepsin K 114F08-114F08 ADAMTS9 114F08-114F08 MMP12 114F08-114F08 Cathepsin L1 114F08-114F08 ADAMTS10 114F08-114F08 MMP13 114F08-114F08 Cathepsin L2
(or V) 114F08-114F08 ADAMTS11 114F08-114F08 MMP14 114F08-114F08 Cathepsin O 114F08-114F08 ADAMTS12 114F08-114F08 MMP15 114F08-114F08 Cathepsin C 114F08-114F08 ADAMTS13 114F08-114F08 MMP16 114F08-114F08 Cathepsin W 114F08-114F08 ADAMTS14 114F08-114F08 MMP17 114F08-114F08 Cathepsin Z
(or X) 114F08-114F08 ADAMTS15 114F08-114F08 MMP18 114F08-114F08 ADAMTS16 114F08-114F08 MMP19 114F08-114F08 ADAMTS17 114F08-114F08 MMP20 114F08-114F08 ADAMTS18 114F08-114F08 MMP21 114F08-114F08 ADAMTS19 114F08-114F08 MMP23A 114F08-114F08 ADAMTS20 114F08-114F08 MMP23B 114F08-114F08 MMP24 114F08-114F08 MMP25 114F08-114F08 MMP26 114F08-114F08 MMP27 114F08-114F08 MMP28 114F08-114F08

--->

Sequence list

<110> Ablynx NV

<120> Aggrecan binding Immunoglobulins

<130> 206 379

<150> US 62/514,180

<151> 2017-06-02

<160> 172

<170>PatentIn version 3.5

<210> 1

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Gly Arg Leu Tyr Arg Ala Thr Pro Arg Pro Ala Asp Phe Gly

100 105 110

Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 2

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 2

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ala Phe Ser Asn Tyr

20 25 30

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

35 40 45

Ala Ala Ile Asn Trp Asn Gly Val Thr Thr His Tyr Thr Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ala Arg Gly Thr Val Tyr Ser Arg Thr Tyr Gly Val Ser Glu Glu

100 105 110

Gly Tyr Met Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 3

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 4

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Phe Cys

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 5

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 6

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Asp Phe Leu Gly Gly Arg Asn Ser Arg Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 7

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 7

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Arg Thr Phe Asn Met Met

20 25 30

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

35 40 45

Ile Thr Trp Asn Gly Gly Asp Thr Arg Tyr Ala Glu Ser Val Lys Gly

50 55 60

Arg Phe Thr Val Ser Arg Asp Asp Val Lys Asn Thr Met Ala Leu Gln

65 70 75 80

Met Asn Arg Leu Asp Pro Leu Asp Thr Ala Val Tyr Tyr Cys Gly Val

85 90 95

Arg Ile His Gly Ser Asn Trp Ser Thr Lys Ala Asp Asp Tyr Asp Asn

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 8

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Pro Arg Met Tyr Val Asp Gly Thr Tyr Glu Lys Glu Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Thr Trp Ser Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Ala Ala Arg Ile Pro Val Arg Thr Tyr Thr Ser Glu Trp Asn

100 105 110

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

115 120

<210> 10

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Gly Leu Ser Tyr Tyr Ser Pro His Ala Tyr Tyr Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Tyr Arg Arg Pro Arg Tyr Ser Pro Thr Gly Thr Trp Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ala Tyr Thr Gly Pro Arg Ser Gly Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 13

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Tyr Arg Arg Arg Arg Ala Ser Ser Asn Arg Gly Leu Trp Asp

100 105 110

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

115 120

<210> 14

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Tyr Arg Arg Val Arg Tyr Thr Asn Leu Glu Val Trp Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Thr Trp Ser Ser Ala Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Ala Arg Ile Pro Val Gly Arg Arg Ser Glu Asn Trp Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 16

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Ala Tyr Thr Gly Arg Ser Tyr Gly Ser Tyr Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Arg Ser Gly Tyr Gly Thr Ser Leu Asp Trp Trp Tyr Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Arg Pro Arg Tyr Tyr Tyr Tyr Ser Leu Tyr Ser Tyr Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Glu Val Thr Thr Gly Trp Val Gly Tyr Ser Trp Tyr Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 20

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 20

Gly Arg Ser Phe Ser Ser Tyr Ala Met Gly

1 5 10

<210> 21

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 21

Gly Arg Ala Phe Ser Asn Tyr Ile Met Gly

1 5 10

<210> 22

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 22

Gly Ser Ile Phe Ser Asn Arg Phe Met Tyr

1 5 10

<210> 23

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 23

Gly Phe Thr Phe Ser Gly Ser Trp Met Phe

1 5 10

<210> 24

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 24

Gly Ser Thr Phe Ile Ile Asn Val Val Arg

1 5 10

<210> 25

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 25

Gly Phe Thr Phe Ser Met Tyr Ala Met Lys

1 5 10

<210> 26

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 26

Arg Arg Thr Phe Asn Met Met Gly

fifteen

<210> 27

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 27

Gly Ile Thr Phe Ser Ser Arg Tyr Met Arg

1 5 10

<210> 28

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 28

Gly Pro Thr Phe Ser Arg Tyr Ala Met Gly

1 5 10

<210> 29

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 29

Gly Arg Thr Phe Ser Ser Tyr Ala Val Gly

1 5 10

<210> 30

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 30

Gly Leu Thr Phe Ser Thr Tyr Ala Met Gly

1 5 10

<210> 31

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 31

Gly Arg Thr Phe Ser Ile Tyr Thr Met Ala

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 32

Gly Arg Thr Phe Ser Ser Tyr Thr Met Gly

1 5 10

<210> 33

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 33

Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly

1 5 10

<210> 34

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 34

Gly Arg Thr Phe Ser Ile Tyr Thr Met Gly

1 5 10

<210> 35

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 35

Gly Arg Thr Phe Ser Ile Tyr Gly Met Gly

1 5 10

<210> 36

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 36

Gly Arg Thr Phe Ser Thr Tyr Thr Met Gly

1 5 10

<210> 37

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 37

Gly Thr Ile Phe Ser Ile Asn Val Met Gly

1 5 10

<210> 38

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 38

Ile Ile Ser Trp Ser Gly Gly Ser Thr Val

1 5 10

<210> 39

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 39

Ala Ile Asn Trp Asn Gly Val Thr Thr His

1 5 10

<210> 40

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 40

Ser Ile Thr Leu Ser Gly Ser Thr Asn

fifteen

<210> 41

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 41

Ser Ile Asn Ser Ser Gly Gly Arg Thr Tyr

1 5 10

<210> 42

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 42

Thr Ile Ser Ser Gly Gly Asn Ala Asn

fifteen

<210> 43

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 43

Gly Ile Asn Ser Ser Gly Gly Arg Thr Asn

1 5 10

<210> 44

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 44

Tyr Ile Thr Trp Asn Gly Gly Asp Thr Arg

1 5 10

<210> 45

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 45

Ala Ile Ser Ser Gly Gly Arg Thr Asp

fifteen

<210> 46

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 46

Ala Ile Thr Trp Ser Ser Gly Gly Arg Thr Tyr

1 5 10

<210> 47

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 47

Ala Ile Ser Arg Ser Gly Arg Ser Thr Tyr

1 5 10

<210> 48

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 48

Ala Ile Ser Trp Ser Gly Ser Arg Thr Tyr

1 5 10

<210> 49

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 49

Ala Ile Ser Trp Ser Ser Gly Arg Thr Tyr

1 5 10

<210> 50

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 50

Ala Ile Ser Trp Ser Gly Gly Arg Thr Tyr

1 5 10

<210> 51

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 51

Ala Ile Ser Trp Ser Gly Arg Thr Thr Tyr

1 5 10

<210> 52

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 52

Ala Ile Thr Trp Ser Ser Ala Thr Thr Tyr

1 5 10

<210> 53

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 53

Ala Ile Asn Gly Gly Ser Arg Thr Tyr

fifteen

<210> 54

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 54

Ala Ile Ser Trp Ser Gly Gly Thr Thr Tyr

1 5 10

<210> 55

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 55

Ala Ile Thr Thr Gly Gly Arg Thr Asn

fifteen

<210> 56

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 56

Gly Arg Leu Tyr Arg Ala Thr Pro Arg Pro Ala Asp Phe Gly Ser

1 5 10 15

<210> 57

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 57

Arg Gly Thr Val Tyr Ser Arg Thr Tyr Gly Val Ser Glu Glu Gly Tyr

1 5 10 15

Met Tyr

<210> 58

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 58

Phe Leu Gln Asn Ser Phe Tyr

fifteen

<210> 59

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 59

Ser Pro Arg Val Gly Ser

fifteen

<210> 60

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 60

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

1 5 10

<210> 61

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 61

Asp Phe Leu Gly Gly Arg Asn Ser

fifteen

<210> 62

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 62

Arg Ile His Gly Ser Asn Trp Ser Thr Lys Ala Asp Asp Tyr Asp Asn

1 5 10 15

<210> 63

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 63

Pro Arg Met Tyr Val Asp Gly Thr Tyr Glu Lys Glu Leu

1 5 10

<210> 64

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 64

Ala Arg Ile Pro Val Arg Thr Tyr Thr Ser Glu Trp Asn Tyr

1 5 10

<210> 65

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 65

Gly Leu Ser Tyr Tyr Ser Pro His Ala Tyr Tyr Asp Tyr

1 5 10

<210> 66

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 66

Tyr Arg Arg Pro Arg Tyr Ser Pro Thr Gly Thr Trp Asp Tyr

1 5 10

<210> 67

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 67

Tyr Thr Gly Pro Arg Ser Gly Tyr Asp Tyr

1 5 10

<210> 68

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 68

Tyr Arg Arg Arg Arg Ala Ser Ser Asn Arg Gly Leu Trp Asp Tyr

1 5 10 15

<210> 69

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 69

Tyr Arg Arg Val Arg Tyr Thr Asn Leu Glu Val Trp Asp Tyr

1 5 10

<210> 70

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 70

Ala Arg Ile Pro Val Gly Arg Arg Ser Glu Asn Trp Asp Tyr

1 5 10

<210> 71

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 71

Tyr Thr Gly Arg Ser Tyr Gly Ser Tyr Asp Tyr

1 5 10

<210> 72

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 72

Asp Arg Ser Gly Tyr Gly Thr Ser Leu Asp Trp Trp Tyr Asp Tyr

1 5 10 15

<210> 73

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 73

Arg Pro Arg Tyr Tyr Tyr Tyr Ser Leu Tyr Ser Tyr Asp Tyr

1 5 10

<210> 74

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 74

Glu Val Thr Thr Gly Trp Val Gly Tyr Ser Trp Tyr Asp Tyr

1 5 10

<210> 75

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 75

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 76

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 76

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser

20 25

<210> 77

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 77

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 78

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 78

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser

20 25

<210> 79

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 79

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 80

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 80

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 81

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 81

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 82

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 82

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 83

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 83

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser

20 25

<210> 84

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 84

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser

20 25

<210> 85

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 85

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

1 5 10

<210> 86

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 86

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

1 5 10

<210> 87

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 87

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

1 5 10

<210> 88

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 88

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

1 5 10

<210> 89

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 89

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

1 5 10

<210> 90

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 90

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

1 5 10

<210> 91

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 91

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

1 5 10

<210> 92

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 92

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

1 5 10

<210> 93

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 93

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

1 5 10

<210> 94

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 94

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

1 5 10 15

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

20 25 30

Ala Ile Tyr Tyr Cys Ala Ala

35

<210> 95

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 95

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

1 5 10 15

Lys Ser Thr Ser Tyr Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr

20 25 30

Ala Val Tyr Phe Cys Ala Ala

35

<210> 96

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 96

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Asn Thr

35

<210> 97

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 97

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

1 5 10 15

Lys Asn Thr Leu Tyr Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr

20 25 30

Ala Leu Tyr Phe Cys Ala Arg

35

<210> 98

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 98

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Asn Val

35

<210> 99

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 99

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Thr

35

<210> 100

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 100

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

1 5 10 15

Lys Asn Thr Met Ala Leu Gln Met Asn Arg Leu Asp Pro Leu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Gly Val

35

<210> 101

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 101

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Tyr Arg

35

<210> 102

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 102

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 103

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 103

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Thr Ala

35

<210> 104

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 104

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Asn Ala

35

<210> 105

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 105

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 106

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 106

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 107

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 107

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 108

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> FR4

<400> 108

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

1 5 10

<210> 109

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 109

Gly Ser Thr Phe Ile Ile Ser Val Met Arg

1 5 10

<210> 110

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 110

Ala Ile Arg Thr Gly Gly Asn Thr Asp

fifteen

<210> 111

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 111

Pro Thr Thr Arg Tyr Gly Gly Asp Tyr Tyr Gly Pro Tyr

1 5 10

<210> 112

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 112

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Asn Val

35

<210> 113

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 113

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Asn Val

35

<210> 114

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 114

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Pro Thr Thr Arg Tyr Gly Gly Asp Tyr Tyr Gly Pro Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 115

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 115

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Pro Thr Thr Arg Tyr Gly Gly Asp Tyr Tyr Gly Pro Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 116

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 116

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 117

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 117

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 118

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 118

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Tyr Arg Arg Arg Arg Ala Ser Ser Asn Arg Gly Leu Trp Asp

100 105 110

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

115 120 125

<210> 119

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 119

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 120

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> FR1

<400> 120

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 121

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 121

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

1 5 10

<210> 122

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 122

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Asn Val

35

<210> 123

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 123

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

1 5 10 15

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

20 25 30

Ala Leu Tyr Tyr Cys Asn Val

35

<210> 124

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 124

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

1 5 10 15

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

20 25 30

Ala Leu Tyr Tyr Cys Ala Ala

35

<210> 125

<211> 2415

<212> PRT

<213> Homo sapiens

<400> 125

Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Thr Ala

1 5 10 15

Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

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

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Val Gln Ser Leu Arg Ser Asn Asp Ser

115 120 125

Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

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

275 280 285

Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Val His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu

370 375 380

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

385 390 395 400

Val Lys Pro Ile Phe Glu Val Ser Pro Ser Pro Leu Glu Pro Glu Glu

405 410 415

Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Ala Glu Val

420 425 430

Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro

435 440 445

Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln

450 455 460

Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe

465 470 475 480

His Tyr Arg Pro Gly Pro Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala

485 490 495

Gln Gln Ala Cys Pro Gly Thr Gly Ala Val Ile Ala Ser Pro Glu Gln

500 505 510

Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp

515 520 525

Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro

530 535 540

Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val

545 550 555 560

Arg Pro Ser Thr Glu Thr Tyr Asp Val Tyr Cys Phe Val Asp Arg Leu

565 570 575

Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln

580 585 590

Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Ala Thr Thr Gly

595 600 605

Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala Gly

610 615 620

Trp Leu Ala Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr Pro Arg Pro

625 630 635 640

Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro

645 650 655

Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe Cys

660 665 670

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

675 680 685

Thr Pro Thr Ser Pro Ser Gly Val Glu Glu Trp Ile Val Thr Gln Val

690 695 700

Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala Val

705 710 715 720

Pro Ser Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro Glu

725 730 735

Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Val Gly Thr Ser Pro

740 745 750

Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Ala Glu Thr Glu

755 760 765

Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Pro Ser Ala Ser Glu

770 775 780

Glu Pro Ser Pro Ser Glu Val Pro Phe Pro Ser Glu Glu Pro Ser Pro

785 790 795 800

Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu Leu

805 810 815

Phe Pro Ser Glu Glu Pro Phe Pro Ser Lys Glu Pro Ser Pro Ser Glu

820 825 830

Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Glu Pro

835 840 845

Ser Trp Thr Glu Leu Pro Ser Ser Gly Glu Glu Ser Gly Ala Pro Asp

850 855 860

Val Ser Gly Asp Phe Thr Gly Ser Gly Asp Val Ser Gly His Leu Asp

865 870 875 880

Phe Ser Gly Gln Leu Ser Gly Asp Arg Ala Ser Gly Leu Pro Ser Gly

885 890 895

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

900 905 910

Val Glu Ser Gly Leu Pro Ser Gly Asp Glu Glu Arg Ile Glu Trp Pro

915 920 925

Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu Glu

930 935 940

Gly Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu

945 950 955 960

Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro

965 970 975

Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser

980 985 990

Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu

995 1000 1005

Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala

1010 1015 1020

Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu

1025 1030 1035

Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser

1040 1045 1050

Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser

1055 1060 1065

Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val

1070 1075 1080

Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala

1085 1090 1095

Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val

1100 1105 1110

Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro

1115 1120 1125

Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile

1130 1135 1140

Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly

1145 1150 1155

Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr

1160 1165 1170

Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu

1175 1180 1185

Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu

1190 1195 1200

Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp

1205 1210 1215

Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro

1220 1225 1230

Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu

1235 1240 1245

Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly

1250 1255 1260

Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly

1265 1270 1275

Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu

1280 1285 1290

Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala

1295 1300 1305

Pro Gly Val Asp Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu

1310 1315 1320

Glu Thr Thr Ala Pro Gly Val Glu Glu Ile Ser Gly Leu Pro Ser

1325 1330 1335

Gly Glu Val Leu Glu Thr Ser Thr Ser Ala Val Gly Asp Leu Ser

1340 1345 1350

Gly Leu Pro Ser Gly Gly Glu Val Leu Glu Ile Ser Val Ser Gly

1355 1360 1365

Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Val Glu Thr

1370 1375 1380

Ser Ala Ser Gly Ile Glu Asp Val Ser Glu Leu Pro Ser Gly Glu

1385 1390 1395

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

1400 1405 1410

Pro Ser Gly Glu Glu Val Leu Glu Ile Ser Ala Ser Gly Phe Gly

1415 1420 1425

Asp Leu Ser Gly Val Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser

1430 1435 1440

Ala Ser Glu Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Arg

1445 1450 1455

Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser Gly

1460 1465 1470

Leu Pro Ser Gly Lys Glu Asp Leu Val Gly Ser Ala Ser Gly Asp

1475 1480 1485

Leu Asp Leu Gly Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln

1490 1495 1500

Ala Pro Glu Thr Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr

1505 1510 1515

Ser Gly Val Asp Leu Gly Ser Gly Pro Pro Ser Gly Leu Pro Asp

1520 1525 1530

Phe Ser Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp

1535 1540 1545

Ser Thr Leu Val Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu

1550 1555 1560

Glu Gly Arg Gly Thr Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser

1565 1570 1575

Gly Leu Pro Ser Ser Glu Leu Asp Ile Ser Gly Arg Ala Ser Gly

1580 1585 1590

Leu Pro Ser Gly Thr Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro

1595 1600 1605

Asp Val Ser Gly Glu Ile Pro Gly Leu Phe Gly Val Ser Gly Gln

1610 1615 1620

Pro Ser Gly Phe Pro Asp Thr Ser Gly Glu Thr Ser Gly Val Thr

1625 1630 1635

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

1640 1645 1650

Ala Ser Gly Val Leu Tyr Gly Thr Ser Gln Pro Phe Gly Ile Thr

1655 1660 1665

Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp Leu Ser Gly Gln

1670 1675 1680

Pro Ser Gly Leu Pro Gly Phe Ser Gly Ala Thr Ser Gly Val Pro

1685 1690 1695

Asp Leu Val Ser Gly Thr Thr Ser Gly Ser Gly Glu Ser Ser Gly

1700 1705 1710

Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Ala Pro Thr Thr

1715 1720 1725

Phe Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu

1730 1735 1740

Pro Ser Gly Glu Ala Asp Leu Ser Gly Lys Ser Gly Met Val Asp

1745 1750 1755

Val Ser Gly Gln Phe Ser Gly Thr Val Asp Ser Ser Gly Phe Thr

1760 1765 1770

Ser Gln Thr Pro Glu Phe Ser Gly Leu Pro Ser Gly Ile Ala Glu

1775 1780 1785

Val Ser Gly Glu Ser Ser Arg Ala Glu Ile Gly Ser Ser Leu Pro

1790 1795 1800

Ser Gly Ala Tyr Tyr Gly Ser Gly Thr Pro Ser Ser Phe Pro Thr

1805 1810 1815

Val Ser Leu Val Asp Arg Thr Leu Val Glu Ser Val Thr Gln Ala

1820 1825 1830

Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro Ser Gly Ile Leu Glu

1835 1840 1845

Leu Ser Gly Ala His Ser Gly Ala Pro Asp Met Ser Gly Glu His

1850 1855 1860

Ser Gly Phe Leu Asp Leu Ser Gly Leu Gln Ser Gly Leu Ile Glu

1865 1870 1875

Pro Ser Gly Glu Pro Pro Gly Thr Pro Tyr Phe Ser Gly Asp Phe

1880 1885 1890

Ala Ser Thr Thr Asn Val Ser Gly Glu Ser Ser Val Ala Met Gly

1895 1900 1905

Thr Ser Gly Glu Ala Ser Gly Leu Pro Glu Val Thr Leu Ile Thr

1910 1915 1920

Ser Glu Phe Val Glu Gly Val Thr Glu Pro Thr Ile Ser Gln Glu

1925 1930 1935

Leu Gly Gln Arg Pro Val Thr His Thr Pro Gln Leu Phe Glu

1940 1945 1950

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

1955 1960 1965

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

1970 1975 1980

Ser Ser Ser Glu Thr Ser Ala Tyr Pro Glu Ala Gly Phe Gly Ala

1985 1990 1995

Ser Ala Ala Pro Glu Ala Ser Arg Glu Asp Ser Gly Ser Pro Asp

2000 2005 2010

Leu Ser Glu Thr Thr Ser Ala Phe His Glu Ala Asn Leu Glu Arg

2015 2020 2025

Ser Ser Gly Leu Gly Val Ser Gly Ser Thr Leu Thr Phe Gln Glu

2030 2035 2040

Gly Glu Ala Ser Ala Ala Pro Glu Val Ser Gly Glu Ser Thr Thr

2045 2050 2055

Thr Ser Asp Val Gly Thr Glu Ala Pro Gly Leu Pro Ser Ala Thr

2060 2065 2070

Pro Thr Ala Ser Gly Asp Arg Thr Glu Ile Ser Gly Asp Leu Ser

2075 2080 2085

Gly His Thr Ser Gln Leu Gly Val Val Ile Ser Thr Ser Ile Pro

2090 2095 2100

Glu Ser Glu Trp Thr Gln Gln Thr Gln Arg Pro Ala Glu Thr His

2105 2110 2115

Leu Glu Ile Glu Ser Ser Ser Leu Leu Tyr Ser Gly Glu Glu Thr

2120 2125 2130

His Thr Val Glu Thr Ala Thr Ser Pro Thr Asp Ala Ser Ile Pro

2135 2140 2145

Ala Ser Pro Glu Trp Lys Arg Glu Ser Glu Ser Thr Ala Ala Ala

2150 2155 2160

Pro Ala Arg Ser Cys Ala Glu Glu Pro Cys Gly Ala Gly Thr Cys

2165 2170 2175

Lys Glu Thr Glu Gly His Val Ile Cys Leu Cys Pro Pro Gly Tyr

2180 2185 2190

Thr Gly Glu His Cys Asn Ile Asp Gln Glu Val Cys Glu Glu Gly

2195 2200 2205

Trp Asn Lys Tyr Gln Gly His Cys Tyr Arg His Phe Pro Asp Arg

2210 2215 2220

Glu Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln Gln Ser

2225 2230 2235

His Leu Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe Val Asn

2240 2245 2250

Asn Asn Ala Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp Arg Thr

2255 2260 2265

Ile Glu Gly Asp Phe Arg Trp Ser Asp Gly His Pro Met Gln Phe

2270 2275 2280

Glu Asn Trp Arg Pro Asn Gln Pro Asp Asn Phe Phe Ala Ala Gly

2285 2290 2295

Glu Asp Cys Val Val Met Ile Trp His Glu Lys Gly Glu Trp Asn

2300 2305 2310

Asp Val Pro Cys Asn Tyr His Leu Pro Phe Thr Cys Lys Lys Gly

2315 2320 2325

Thr Val Ala Cys Gly Glu Pro Pro Val Val Glu His Ala Arg Thr

2330 2335 2340

Phe Gly Gln Lys Lys Asp Arg Tyr Glu Ile Asn Ser Leu Val Arg

2345 2350 2355

Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg His Met Pro Thr Ile

2360 2365 2370

Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro Arg Ile Thr Cys

2375 2380 2385

Thr Asp Ala Thr Thr Tyr Lys Arg Arg Leu Gln Lys Arg Ser Ser

2390 2395 2400

Arg His Pro Arg Arg Ser Arg Pro Ser Thr Ala His

2405 2410 2415

<210> 126

<211> 2333

<212> PRT

<213> Canis lupus

<400> 126

Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Thr Ala

1 5 10 15

Ala Ser Ser Glu Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

Ile Pro Glu Pro Ser Pro Met Arg Val Leu Leu Gly Ser Ser Leu Thr

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Ile Thr Lys

65 70 75 80

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

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser

115 120 125

Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Leu

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu

275 280 285

Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu

370 375 380

Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Asn Val Ile Leu Thr

385 390 395 400

Val Lys Pro Ile Phe Asp Leu Ser Pro Thr Ala Pro Glu Pro Glu Glu

405 410 415

Pro Phe Thr Phe Val Pro Glu Pro Glu Lys Pro Phe Thr Phe Ala Thr

420 425 430

Asp Val Gly Val Thr Ala Phe Pro Glu Ala Glu Asn Arg Thr Gly Glu

435 440 445

Ala Thr Arg Pro Trp Gly Val Pro Glu Glu Ser Thr Pro Gly Pro Ala

450 455 460

Phe Thr Ala Phe Thr Ser Glu Asp His Val Val Gln Val Thr Ala Val

465 470 475 480

Pro Gly Ala Ala Glu Val Pro Gly Gln Pro Arg Leu Pro Gly Gly Val

485 490 495

Val Phe His Tyr Arg Pro Gly Ser Ala Arg Tyr Ser Leu Thr Phe Glu

500 505 510

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

515 520 525

Glu Gln Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala

530 535 540

Gly Trp Leu Gln Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg

545 550 555 560

Thr Pro Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr

565 570 575

Gly Val Arg Pro Ser Glu Thr Tyr Asp Val Tyr Cys Tyr Val Asp

580 585 590

Lys Leu Glu Gly Glu Val Phe Phe Ile Thr Arg Leu Glu Gln Phe Thr

595 600 605

Phe Gln Glu Ala Leu Ala Phe Cys Glu Ser His Asn Ala Thr Leu Ala

610 615 620

Ser Thr Gly Gln Leu Tyr Ala Ala Trp Arg Gln Gly Leu Asp Lys Cys

625 630 635 640

Tyr Ala Gly Trp Leu Ser Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr

645 650 655

Pro Arg Pro Ser Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr

660 665 670

Leu Tyr Pro Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His

675 680 685

Val Phe Cys Phe Arg Gly Val Ser Gly Val Pro Ser Pro Gly Glu Glu

690 695 700

Glu Gly Gly Thr Pro Thr Pro Ser Val Val Glu Asp Trp Ile Pro Thr

705 710 715 720

Gln Val Gly Pro Val Val Pro Ser Val Pro Met Gly Glu Glu Thr Thr

725 730 735

Ala Ile Leu Asp Phe Thr Ile Glu Pro Glu Asn Gln Thr Glu Trp Glu

740 745 750

Pro Ala Tyr Ser Pro Ala Gly Thr Ser Pro Leu Pro Gly Ile Pro Pro

755 760 765

Thr Trp Pro Pro Thr Ser Thr Ala Thr Glu Glu Ser Thr Glu Gly Pro

770 775 780

Ser Gly Thr Glu Val Pro Ser Val Ser Glu Glu Pro Ser Pro Ser Glu

785 790 795 800

Glu Pro Phe Pro Trp Glu Glu Leu Ser Thr Leu Ser Pro Pro Gly Pro

805 810 815

Ser Gly Thr Glu Leu Pro Gly Ser Gly Glu Ala Ser Gly Val Pro Glu

820 825 830

Val Ser Gly Asp Phe Thr Gly Ser Gly Glu Val Ser Gly His Pro Asp

835 840 845

Ser Ser Gly Gln Leu Ser Gly Glu Ser Ala Ser Gly Leu Pro Ser Glu

850 855 860

Asp Leu Asp Ser Ser Gly Leu Thr Ser Ala Val Gly Ser Gly Leu Ala

865 870 875 880

Ser Gly Asp Glu Asp Arg Ile Thr Leu Ser Ser Ile Pro Lys Val Glu

885 890 895

Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Leu Ser Gly

900 905 910

Leu Pro Ser Gly Arg Glu Gly Leu Glu Thr Ser Thr Ser Gly Val Gly

915 920 925

Asp Leu Ser Gly Leu Pro Ser Gly Glu Gly Leu Glu Val Ser Ala Ser

930 935 940

Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Gly Pro Glu Thr

945 950 955 960

Ser Thr Ser Gly Val Gly Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly

965 970 975

Pro Glu Val Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser

980 985 990

Gly Arg Glu Gly Leu Glu Thr Ser Thr Ser Gly Val Glu Asp Leu Ser

995 1000 1005

Gly Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Thr Ser Gly Val

1010 1015 1020

Gly Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val Ser

1025 1030 1035

Ala Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Gly

1040 1045 1050

Leu Glu Ala Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro

1055 1060 1065

Ser Gly Glu Gly Pro Glu Ala Ser Ala Ser Gly Val Gly Asp Leu

1070 1075 1080

Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser Gly

1085 1090 1095

Val Glu Asp Leu Ser Gly Leu Ser Ser Gly Glu Ser Pro Glu Ala

1100 1105 1110

Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Arg

1115 1120 1125

Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly

1130 1135 1140

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

1145 1150 1155

Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Ala

1160 1165 1170

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

1175 1180 1185

Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro

1190 1195 1200

Ser Gly Glu Gly Pro Glu Ala Phe Ala Ser Gly Val Glu Asp Leu

1205 1210 1215

Ser Ile Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Ala Ser Gly

1220 1225 1230

Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu

1235 1240 1245

Thr Ser Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly

1250 1255 1260

Arg Glu Gly Leu Glu Thr Ser Thr Ser Gly Val Gly Asp Leu Ser

1265 1270 1275

Gly Leu Pro Ser Gly Glu Gly Pro Glu Ala Ser Ala Ser Gly Ile

1280 1285 1290

Gly Asp Ile Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu Thr

1295 1300 1305

Ser Ser Ser Gly Val Glu Asp His Pro Glu Thr Ser Ala Ser Gly

1310 1315 1320

Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Val Glu Gly His Pro

1325 1330 1335

Glu Thr Ser Ala Ser Gly Val Glu Asp Leu Ser Asp Leu Ser Ser

1340 1345 1350

Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu

1355 1360 1365

Ser Gly Phe Pro Ser Gly Lys Glu Asp Leu Ile Gly Ser Ala Ser

1370 1375 1380

Gly Ala Leu Asp Phe Gly Arg Ile Pro Ser Gly Thr Leu Gly Ser

1385 1390 1395

Gly Gln Ala Pro Glu Ala Ser Ser Leu Pro Ser Gly Phe Ser Gly

1400 1405 1410

Glu Tyr Ser Gly Val Asp Phe Gly Ser Gly Pro Ile Ser Gly Leu

1415 1420 1425

Pro Asp Phe Ser Gly Leu Pro Ser Gly Phe Pro Thr Ile Ser Leu

1430 1435 1440

Val Asp Thr Thr Leu Val Glu Val Ile Thr Thr Thr Ser Ala Ser

1445 1450 1455

Glu Leu Glu Gly Arg Gly Thr Ile Gly Ile Ser Gly Ala Gly Glu

1460 1465 1470

Thr Ser Gly Leu Pro Val Ser Glu Leu Asp Ile Ser Gly Ala Val

1475 1480 1485

Ser Gly Leu Pro Ser Gly Ala Glu Leu Ser Gly Gln Ala Ser Gly

1490 1495 1500

Ser Pro Asp Met Ser Gly Glu Thr Ser Gly Phe Phe Gly Val Ser

1505 1510 1515

Gly Gln Pro Ser Gly Phe Pro Asp Ile Ser Gly Gly Thr Ser Gly

1520 1525 1530

Leu Phe Glu Val Ser Gly Gln Pro Ser Gly Phe Ser Gly Glu Thr

1535 1540 1545

Ser Gly Val Thr Glu Leu Ser Gly Leu Tyr Ser Gly Gln Pro Asp

1550 1555 1560

Val Ser Gly Glu Ala Ser Gly Val Pro Ser Gly Ser Gly Gln Pro

1565 1570 1575

Phe Gly Met Thr Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp

1580 1585 1590

Ile Ser Gly Gln Pro Ser Gly Leu Pro Glu Phe Ser Gly Thr Thr

1595 1600 1605

Ser Gly Ile Pro Asp Leu Val Ser Ser Thr Met Ser Gly Ser Gly

1610 1615 1620

Glu Ser Ser Gly Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val

1625 1630 1635

Thr Pro Thr Thr Phe Lys Glu Lys Lys Arg Leu Gly Ser Val Glu

1640 1645 1650

Leu Ser Gly Leu Pro Ser Gly Glu Val Asp Leu Ser Gly Ala Ser

1655 1660 1665

Gly Thr Met Asp Ile Ser Gly Gln Ser Ser Gly Ala Thr Asp Ser

1670 1675 1680

Ser Gly Leu Thr Ser His Leu Pro Lys Phe Ser Gly Leu Pro Ser

1685 1690 1695

Gly Ala Ala Glu Val Ser Gly Glu Ser Ser Gly Ala Glu Val Gly

1700 1705 1710

Ser Ser Leu Pro Ser Gly Thr Tyr Glu Gly Ser Gly Asn Phe His

1715 1720 1725

Pro Ala Phe Pro Thr Val Phe Leu Val Asp Arg Thr Leu Val Glu

1730 1735 1740

Ser Val Thr Gln Ala Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro

1745 1750 1755

Ser Gly Ile Leu Glu Leu Ser Gly Ala His Ser Gly Ala Pro Asp

1760 1765 1770

Val Ser Gly Asp His Ser Gly Ser Leu Asp Leu Ser Gly Met Gln

1775 1780 1785

Ser Gly Leu Val Glu Pro Ser Gly Glu Pro Ser Ser Thr Pro Tyr

1790 1795 1800

Phe Ser Gly Asp Phe Ser Gly Thr Met Asp Val Thr Gly Glu Pro

1805 1810 1815

Ser Thr Ala Met Ser Ala Ser Gly Glu Ala Ser Gly Leu Leu Glu

1820 1825 1830

Val Thr Leu Ile Thr Ser Glu Phe Val Glu Gly Val Thr Glu Pro

1835 1840 1845

Thr Val Ser Gln Glu Leu Ala Gln Arg Pro Pro Val Thr His Thr

1850 1855 1860

Pro Gln Leu Phe Glu Ser Ser Gly Glu Ala Ser Ala Ser Gly Glu

1865 1870 1875

Ile Ser Gly Ala Thr Pro Ala Phe Pro Gly Ser Gly Leu Glu Ala

1880 1885 1890

Ser Ser Val Pro Glu Ser Ser Ser Glu Thr Ser Asp Phe Pro Glu

1895 1900 1905

Arg Ala Val Gly Val Ser Ala Ala Pro Glu Ala Ser Gly Gly Ala

1910 1915 1920

Ser Gly Ala Pro Asp Val Ser Glu Ala Thr Ser Thr Phe Pro Glu

1925 1930 1935

Ala Asp Val Glu Gly Ala Ser Gly Leu Gly Val Ser Gly Gly Thr

1940 1945 1950

Ser Ala Phe Pro Glu Ala Pro Arg Glu Gly Ser Ala Thr Pro Glu

1955 1960 1965

Val Gln Glu Glu Pro Thr Thr Ser Tyr Asp Val Gly Arg Glu Ala

1970 1975 1980

Leu Gly Trp Pro Ser Ala Thr Pro Thr Ala Ser Gly Asp Arg Ile

1985 1990 1995

Glu Val Ser Gly Asp Leu Ser Gly His Thr Ser Gly Leu Asp Val

2000 2005 2010

Val Ile Ser Thr Ser Val Pro Glu Ser Glu Trp Ile Gln Gln Thr

2015 2020 2025

Gln Arg Pro Ala Glu Ala His Leu Glu Ile Glu Ala Ser Ser Pro

2030 2035 2040

Leu His Ser Gly Glu Glu Thr Gln Thr Ala Glu Thr Ala Thr Ser

2045 2050 2055

Pro Thr Asp Asp Ala Ser Ile Pro Thr Ser Pro Ser Gly Thr Asp

2060 2065 2070

Glu Ser Ala Pro Ala Ile Pro Asp Ile Asp Glu Cys Leu Ser Ser

2075 2080 2085

Pro Cys Leu Asn Gly Ala Thr Cys Val Asp Ala Ile Asp Ser Phe

2090 2095 2100

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

2105 2110 2115

Asp Gln Glu Leu Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly His

2120 2125 2130

Cys Tyr Arg Tyr Phe Pro Asp Arg Glu Ser Trp Val Asp Ala Glu

2135 2140 2145

Ser Arg Cys Arg Ala Gln Gln Ser His Leu Ser Ser Ser Ile Val Thr

2150 2155 2160

Pro Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr Gln

2165 2170 2175

Trp Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg Trp

2180 2185 2190

Ser Asp Gly His Ser Leu Gln Phe Glu Asn Trp Arg Pro Asn Gln

2195 2200 2205

Pro Asp Asn Phe Phe Val Ser Gly Glu Asp Cys Val Val Met Ile

2210 2215 2220

Trp His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr Tyr

2225 2230 2235

Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Asp Pro

2240 2245 2250

Pro Val Val Glu His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg

2255 2260 2265

Tyr Glu Ile Asn Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly Phe

2270 2275 2280

Val Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Gly His

2285 2290 2295

Trp Glu Lys Pro Arg Ile Thr Cys Thr Asp Pro Ser Thr Tyr Lys

2300 2305 2310

Arg Arg Leu Gln Lys Arg Ser Ser Arg Ala Pro Arg Arg Ser Arg

2315 2320 2325

Pro Ser Thr Ala His

2330

<210> 127

<211> 2364

<212> PRT

<213> Bos taurus

<400> 127

Met Thr Thr Leu Leu Leu Val Phe Val Thr Leu Arg Val Ile Thr Ala

1 5 10 15

Ala Ile Ser Val Glu Val Ser Glu Pro Asp Asn Ser Leu Ser Val Ser

20 25 30

Ile Pro Glu Pro Ser Pro Leu Arg Val Leu Leu Gly Ser Ser Leu Thr

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Ile Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Thr Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Met Arg Ser Asn Asp Ser

115 120 125

Gly Ile Leu Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Gln Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu

275 280 285

Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Ser Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu

370 375 380

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

385 390 395 400

Ala Lys Pro Asp Phe Glu Val Ser Pro Thr Ala Pro Glu Pro Glu Glu

405 410 415

Pro Phe Thr Phe Val Pro Glu Val Arg Ala Thr Ala Phe Pro Glu Val

420 425 430

Glu Asn Arg Thr Glu Glu Ala Thr Arg Pro Trp Ala Phe Pro Arg Glu

435 440 445

Ser Thr Pro Gly Leu Gly Ala Pro Thr Ala Phe Thr Ser Glu Asp Leu

450 455 460

Val Val Gln Val Thr Leu Ala Pro Gly Ala Ala Glu Val Pro Gly Gln

465 470 475 480

Pro Arg Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Ser Ser

485 490 495

Arg Tyr Ser Leu Thr Phe Glu Glu Ala Lys Gln Ala Cys Leu Arg Thr

500 505 510

Gly Ala Ile Ile Ala Ser Pro Glu Gln Leu Gln Ala Ala Tyr Glu Ala

515 520 525

Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg

530 535 540

Tyr Pro Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser

545 550 555 560

Ser Pro Gly Val Arg Thr Tyr Gly Val Arg Pro Ser Glu Thr Tyr

565 570 575

Asp Val Tyr Cys Tyr Val Asp Arg Leu Glu Gly Glu Val Phe Phe Ala

580 585 590

Thr Arg Leu Glu Gln Phe Thr Phe Trp Glu Ala Gln Glu Phe Cys Glu

595 600 605

Ser Gln Asn Ala Thr Leu Ala Thr Thr Gly Gln Leu Tyr Ala Ala Trp

610 615 620

Ser Arg Gly Leu Asp Lys Cys Tyr Ala Gly Trp Leu Ala Asp Gly Ser

625 630 635 640

Leu Arg Tyr Pro Ile Val Thr Pro Arg Pro Ala Cys Gly Gly Asp Lys

645 650 655

Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Leu

660 665 670

Asp Pro Leu Ser Arg His His Ala Phe Cys Phe Arg Gly Val Ser Ala

675 680 685

Ala Pro Ser Pro Glu Glu Glu Glu Gly Ser Ala Pro Thr Ala Gly Pro

690 695 700

Asp Val Glu Glu Trp Met Val Thr Gln Val Gly Pro Gly Val Ala Ala

705 710 715 720

Val Pro Ile Gly Glu Glu Thr Thr Ala Ile Pro Gly Phe Thr Val Glu

725 730 735

Pro Glu Asn Lys Thr Glu Trp Glu Leu Ala Tyr Thr Pro Ala Gly Thr

740 745 750

Leu Pro Leu Pro Gly Ile Pro Thr Trp Pro Pro Thr Gly Glu Ala

755 760 765

Thr Glu Glu His Thr Glu Gly Pro Ser Ala Thr Glu Val Pro Ser Ala

770 775 780

Ser Glu Lys Pro Phe Pro Ser Glu Glu Pro Phe Pro Pro Glu Glu Pro

785 790 795 800

Phe Pro Ser Glu Lys Pro Phe Pro Pro Glu Glu Leu Phe Pro Ser Glu

805 810 815

Lys Pro Phe Pro Ser Glu Lys Pro Phe Pro Ser Glu Glu Pro Phe Pro

820 825 830

Ser Glu Lys Pro Phe Pro Pro Glu Glu Leu Phe Pro Ser Glu Lys Pro

835 840 845

Ile Pro Ser Glu Glu Pro Phe Pro Ser Glu Glu Pro Phe Pro Ser Glu

850 855 860

Lys Pro Phe Pro Pro Glu Glu Pro Phe Pro Ser Glu Lys Pro Ile Pro

865 870 875 880

Ser Glu Glu Pro Phe Pro Ser Glu Lys Pro Phe Pro Ser Glu Glu Pro

885 890 895

Phe Pro Ser Glu Glu Pro Ser Thr Leu Ser Ala Pro Val Pro Ser Arg

900 905 910

Thr Glu Leu Pro Ser Ser Gly Glu Val Ser Gly Val Pro Glu Ile Ser

915 920 925

Gly Asp Phe Thr Gly Ser Gly Glu Ile Ser Gly His Leu Asp Phe Ser

930 935 940

Gly Gln Pro Ser Gly Glu Ser Ala Ser Gly Leu Pro Ser Glu Asp Leu

945 950 955 960

Asp Ser Ser Gly Leu Thr Ser Thr Val Gly Ser Gly Leu Pro Val Glu

965 970 975

Ser Gly Leu Pro Ser Gly Glu Glu Glu Arg Ile Thr Trp Thr Ser Ala

980 985 990

Pro Lys Val Asp Arg Leu Pro Ser Gly Gly Glu Gly Pro Glu Val Ser

995 1000 1005

Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Gly Glu Val His

1010 1015 1020

Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Ile Ser Gly Leu Pro

1025 1030 1035

Ser Gly Gly Glu Val His Leu Glu Ile Ser Ala Ser Gly Val Glu

1040 1045 1050

Asp Leu Ser Arg Ile Pro Ser Gly Glu Gly Pro Glu Ile Ser Ala

1055 1060 1065

Ser Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Glu Gly

1070 1075 1080

His Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Leu Ser Gly Ile

1085 1090 1095

Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser Gly Val Glu Asp

1100 1105 1110

Leu Ile Gly Leu Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser

1115 1120 1125

Gly Val Glu Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu

1130 1135 1140

Val Ser Ala Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly

1145 1150 1155

Glu Gly Pro Glu Val Ser Val Ser Gly Val Glu Asp Leu Ser Arg

1160 1165 1170

Leu Pro Ser Gly Glu Gly Pro Glu Val Ser Ala Ser Gly Val Glu

1175 1180 1185

Asp Leu Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Ile Ser Val

1190 1195 1200

Ser Gly Val Glu Asp Ile Ser Ile Leu Pro Ser Gly Glu Gly Pro

1205 1210 1215

Glu Val Ser Ala Ser Gly Val Glu Asp Leu Ser Val Leu Pro Ser

1220 1225 1230

Gly Glu Gly His Leu Glu Ile Ser Thr Ser Gly Val Glu Asp Leu

1235 1240 1245

Ser Val Leu Pro Ser Gly Glu Gly His Leu Glu Thr Ser Ser Gly

1250 1255 1260

Val Glu Asp Ile Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val

1265 1270 1275

Ser Ala Ser Gly Val Glu Asp Leu Ser Val Leu Pro Ser Gly Glu

1280 1285 1290

Asp His Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Leu Gly Val

1295 1300 1305

Leu Pro Ser Gly Glu Asp His Leu Glu Ile Ser Ala Ser Gly Val

1310 1315 1320

Glu Asp Ile Ser Arg Leu Pro Ser Gly Glu Gly Pro Glu Val Ser

1325 1330 1335

Ala Ser Gly Val Glu Asp Leu Ser Val Leu Pro Ser Gly Glu Gly

1340 1345 1350

His Leu Glu Ile Ser Ala Ser Gly Val Glu Asp Leu Ser Arg Leu

1355 1360 1365

Pro Ser Gly Gly Glu Asp His Leu Glu Thr Ser Ala Ser Gly Val

1370 1375 1380

Gly Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu Ile

1385 1390 1395

Ser Ala Ser Gly Ala Gly Asp Leu Ser Gly Leu Thr Ser Gly Lys

1400 1405 1410

Glu Asp Leu Thr Gly Ser Ala Ser Gly Ala Leu Asp Leu Gly Arg

1415 1420 1425

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

1430 1435 1440

Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly Val Asp Leu

1445 1450 1455

Glu Ser Gly Pro Ser Ser Gly Leu Pro Asp Phe Ser Gly Leu Pro

1460 1465 1470

Ser Gly Phe Pro Thr Val Ser Leu Val Asp Thr Thr Leu Val Glu

1475 1480 1485

Val Val Thr Ala Thr Thr Ala Gly Glu Leu Glu Gly Arg Gly Thr

1490 1495 1500

Ile Asp Ile Ser Gly Ala Gly Glu Thr Ser Gly Leu Pro Phe Ser

1505 1510 1515

Glu Leu Asp Ile Ser Gly Gly Ala Ser Gly Leu Ser Ser Gly Ala

1520 1525 1530

Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro Asp Ile Ser Gly Glu

1535 1540 1545

Thr Ser Gly Leu Phe Gly Val Ser Gly Gln Pro Ser Gly Phe Pro

1550 1555 1560

Asp Ile Ser Gly Glu Thr Ser Gly Leu Leu Glu Val Ser Gly Gln

1565 1570 1575

Pro Ser Gly Phe Tyr Gly Glu Ile Ser Gly Val Thr Glu Leu Ser

1580 1585 1590

Gly Leu Ala Ser Gly Gln Pro Glu Ile Ser Gly Glu Ala Ser Gly

1595 1600 1605

Ile Leu Ser Gly Leu Gly Pro Pro Phe Gly Ile Thr Asp Leu Ser

1610 1615 1620

Gly Glu Ala Pro Gly Ile Pro Asp Leu Ser Gly Gln Pro Ser Gly

1625 1630 1635

Leu Pro Glu Phe Ser Gly Thr Ala Ser Gly Ile Pro Asp Leu Val

1640 1645 1650

Ser Ser Ala Val Ser Gly Ser Gly Glu Ser Ser Gly Ile Thr Phe

1655 1660 1665

Val Asp Thr Ser Leu Val Glu Val Thr Pro Thr Thr Phe Lys Glu

1670 1675 1680

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

1685 1690 1695

Glu Leu Gly Val Ser Gly Thr Ser Gly Leu Ala Asp Val Ser Gly

1700 1705 1710

Leu Ser Ser Gly Ala Ile Asp Ser Ser Gly Phe Thr Ser Gln Pro

1715 1720 1725

Pro Glu Phe Ser Gly Leu Pro Ser Gly Val Thr Glu Val Ser Gly

1730 1735 1740

Glu Ala Ser Gly Ala Glu Ser Gly Ser Ser Leu Pro Ser Gly Ala

1745 1750 1755

Tyr Asp Ser Ser Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Phe

1760 1765 1770

Val Asp Arg Thr Leu Val Glu Ser Val Thr Gln Ala Pro Thr Ala

1775 1780 1785

Gln Glu Ala Gly Glu Gly Pro Ser Gly Ile Leu Glu Leu Ser Gly

1790 1795 1800

Ala Pro Ser Gly Ala Pro Asp Met Ser Gly Asp His Leu Gly Ser

1805 1810 1815

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

1820 1825 1830

Glu Pro Ala Ser Thr Pro Tyr Phe Ser Gly Asp Phe Ser Gly Thr

1835 1840 1845

Thr Asp Val Ser Gly Glu Ser Ser Ala Ala Thr Ser Thr Ser Gly

1850 1855 1860

Glu Ala Ser Gly Leu Pro Glu Val Thr Leu Ile Thr Ser Glu Leu

1865 1870 1875

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

1880 1885 1890

Arg Pro Pro Val Thr Tyr Thr Pro Gln Leu Phe Glu Ser Ser Gly

1895 1900 1905

Glu Ala Ser Ala Ser Gly Asp Val Pro Arg Phe Pro Gly Ser Gly

1910 1915 1920

Val Glu Val Ser Ser Val Pro Glu Ser Ser Gly Glu Thr Ser Ala

1925 1930 1935

Tyr Pro Glu Ala Glu Val Gly Ala Ser Ala Ala Pro Glu Ala Ser

1940 1945 1950

Gly Gly Ala Ser Gly Ser Pro Asn Leu Ser Glu Thr Thr Ser Thr

1955 1960 1965

Phe His Glu Ala Asp Leu Glu Gly Thr Ser Gly Leu Gly Val Ser

1970 1975 1980

Gly Ser Pro Ser Ala Phe Pro Glu Gly Pro Thr Glu Gly Leu Ala

1985 1990 1995

Thr Pro Glu Val Ser Gly Glu Ser Thr Thr Ala Phe Asp Val Ser

2000 2005 2010

Val Glu Ala Ser Gly Ser Pro Ser Ala Thr Pro Leu Ala Ser Gly

2015 2020 2025

Asp Arg Thr Asp Thr Ser Gly Asp Leu Ser Gly His Thr Ser Gly

2030 2035 2040

Leu Asp Ile Val Ile Ser Thr Thr Ile Pro Glu Ser Glu Trp Thr

2045 2050 2055

Gln Gln Thr Gln Arg Pro Ala Glu Ala Arg Leu Glu Ile Glu Ser

2060 2065 2070

Ser Ser Pro Val His Ser Gly Glu Glu Ser Gln Thr Ala Asp Thr

2075 2080 2085

Ala Thr Ser Pro Thr Asp Ala Ser Ile Pro Ala Ser Ala Gly Gly

2090 2095 2100

Thr Asp Asp Ser Glu Ala Thr Thr Thr Asp Ile Asp Glu Cys Leu

2105 2110 2115

Ser Ser Pro Cys Leu Asn Gly Ala Thr Cys Val Asp Ala Ile Asp

2120 2125 2130

Ser Phe Thr Cys Leu Cys Leu Pro Ser Tyr Gln Gly Asp Val Cys

2135 2140 2145

Glu Ile Gln Lys Leu Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly

2150 2155 2160

His Cys Tyr Arg His Phe Pro Asp Arg Ala Thr Trp Val Asp Ala

2165 2170 2175

Glu Ser Gln Cys Arg Lys Gln Gln Ser His Leu Ser Ser Ile Val

2180 2185 2190

Thr Pro Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr

2195 2200 2205

Gln Trp Ile Gly Leu Asn Asp Lys Thr Ile Glu Gly Asp Phe Arg

2210 2215 2220

Trp Ser Asp Gly His Ser Leu Gln Phe Glu Asn Trp Arg Pro Asn

2225 2230 2235

Gln Pro Asp Asn Phe Phe Ala Thr Gly Glu Asp Cys Val Val Met

2240 2245 2250

Ile Trp His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr

2255 2260 2265

Gln Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu

2270 2275 2280

Pro Pro Val Val Glu His Ala Arg Ile Phe Gly Gln Lys Lys Asp

2285 2290 2295

Arg Tyr Glu Ile Asn Ala Leu Val Arg Tyr Gln Cys Thr Glu Gly

2300 2305 2310

Phe Ile Gln Gly His Val Pro Thr Ile Arg Cys Gln Pro Ser Gly

2315 2320 2325

His Trp Glu Glu Pro Arg Ile Thr Cys Thr Asp Pro Ala Thr Tyr

2330 2335 2340

Lys Arg Arg Leu Gln Lys Arg Ser Ser Arg Pro Leu Arg Arg Ser

2345 2350 2355

His Pro Ser Thr Ala His

2360

<210> 128

<211> 2124

<212> PRT

<213> Rattus norvegicus

<400> 128

Met Thr Thr Leu Leu Leu Val Phe Val Thr Leu Arg Val Ile Ala Ala

1 5 10 15

Val Ile Ser Glu Glu Val Pro Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

Ile Pro Gln Pro Ser Pro Leu Lys Ala Leu Leu Gly Thr Ser Leu Thr

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Thr Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gln Val Arg Val

85 90 95

Asn Ser Ile Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser

115 120 125

Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Ile Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

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

275 280 285

Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Leu Glu Leu Pro Leu

370 375 380

Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Asn Val Ile Leu Thr

385 390 395 400

Ala Lys Pro Ile Phe Asp Met Ser Pro Thr Val Ser Glu Pro Gly Glu

405 410 415

Ala Leu Thr Leu Ala Pro Glu Val Gly Thr Thr Val Phe Pro Glu Ala

420 425 430

Gly Glu Arg Thr Glu Lys Thr Thr Arg Pro Trp Gly Phe Pro Glu Glu

435 440 445

Ala Thr Arg Gly Pro Asp Ser Ala Thr Ala Phe Ala Ser Glu Asp Leu

450 455 460

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

465 470 475 480

Pro Arg Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Ser Thr

485 490 495

Arg Tyr Ser Leu Thr Phe Glu Glu Ala Gln Gln Ala Cys Ile Arg Thr

500 505 510

Gly Ala Ala Ile Ala Ser Pro Glu Gln Leu Gln Ala Ala Tyr Glu Ala

515 520 525

Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg

530 535 540

Tyr Pro Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser

545 550 555 560

Ser Pro Gly Val Arg Thr Tyr Gly Val Arg Pro Ser Ser Glu Thr Tyr

565 570 575

Asp Val Tyr Cys Tyr Val Asp Lys Leu Glu Gly Glu Val Phe Phe Ala

580 585 590

Thr Gln Met Glu Gln Phe Thr Phe Gln Glu Ala Gln Ala Phe Cys Ala

595 600 605

Ala Gln Asn Ala Thr Leu Ala Ser Thr Gly Gln Leu Tyr Ala Ala Trp

610 615 620

Ser Gln Gly Leu Asp Lys Cys Tyr Ala Gly Trp Leu Ala Asp Gly Thr

625 630 635 640

Leu Arg Tyr Pro Ile Val Asn Pro Arg Pro Ala Cys Gly Gly Asp Lys

645 650 655

Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Pro

660 665 670

Asp Pro Leu Ser Lys His His Ala Phe Cys Phe Arg Gly Val Ser Val

675 680 685

Val Pro Ser Pro Gly Gly Thr Pro Thr Ser Pro Ser Asp Ile Glu Asp

690 695 700

Trp Ile Val Thr Arg Val Glu Pro Gly Val Asp Ala Val Pro Leu Glu

705 710 715 720

Pro Glu Thr Thr Glu Val Pro Tyr Phe Thr Thr Glu Pro Glu Lys Gln

725 730 735

Thr Glu Trp Glu Pro Ala Tyr Thr Pro Val Gly Thr Ser Pro Leu Pro

740 745 750

Gly Ile Pro Pro Thr Trp Leu Pro Thr Val Pro Ala Ala Glu Glu His

755 760 765

Thr Glu Ser Pro Ser Ala Ser Gln Glu Pro Ser Ala Ser Gln Val Pro

770 775 780

Ser Thr Ser Glu Glu Pro Tyr Thr Pro Ser Leu Ala Val Pro Ser Gly

785 790 795 800

Thr Glu Leu Pro Ser Ser Gly Asp Thr Ser Gly Ala Pro Asp Leu Ser

805 810 815

Gly Asp Phe Thr Gly Ser Thr Asp Thr Ser Gly Arg Leu Asp Ser Ser

820 825 830

Gly Glu Pro Ser Gly Gly Ser Glu Ser Gly Leu Pro Ser Gly Asp Leu

835 840 845

Asp Ser Ser Gly Leu Gly Pro Thr Val Ser Ser Gly Leu Pro Val Glu

850 855 860

Ser Gly Ser Ala Ser Gly Asp Gly Glu Ile Pro Trp Ser Ser Thr Pro

865 870 875 880

Thr Val Asp Arg Leu Pro Ser Gly Gly Glu Ser Leu Glu Gly Ser Ala

885 890 895

Ser Ala Ser Gly Thr Gly Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu

900 905 910

Ile Thr Glu Thr Ser Ala Ser Gly Thr Glu Glu Ile Ser Gly Leu Pro

915 920 925

Ser Gly Gly Asp Asp Leu Glu Thr Ser Thr Ser Gly Ile Asp Gly Ala

930 935 940

Ser Val Leu Pro Thr Gly Arg Gly Gly Leu Glu Thr Ser Ala Ser Gly

945 950 955 960

Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Glu Gly Ser Glu Thr

965 970 975

Ser Thr Ser Gly Ile Glu Asp Ile Ser Val Leu Pro Thr Gly Glu Ser

980 985 990

Pro Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser

995 1000 1005

Gly Gly Glu Ser Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Val

1010 1015 1020

Thr Gln Leu Pro Thr Glu Arg Gly Gly Leu Glu Thr Ser Ala Ser

1025 1030 1035

Gly Ile Glu Asp Ile Thr Val Leu Pro Thr Gly Arg Glu Asn Leu

1040 1045 1050

Glu Thr Ser Ala Ser Gly Val Glu Asp Val Ser Gly Leu Pro Ser

1055 1060 1065

Gly Lys Glu Gly Leu Glu Thr Ser Ala Ser Gly Ile Glu Asp Ile

1070 1075 1080

Ser Val Phe Pro Thr Glu Ala Glu Gly Leu Glu Thr Ser Ala Ser

1085 1090 1095

Gly Gly Tyr Val Ser Gly Ile Pro Ser Gly Glu Asp Gly Thr Glu

1100 1105 1110

Thr Ser Thr Ser Gly Val Glu Gly Val Ser Gly Leu Pro Ser Gly

1115 1120 1125

Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Glu Asp Leu Gly

1130 1135 1140

Leu Pro Thr Arg Asp Ser Leu Glu Thr Ser Ala Ser Gly Val Asp

1145 1150 1155

Val Thr Gly Tyr Pro Ser Gly Arg Glu Asp Thr Glu Thr Ser Val

1160 1165 1170

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

1175 1180 1185

Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu Gly Gly Leu

1190 1195 1200

Pro Ser Gly Lys Glu Asp Leu Val Gly Ser Ala Ser Gly Ala Leu

1205 1210 1215

Asp Phe Gly Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln Thr

1220 1225 1230

Pro Glu Ala Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser

1235 1240 1245

Gly Val Asp Ile Gly Ser Gly Pro Ser Ser Gly Leu Pro Asp Phe

1250 1255 1260

Ser Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser

1265 1270 1275

Thr Leu Val Glu Val Ile Thr Ala Thr Thr Ala Ser Glu Leu Glu

1280 1285 1290

Gly Arg Gly Thr Ile Ser Val Ser Gly Ser Gly Glu Glu Ser Gly

1295 1300 1305

Pro Pro Leu Ser Glu Leu Asp Ser Ser Ala Asp Ile Ser Gly Leu

1310 1315 1320

Pro Ser Gly Thr Glu Leu Ser Gly Gln Thr Ser Gly Ser Leu Asp

1325 1330 1335

Val Ser Gly Glu Thr Ser Gly Phe Phe Asp Val Ser Gly Gln Pro

1340 1345 1350

Phe Gly Ser Ser Gly Thr Gly Glu Gly Thr Ser Gly Ile Pro Glu

1355 1360 1365

Val Ser Gly Gln Ala Val Arg Ser Pro Asp Thr Thr Glu Ile Ser

1370 1375 1380

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

1385 1390 1395

Gly Ser Gly Ile Leu Phe Gly Ser Gly Gln Ser Ser Gly Ile Thr

1400 1405 1410

Ser Val Ser Gly Glu Thr Ser Gly Ile Ser Asp Leu Ser Gly Gln

1415 1420 1425

Pro Ser Gly Phe Pro Val Leu Ser Gly Thr Thr Pro Gly Thr Pro

1430 1435 1440

Asp Leu Ala Ser Gly Ala Met Ser Gly Ser Gly Asp Ser Ser Gly

1445 1450 1455

Ile Thr Phe Val Asp Thr Ser Leu Ile Glu Val Thr Pro Thr Thr

1460 1465 1470

Phe Arg Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu

1475 1480 1485

Pro Ser Gly Glu Thr Asp Leu Ser Gly Thr Ser Gly Met Val Asp

1490 1495 1500

Val Ser Gly Glyn Ser Ser Gly Ala Ile Asp Ser Ser Gly Leu Ile

1505 1510 1515

Ser Pro Thr Pro Glu Phe Ser Gly Leu Pro Ser Gly Val Ala Glu

1520 1525 1530

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

1535 1540 1545

Ser Gly Ala Phe Asp Gly Ser Gly Leu Val Ser Gly Phe Pro Thr

1550 1555 1560

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

1565 1570 1575

Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro Ser Ser Ile Leu Glu

1580 1585 1590

Phe Ser Gly Ala His Ser Gly Thr Pro Asp Ile Ser Gly Asp Leu

1595 1600 1605

Ser Gly Ser Leu Asp Gln Ser Thr Trp Gln Pro Gly Trp Thr Glu

1610 1615 1620

Ala Ser Thr Glu Pro Pro Ser Ser Pro Tyr Phe Ser Gly Asp Phe

1625 1630 1635

Ser Ser Thr Thr Asp Ala Ser Gly Glu Ser Ile Thr Ala Pro Thr

1640 1645 1650

Gly Ser Gly Glu Thr Ser Gly Leu Pro Glu Val Thr Leu Ile Thr

1655 1660 1665

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

1670 1675 1680

Leu Gly His Gly Pro Ser Met Thr Tyr Thr Pro Arg Leu Phe Glu

1685 1690 1695

Ala Ser Gly Glu Ala Ser Ala Ser Gly Asp Leu Gly Gly Pro Val

1700 1705 1710

Thr Ile Phe Pro Gly Ser Gly Val Glu Ala Ser Val Pro Glu Gly

1715 1720 1725

Ser Ser Asp Pro Ser Ala Tyr Pro Glu Ala Gly Val Gly Val Ser

1730 1735 1740

Ala Ala Pro Glu Ala Ser Ser Gln Leu Ser Glu Phe Pro Asp Leu

1745 1750 1755

His Gly Ile Thr Ser Ala Ser Arg Glu Thr Asp Leu Glu Met Thr

1760 1765 1770

Thr Pro Gly Thr Glu Val Ser Ser Asn Pro Trp Thr Phe Gln Glu

1775 1780 1785

Gly Thr Arg Glu Gly Ser Ala Ala Pro Glu Val Ser Gly Glu Ser

1790 1795 1800

Ser Thr Thr Ser Asp Ile Asp Ala Gly Thr Ser Gly Val Pro Phe

1805 1810 1815

Ala Thr Pro Met Thr Ser Gly Asp Arg Thr Glu Ile Ser Gly Glu

1820 1825 1830

Trp Ser Asp His Thr Ser Glu Val Asn Val Thr Val Ser Thr Thr

1835 1840 1845

Val Pro Glu Ser Arg Trp Ala Gln Ser Thr Gln His Pro Thr Glu

1850 1855 1860

Thr Leu Gln Glu Ile Gly Ser Pro Asn Pro Ser Tyr Ser Gly Glu

1865 1870 1875

Glu Thr Gln Thr Ala Glu Thr Ala Lys Ser Leu Thr Asp Thr Pro

1880 1885 1890

Thr Leu Ala Ser Pro Glu Gly Ser Gly Glu Thr Glu Ser Thr Ala

1895 1900 1905

Ala Asp Gln Glu Gln Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly

1910 1915 1920

His Cys Tyr Arg His Phe Pro Asp Arg Glu Thr Trp Val Asp Ala

1925 1930 1935

Glu Arg Arg Cys Arg Glu Gln Gln Ser His Leu Ser Ser Ile Val

1940 1945 1950

Thr Pro Glu Glu Gln Glu Phe Val Asn Lys Asn Ala Gln Asp Tyr

1955 1960 1965

Gln Trp Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg

1970 1975 1980

Trp Ser Asp Gly His Ser Leu Gln Phe Glu Lys Trp Arg Pro Asn

1985 1990 1995

Gln Pro Asp Asn Phe Phe Ala Thr Gly Glu Asp Cys Val Val Met

2000 2005 2010

Ile Trp His Glu Arg Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr

2015 2020 2025

Gln Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu

2030 2035 2040

Pro Pro Ala Val Glu His Ala Arg Thr Leu Gly Gln Lys Lys Asp

2045 2050 2055

Arg Tyr Glu Ile Ser Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly

2060 2065 2070

Phe Val Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Ala

2075 2080 2085

Asp Trp Glu Glu Pro Arg Ile Thr Cys Thr Asp Pro Asn Thr Tyr

2090 2095 2100

Lys His Arg Leu Gln Lys Arg Thr Met Arg Pro Thr Arg Arg Ser

2105 2110 2115

Arg Pro Ser Met Ala His

2120

<210> 129

<211> 537

<212> PRT

<213> Sus scrofa

<220>

<221> VARIANT

<222> (45)..(45)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> VARIANT

<222> (49)..(49)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> VARIANT

<222> (146)..(146)

<223> Xaa can be any naturally occurring amino acids

<400> 129

Ala Ile Ser Val Glu Val Ser Glu Pro Asp Asn Ser Leu Ser Val Ser

1 5 10 15

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

20 25 30

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Xaa Thr Ala Pro

35 40 45

Xaa Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

50 55 60

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gln Val Arg Val

65 70 75 80

Asn Ser Ala Tyr Gln Asp Arg Val Thr Leu Pro Asn Tyr Pro Ala Ile

85 90 95

Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser

100 105 110

Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

115 120 125

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

130 135 140

Ser Xaa Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

145 150 155 160

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

165 170 175

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

180 185 190

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

195 200 205

Asp Glu Phe Pro Gly Val Ile Thr Tyr Gly Ile Arg Asp Thr Asn Glu

210 215 220

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

225 230 235 240

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

245 250 255

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu

260 265 270

Ala Trp Arg Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

275 280 285

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

290 295 300

Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly

305 310 315 320

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

325 330 335

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

340 345 350

Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu

355 360 365

Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Thr Val Ile Leu Thr

370 375 380

Val Lys Pro Val Phe Glu Phe Ser Pro Thr Ala Pro Glu Pro Glu Glu

385 390 395 400

Pro Phe Thr Phe Ala Pro Gly Thr Gly Ala Thr Ala Phe Pro Glu Ala

405 410 415

Glu Asn Arg Thr Gly Glu Ala Thr Arg Pro Trp Ala Phe Pro Glu Glu

420 425 430

Ser Thr Pro Gly Leu Gly Ala Pro Thr Ala Phe Thr Ser Glu Asp Leu

435 440 445

Val Val Gln Val Thr Ser Ala Ala Thr Glu Glu Gly Thr Glu Gly Pro

450 455 460

Ser Ala Thr Glu Ala Pro Ser Thr Ser Glu Glu Pro Phe Pro Ser Glu

465 470 475 480

Lys Pro Phe Pro Ser Glu Glu Pro Phe Pro Ser Glu Glu Pro Phe Pro

485 490 495

Ser Glu Lys Pro Ser Ala Ser Glu Glu Pro Phe Pro Ser Glu Gln Pro

500 505 510

Ser Thr Leu Ser Ala Pro Val Pro Ser Arg Thr Glu Leu Pro Gly Ser

515 520 525

Gly Glu Val Ser Gly Ala Pro Glu Val

530 535

<210> 130

<211> 2132

<212> PRT

<213> Mus musculus

<400> 130

Met Thr Thr Leu Leu Leu Val Phe Val Thr Leu Arg Val Ile Ala Ala

1 5 10 15

Val Ile Ser Glu Glu Val Pro Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

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

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Thr Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gln Val Arg Val

85 90 95

Asn Ser Ile Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Ile Gln Asn Leu Arg Ser Asn Asp Ser

115 120 125

Gly Ile Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Ile Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu

275 280 285

Ala Trp Gln Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Asp

355 360 365

Asp Ile Thr Ile Gln Thr Val Thr Trp Pro Asp Leu Glu Leu Pro Leu

370 375 380

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

385 390 395 400

Ala Lys Pro Ile Phe Asp Leu Ser Pro Thr Ile Ser Glu Pro Gly Glu

405 410 415

Ala Leu Thr Leu Ala Pro Glu Val Gly Ser Thr Ala Phe Pro Glu Ala

420 425 430

Glu Glu Arg Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Ala Glu

435 440 445

Val Thr Arg Gly Pro Asp Ser Ala Thr Ala Phe Ala Ser Glu Asp Leu

450 455 460

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

465 470 475 480

Pro Arg Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Ser Thr

485 490 495

Arg Tyr Ser Leu Thr Phe Glu Glu Ala Gln Gln Ala Cys Met His Thr

500 505 510

Gly Ala Val Ile Ala Ser Pro Glu Gln Leu Gln Ala Ala Tyr Glu Ala

515 520 525

Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg

530 535 540

Tyr Pro Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser

545 550 555 560

Ser Pro Gly Val Arg Thr Tyr Gly Val Arg Pro Ser Ser Glu Thr Tyr

565 570 575

Asp Val Tyr Cys Tyr Val Asp Lys Leu Glu Gly Glu Val Phe Phe Ala

580 585 590

Thr Arg Leu Glu Gln Phe Thr Phe Gln Glu Ala Arg Ala Phe Cys Ala

595 600 605

Ala Gln Asn Ala Thr Leu Ala Ser Thr Gly Gln Leu Tyr Ala Ala Trp

610 615 620

Ser Gln Gly Leu Asp Lys Cys Tyr Ala Gly Trp Leu Ala Asp Gly Thr

625 630 635 640

Leu Arg Tyr Pro Ile Ile Thr Pro Arg Pro Ala Cys Gly Gly Asp Lys

645 650 655

Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Pro

660 665 670

Asp Pro Leu Ser Lys His His Ala Phe Cys Phe Arg Gly Val Ser Val

675 680 685

Ala Pro Ser Pro Gly Glu Glu Gly Gly Ser Thr Pro Thr Ser Pro Ser

690 695 700

Asp Ile Glu Asp Trp Ile Val Thr Gln Val Gly Pro Gly Val Asp Ala

705 710 715 720

Val Pro Leu Glu Pro Lys Thr Thr Glu Val Pro Tyr Phe Thr Thr Glu

725 730 735

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

740 745 750

Ser Pro Gln Pro Gly Ile Pro Thr Trp Leu Pro Thr Leu Pro Ala

755 760 765

Ala Glu Glu His Thr Glu Ser Pro Ser Ala Ser Glu Glu Pro Ser Ala

770 775 780

Ser Ala Val Pro Ser Thr Ser Glu Glu Pro Tyr Thr Ser Ser Phe Ala

785 790 795 800

Val Pro Ser Met Thr Glu Leu Pro Gly Ser Gly Glu Ala Ser Gly Ala

805 810 815

Pro Asp Leu Ser Gly Asp Phe Thr Gly Ser Gly Asp Ala Ser Gly Arg

820 825 830

Leu Asp Ser Ser Gly Gln Pro Ser Gly Gly Ile Glu Ser Gly Leu Pro

835 840 845

Ser Gly Asp Leu Asp Ser Ser Gly Leu Ser Pro Thr Val Ser Ser Gly

850 855 860

Leu Pro Val Glu Ser Gly Ser Ala Ser Gly Asp Gly Glu Val Pro Trp

865 870 875 880

Ser His Thr Pro Thr Val Gly Arg Leu Pro Ser Gly Gly Glu Ser Pro

885 890 895

Glu Gly Ser Ala Ser Ala Ser Gly Thr Gly Asp Leu Ser Gly Leu Pro

900 905 910

Ser Gly Gly Glu Ile Thr Glu Thr Ser Thr Ser Gly Ala Glu Glu Thr

915 920 925

Ser Gly Leu Pro Ser Gly Gly Asp Gly Leu Glu Thr Ser Thr Ser Gly

930 935 940

Val Asp Asp Val Ser Gly Ile Pro Thr Gly Arg Glu Gly Leu Glu Thr

945 950 955 960

Ser Ala Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Glu Glu

965 970 975

Gly Ser Glu Thr Ser Thr Ser Gly Ile Glu Asp Ile Ser Val Leu Pro

980 985 990

Thr Gly Gly Glu Ser Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu

995 1000 1005

Ser Gly Leu Pro Ser Gly Gly Glu Ser Leu Glu Thr Ser Ala Ser

1010 1015 1020

Gly Ala Glu Asp Val Thr Gln Leu Pro Thr Glu Arg Gly Gly Leu

1025 1030 1035

Glu Thr Ser Ala Ser Gly Val Glu Asp Ile Thr Val Leu Pro Thr

1040 1045 1050

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

1055 1060 1065

Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu Thr Ser Ala Ser

1070 1075 1080

Gly Ile Glu Asp Ile Ser Val Phe Pro Thr Glu Ala Glu Gly Leu

1085 1090 1095

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

1100 1105 1110

Gly Asp Gly Thr Glu Thr Ser Ala Ser Gly Val Glu Asp Val Ser

1115 1120 1125

Gly Leu Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly

1130 1135 1140

Val Glu Asp Leu Gly Pro Ser Thr Arg Asp Ser Leu Glu Thr Ser

1145 1150 1155

Ala Ser Gly Val Asp Val Thr Gly Phe Pro Ser Gly Arg Gly Asp

1160 1165 1170

Pro Glu Thr Ser Val Ser Gly Val Gly Asp Asp Phe Ser Gly Leu

1175 1180 1185

Pro Ser Gly Lys Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu

1190 1195 1200

Asp Leu Ser Gly Leu Pro Ser Gly Lys Glu Asp Leu Val Gly Ser

1205 1210 1215

Ala Ser Gly Ala Leu Asp Phe Gly Lys Leu Pro Pro Gly Thr Leu

1220 1225 1230

Gly Ser Gly Gln Thr Pro Glu Val Asn Gly Phe Pro Ser Gly Phe

1235 1240 1245

Ser Gly Glu Tyr Ser Gly Ala Asp Ile Gly Ser Gly Pro Ser Ser

1250 1255 1260

Gly Leu Pro Asp Phe Ser Gly Leu Pro Ser Gly Phe Pro Thr Val

1265 1270 1275

Ser Leu Val Asp Ser Thr Leu Val Glu Val Ile Thr Ala Thr Thr

1280 1285 1290

Ser Ser Glu Leu Glu Gly Arg Gly Thr Ile Gly Ile Ser Gly Ser

1295 1300 1305

Gly Glu Val Ser Gly Leu Pro Leu Gly Glu Leu Asp Ser Ser Ala

1310 1315 1320

Asp Ile Ser Gly Leu Pro Ser Gly Thr Glu Leu Ser Gly Gln Ala

1325 1330 1335

Ser Gly Ser Pro Asp Ser Ser Gly Glu Thr Ser Gly Phe Phe Asp

1340 1345 1350

Val Ser Gly Gln Pro Phe Gly Ser Ser Gly Val Ser Glu Glu Thr

1355 1360 1365

Ser Gly Ile Pro Glu Ile Ser Gly Gln Pro Ser Gly Thr Pro Asp

1370 1375 1380

Thr Thr Ala Thr Ser Gly Val Thr Glu Leu Asn Glu Leu Ser Ser

1385 1390 1395

Gly Gln Pro Asp Val Ser Gly Asp Gly Ser Gly Ile Leu Phe Gly

1400 1405 1410

Ser Gly Gln Ser Ser Gly Ile Thr Ser Val Ser Gly Glu Thr Ser

1415 1420 1425

Gly Ile Ser Asp Leu Ser Gly Gln Pro Ser Gly Phe Pro Val Phe

1430 1435 1440

Ser Gly Thr Ala Thr Arg Thr Pro Asp Leu Ala Ser Gly Thr Ile

1445 1450 1455

Ser Gly Ser Gly Glu Ser Ser Gly Ile Thr Phe Val Asp Thr Ser

1460 1465 1470

Phe Val Glu Val Thr Pro Thr Thr Phe Arg Glu Glu Glu Gly Leu

1475 1480 1485

Gly Ser Val Glu Leu Ser Gly Phe Pro Ser Gly Glu Thr Glu Leu

1490 1495 1500

Ser Gly Thr Ser Gly Thr Val Asp Val Ser Glu Gln Ser Ser Gly

1505 1510 1515

Ala Ile Asp Ser Ser Gly Leu Thr Ser Pro Thr Pro Glu Phe Ser

1520 1525 1530

Gly Leu Pro Ser Gly Val Ala Glu Val Ser Gly Glu Phe Ser Gly

1535 1540 1545

Val Glu Thr Gly Ser Ser Leu Pro Ser Gly Ala Phe Asp Gly Ser

1550 1555 1560

Gly Leu Val Ser Gly Phe Pro Thr Val Ser Leu Val Asp Arg Thr

1565 1570 1575

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

1580 1585 1590

Glu Gly Pro Ser Gly Ile Leu Glu Phe Ser Gly Ala His Ser Gly

1595 1600 1605

Thr Pro Asp Ile Ser Gly Glu Leu Ser Gly Ser Leu Asp Leu Ser

1610 1615 1620

Thr Leu Gln Ser Gly Gln Met Glu Thr Ser Thr Glu Thr Pro Ser

1625 1630 1635

Ser Pro Tyr Phe Ser Gly Asp Phe Ser Ser Thr Thr Asp Val Ser

1640 1645 1650

Gly Glu Ser Ile Ala Ala Thr Thr Gly Ser Gly Glu Ser Ser Gly

1655 1660 1665

Leu Pro Glu Val Thr Leu Asn Thr Ser Glu Leu Val Glu Gly Val

1670 1675 1680

Thr Glu Pro Thr Val Ser Gln Glu Leu Gly His Gly Pro Ser Met

1685 1690 1695

Thr Tyr Thr Pro Arg Leu Phe Glu Ala Ser Gly Asp Ala Ser Ala

1700 1705 1710

Ser Gly Asp Leu Gly Gly Ala Val Thr Asn Phe Pro Gly Ser Gly

1715 1720 1725

Ile Glu Ala Ser Val Pro Glu Ala Ser Ser Asp Leu Ser Ala Tyr

1730 1735 1740

Pro Glu Ala Gly Val Gly Val Ser Ala Ala Pro Glu Ala Ser Ser

1745 1750 1755

Lys Leu Ser Glu Phe Pro Asp Leu His Gly Ile Thr Ser Ala Phe

1760 1765 1770

His Glu Thr Asp Leu Glu Met Thr Pro Ser Thr Glu Val Asn

1775 1780 1785

Ser Asn Pro Trp Thr Phe Gln Glu Gly Thr Arg Glu Gly Ser Ala

1790 1795 1800

Ala Pro Glu Val Ser Gly Glu Ser Ser Thr Thr Ser Asp Ile Asp

1805 1810 1815

Thr Gly Thr Ser Gly Val Pro Ser Ala Thr Pro Met Ala Ser Gly

1820 1825 1830

Asp Arg Thr Glu Ile Ser Gly Glu Trp Ser Asp His Thr Ser Glu

1835 1840 1845

Val Asn Val Ala Ile Ser Ser Thr Ile Thr Glu Ser Glu Trp Ala

1850 1855 1860

Gln Pro Thr Arg Tyr Pro Thr Glu Thr Leu Gln Glu Ile Glu Ser

1865 1870 1875

Pro Asn Pro Ser Tyr Ser Gly Glu Glu Thr Gln Thr Ala Glu Thr

1880 1885 1890

Thr Met Ser Leu Thr Asp Ala Pro Thr Leu Ser Ser Ser Glu Gly

1895 1900 1905

Ser Gly Glu Thr Glu Ser Thr Val Ala Asp Gln Glu Gln Cys Glu

1910 1915 1920

Glu Gly Trp Thr Lys Phe Gln Gly His Cys Tyr Arg His Phe His

1925 1930 1935

Asp Arg Glu Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln

1940 1945 1950

Gln Ser His Leu Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe

1955 1960 1965

Val Asn Lys Asn Ala Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp

1970 1975 1980

Arg Thr Ile Glu Gly Asp Phe Arg Trp Ser Asp Gly His Ser Leu

1985 1990 1995

Gln Phe Glu Lys Trp Arg Pro Asn Gln Pro Asp Asn Phe Phe Ala

2000 2005 2010

Thr Gly Glu Asp Cys Val Val Met Ile Trp His Glu Arg Gly Glu

2015 2020 2025

Trp Asn Asp Val Pro Cys Asn Tyr Gln Leu Pro Phe Thr Cys Lys

2030 2035 2040

Lys Gly Thr Val Ala Cys Gly Asp Pro Pro Val Val Glu His Ala

2045 2050 2055

Arg Thr Leu Gly Gln Lys Lys Asp Arg Tyr Glu Ile Ser Ser Leu

2060 2065 2070

Val Arg Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg His Val Pro

2075 2080 2085

Thr Ile Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro Arg Ile

2090 2095 2100

Thr Cys Thr Asp Pro Asn Thr Tyr Lys His Arg Leu Gln Lys Arg

2105 2110 2115

Ser Met Arg Pro Thr Arg Arg Ser Arg Pro Ser Met Ala His

2120 2125 2130

<210> 131

<211> 2167

<212> PRT

<213> Oryctolagus cuniculus

<400> 131

Met Thr Thr Leu Leu Leu Val Leu Val Ala Leu Arg Val Ile Ala Ala

1 5 10 15

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

20 25 30

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

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Val His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Thr Pro Arg Ile Lys Trp Ser Arg Ile Ser Lys

65 70 75 80

Asp Lys Glu Val Val Leu Leu Val Ala Asn Glu Gly Arg Val Arg Ile

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Ile Gln Ser Leu Arg Ser Asn Asp Ser

115 120 125

Gly Ile Tyr Arg Cys Glu Val Met His Gly Leu Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Val Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Ser Glu

260 265 270

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu

275 280 285

Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Val His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Met Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Val

370 375 380

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

385 390 395 400

Ala Lys Pro Val Leu Asp Val Ser Pro Thr Ala Pro Gln Pro Glu Glu

405 410 415

Thr Phe Ala Pro Gly Val Gly Ala Thr Ala Phe Pro Gly Val Glu Asn

420 425 430

Gly Thr Glu Glu Ala Thr Arg Pro Arg Gly Phe Ala Asp Glu Ala Thr

435 440 445

Leu Gly Pro Ser Ser Ala Thr Ala Phe Thr Ser Ala Asp Leu Val Val

450 455 460

Gln Val Thr Ala Ala Pro Gly Val Ala Glu Val Pro Gly Gln Pro Arg

465 470 475 480

Leu Pro Gly Gly Val Val Phe His Tyr Arg Pro Gly Pro Thr Arg Tyr

485 490 495

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

500 505 510

Ala Met Ala Ser Ala Glu Gln Leu Gln Ala Ala Tyr Glu Ala Gly Tyr

515 520 525

Glu Gln Cys Asp Ala Gly Trp Leu Gln Asp Gln Thr Val Arg Tyr Pro

530 535 540

Ile Val Ser Pro Arg Thr Pro Cys Val Gly Asp Lys Asp Ser Ser Pro

545 550 555 560

Gly Val Arg Thr Tyr Gly Val Arg Pro Ser Glu Thr Tyr Asp Val

565 570 575

Tyr Cys Tyr Val Asp Arg Leu Glu Gly Glu Val Phe Phe Ala Thr Arg

580 585 590

Leu Glu Gln Phe Thr Phe Gln Glu Ala Leu Glu Phe Cys Glu Ser His

595 600 605

Asn Ala Thr Leu Ala Ser Thr Gly Gln Leu Tyr Ala Ala Trp Ser Arg

610 615 620

Gly Leu Asp Arg Cys Tyr Ala Gly Trp Leu Ala Asp Gly Ser Leu Arg

625 630 635 640

Tyr Pro Ile Val Thr Pro Arg Pro Ala Cys Gly Gly Asp Lys Pro Gly

645 650 655

Val Arg Thr Val Tyr Leu Tyr Pro Asn Gln Thr Gly Leu Pro Asp Pro

660 665 670

Leu Ser Arg His His Ala Phe Cys Phe Arg Gly Thr Ser Glu Ala Pro

675 680 685

Ser Pro Gly Pro Glu Glu Gly Gly Thr Ala Thr Pro Ala Ser Gly Leu

690 695 700

Glu Asp Trp Ile Val Thr Gln Val Gly Pro Gly Val Ala Ala Thr Pro

705 710 715 720

Arg Ala Glu Glu Arg Thr Ala Val Pro Ser Phe Ala Thr Glu Pro Gly

725 730 735

Asn Gln Thr Gly Trp Glu Ala Ala Ser Ser Pro Val Gly Thr Ser Leu

740 745 750

Leu Pro Gly Ile Pro Pro Thr Trp Pro Thr Gly Thr Ala Ala Glu

755 760 765

Gly Thr Thr Glu Gly Leu Ser Thr Ala Ala Met Pro Ser Ala Ser Glu

770 775 780

Gly Pro Tyr Thr Pro Ser Ser Leu Val Ala Arg Glu Thr Glu Leu Pro

785 790 795 800

Gly Leu Gly Val Thr Ser Val Pro Pro Asp Ile Ser Gly Asp Leu Thr

805 810 815

Ser Ser Gly Glu Ala Ser Gly Leu Phe Gly Pro Thr Gly Gln Pro Leu

820 825 830

Gly Gly Ser Ala Ser Gly Leu Pro Ser Gly Glu Leu Asp Ser Gly Ser

835 840 845

Leu Thr Pro Thr Val Gly Ser Gly Leu Pro Ile Gly Ser Gly Leu Ala

850 855 860

Ser Gly Asp Glu Asp Arg Ile Gln Trp Ser Ser Ser Ser Thr Glu Val Gly

865 870 875 880

Gly Val Thr Ser Gly Ala Glu Ile Pro Glu Thr Ser Ala Ser Gly Val

885 890 895

Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Pro Glu Thr

900 905 910

Phe Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala

915 920 925

Glu Ile Pro Glu Thr Phe Ala Ser Gly Val Gly Thr Asp Leu Ser Gly

930 935 940

Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser Ala Ser Gly Val Gly

945 950 955 960

Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser

965 970 975

Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu

980 985 990

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

995 1000 1005

Pro Ser Gly Ala Glu Ile Pro Glu Thr Phe Ala Ser Gly Val Gly

1010 1015 1020

Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr

1025 1030 1035

Ser Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly

1040 1045 1050

Ala Glu Ile Pro Glu Thr Ser Ala Ser Gly Val Gly Thr Asp Leu

1055 1060 1065

Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser Ala Ser

1070 1075 1080

Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile

1085 1090 1095

Leu Glu Thr Ser Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu

1100 1105 1110

Pro Ser Gly Ala Glu Ile Leu Glu Thr Ser Ala Ser Gly Val Gly

1115 1120 1125

Thr Asp Leu Ser Gly Leu Pro Ser Gly Ala Glu Ile Leu Glu Thr

1130 1135 1140

Ser Ala Ser Gly Val Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly

1145 1150 1155

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

1160 1165 1170

Ser Gly Leu Pro Ser Gly Gly Glu Ile Pro Glu Thr Phe Ala Ser

1175 1180 1185

Gly Val Gly Asp Leu Ser Gly Leu Pro Pro Gly Arg Glu Asp Leu

1190 1195 1200

Glu Thr Leu Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Ser Ser

1205 1210 1215

Gly Lys Asp Gly Leu Val Gly Ser Ala Ser Gly Ala Leu Asp Phe

1220 1225 1230

Gly Gly Thr Leu Gly Ser Gly Gln Ile Pro Glu Thr Ser Gly Leu

1235 1240 1245

Pro Ser Gly Tyr Ser Gly Glu Tyr Ser Glu Val Asp Leu Gly Ser

1250 1255 1260

Gly Pro Ser Ser Gly Leu Pro Asp Phe Ser Gly Leu Pro Ser Gly

1265 1270 1275

Phe Pro Thr Val Ser Leu Val Asp Thr Pro Leu Val Glu Val Val

1280 1285 1290

Thr Ala Thr Thr Ala Arg Glu Leu Glu Gly Arg Gly Thr Ile Gly

1295 1300 1305

Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser Ser Glu Leu

1310 1315 1320

Asp Val Ser Gly Gly Thr Ser Gly Ala Asp Ile Ser Gly Glu Ala

1325 1330 1335

Asp Val Gly Gly Glu Ala Ser Gly Leu Ile Val Arg Gly Gln Pro

1340 1345 1350

Ser Gly Phe Pro Asp Thr Ser Gly Glu Ala Phe Gly Val Thr Glu

1355 1360 1365

Val Ser Gly Leu Ser Ser Gly Gln Pro Asp Leu Ser Gly Glu Ala

1370 1375 1380

Ser Gly Val Leu Phe Gly Ser Gly Pro Pro Phe Gly Ile Thr Asp

1385 1390 1395

Leu Ser Gly Glu Pro Ser Gly Gln Pro Ser Gly Leu Pro Glu Phe

1400 1405 1410

Ser Gly Thr Thr His Arg Ile Pro Asp Leu Val Ser Gly Ala Thr

1415 1420 1425

Ser Gly Ser Gly Glu Ser Ser Gly Ile Ala Phe Val Asp Thr Ser

1430 1435 1440

Val Val Glu Val Thr Pro Thr Thr Leu Arg Glu Glu Glu Gly Leu

1445 1450 1455

Gly Ser Val Glu Phe Ser Gly Phe Pro Ser Gly Glu Thr Gly Leu

1460 1465 1470

Ser Gly Thr Pro Glu Thr Ile Asp Val Ser Gly Gln Ser Ser Gly

1475 1480 1485

Thr Ile Asp Ser Ser Gly Phe Thr Ser Leu Ala Pro Glu Val Ser

1490 1495 1500

Gly Ser Pro Ser Gly Val Ala Glu Val Ser Gly Glu Ala Ser Gly

1505 1510 1515

Thr Glu Ile Thr Ser Gly Leu Pro Ser Gly Val Phe Asp Ser Ser

1520 1525 1530

Gly Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Arg Thr

1535 1540 1545

Leu Val Glu Ser Val Thr Gln Ala Pro Thr Ala Gln Glu Ala Glu

1550 1555 1560

Gly Pro Ser Asp Ile Leu Glu Leu Ser Gly Val His Ser Gly Leu

1565 1570 1575

Pro Asp Val Ser Gly Ala His Ser Gly Phe Leu Asp Pro Ser Gly

1580 1585 1590

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

1595 1600 1605

Pro Tyr Phe Ser Gly Asp Phe Pro Ser Thr Pro Asp Val Ser Gly

1610 1615 1620

Glu Ala Ser Ala Ala Thr Ser Ser Ser Gly Asp Ile Ser Gly Leu

1625 1630 1635

Pro Glu Val Thr Leu Val Thr Ser Glu Phe Met Glu Gly Val Thr

1640 1645 1650

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

1655 1660 1665

His Val Pro Lys Leu Phe Glu Ser Ser Gly Glu Ala Leu Ala Ser

1670 1675 1680

Gly Asp Thr Ser Gly Ala Ala Pro Ala Phe Pro Gly Ser Gly Leu

1685 1690 1695

Glu Ala Ser Ser Val Pro Glu Ser His Gly Glu Thr Ser Ala Tyr

1700 1705 1710

Ala Glu Pro Gly Thr Lys Ala Ala Ala Ala Pro Asp Ala Ser Gly

1715 1720 1725

Glu Ala Ser Gly Ser Pro Asp Ser Gly Glu Ile Thr Ser Val Phe

1730 1735 1740

Arg Glu Ala Ala Gly Glu Gly Ala Ser Gly Leu Glu Val Ser Ser

1745 1750 1755

Ser Ser Leu Ala Ser Gln Gln Gly Pro Arg Glu Gly Ser Ala Ser

1760 1765 1770

Pro Glu Val Ser Gly Glu Ser Thr Thr Ser Tyr Glu Ile Gly Thr

1775 1780 1785

Glu Thr Ser Gly Leu Pro Leu Ala Thr Pro Ala Ala Ser Glu Asp

1790 1795 1800

Arg Ala Glu Val Ser Gly Asp Leu Ser Gly Arg Thr Pro Val Pro

1805 1810 1815

Val Asp Val Val Thr Asn Val Pro Glu Ala Glu Trp Ile Gln His

1820 1825 1830

Ser Gln Arg Pro Ala Glu Met Trp Pro Glu Thr Lys Ser Ser Ser

1835 1840 1845

Pro Ser Tyr Ser Gly Glu Asp Thr Ala Gly Thr Ala Ala Ser Pro

1850 1855 1860

Ala Ser Ala Asp Thr Pro Gly Glu Pro Gly Pro Thr Thr Ala Ala

1865 1870 1875

Pro Arg Ser Cys Ala Glu Glu Pro Cys Gly Pro Gly Thr Cys Gln

1880 1885 1890

Glu Thr Glu Gly Arg Val Thr Cys Leu Cys Pro Pro Gly His Thr

1895 1900 1905

Gly Glu Tyr Cys Asp Ile Asp Ile Asp Glu Cys Leu Ser Ser Pro

1910 1915 1920

Cys Val Asn Gly Ala Thr Cys Val Asp Ala Ser Asp Ser Phe Thr

1925 1930 1935

Cys Leu Cys Leu Pro Ser Tyr Gly Gly Asp Leu Cys Glu Thr Asp

1940 1945 1950

Gln Glu Val Cys Glu Glu Gly Trp Thr Lys Phe Gln Gly His Cys

1955 1960 1965

Tyr Arg His Phe Pro Asp Arg Glu Thr Trp Val Asp Ala Glu Gly

1970 1975 1980

Arg Cys Arg Glu Gln Gln Ser His Leu Ser Ser Ile Val Thr Pro

1985 1990 1995

Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr Gln Trp

2000 2005 2010

Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg Trp Ser

2015 2020 2025

Asp Gly His Pro Leu Gln Phe Glu Asn Trp Arg Pro Asn Gln Pro

2030 2035 2040

Asp Asn Phe Phe Ala Thr Gly Glu Asp Cys Val Val Met Ile Trp

2045 2050 2055

His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr His Leu

2060 2065 2070

Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Asp Pro Pro

2075 2080 2085

Val Val Glu His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg Tyr

2090 2095 2100

Glu Ile Asn Ser Leu Val Arg Tyr Gln Cys Ala Glu Gly Phe Thr

2105 2110 2115

Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Gly His Trp

2120 2125 2130

Glu Glu Pro Arg Ile Thr Cys Thr His Pro Thr Thr Tyr Lys Arg

2135 2140 2145

Arg Val Gln Lys Arg Ser Ser Arg Thr Leu Gln Arg Ser Gln Ala

2150 2155 2160

Ser Ser Ala Pro

2165

<210> 132

<211> 2266

<212> PRT

<213> Macaca fascicularis

<400> 132

Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Ala Ala

1 5 10 15

Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

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

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Ile Gln Ser Leu Arg Ser Asn Asp Ser

115 120 125

Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu

275 280 285

Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu

370 375 380

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

385 390 395 400

Val Lys Pro Ile Phe Asp Val Ser Pro Ser Pro Leu Glu Pro Glu Glu

405 410 415

Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Pro Glu Val

420 425 430

Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro

435 440 445

Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln

450 455 460

Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe

465 470 475 480

His Tyr Arg Pro Gly Ser Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala

485 490 495

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

500 505 510

Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp

515 520 525

Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro

530 535 540

Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val

545 550 555 560

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

565 570 575

Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln

580 585 590

Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Leu Ala Thr Thr

595 600 605

Gly Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala

610 615 620

Gly Trp Leu Ala Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr Pro Arg

625 630 635 640

Pro Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr

645 650 655

Pro Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe

660 665 670

Cys Phe Arg Gly Val Ser Ala Val Pro Ser Pro Gly Glu Glu Glu Gly

675 680 685

Gly Thr Pro Thr Ser Pro Ser Gly Val Glu Asp Trp Ile Ala Thr Gln

690 695 700

Val Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala

705 710 715 720

Val Pro Leu Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro

725 730 735

Glu Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Met Gly Thr Ser

740 745 750

Pro Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Thr Ala Thr

755 760 765

Glu Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Leu Thr Ala Ser

770 775 780

Lys Glu Pro Ser Pro Pro Glu Val Pro Phe Pro Ser Glu Glu Pro Ser

785 790 795 800

Pro Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu

805 810 815

Pro Ser Pro Ser Glu Glu Pro Phe Pro Ser Val Glu Pro Ser Pro Ser

820 825 830

Glu Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Val

835 840 845

Pro Ser Trp Thr Glu Leu Pro Gly Ser Gly Glu Glu Ser Gly Ala Pro

850 855 860

Asp Val Ser Gly Asp Phe Ile Gly Ser Gly Asp Val Ser Gly His Leu

865 870 875 880

Asp Phe Ser Gly Gln Leu Ser Gly Asp Arg Ile Ser Gly Leu Pro Ser

885 890 895

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

900 905 910

Pro Val Asp Ser Gly Leu Ala Ser Gly Asp Glu Glu Arg Ile Glu Trp

915 920 925

Ser Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu

930 935 940

Glu Gly Ser Ala Ser Glu Val Gly Asp Leu Ser Gly Leu Pro Ser Gly

945 950 955 960

Asp Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu

965 970 975

Pro Ser Gly Glu Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu

980 985 990

Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val

995 1000 1005

Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser

1010 1015 1020

Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Ala Gly Glu

1025 1030 1035

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

1040 1045 1050

Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser Gly Val Glu Asp

1055 1060 1065

Ile Ser Gly Phe Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser

1070 1075 1080

Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu

1085 1090 1095

Thr Thr Ala Ser Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly

1100 1105 1110

Glu Val Leu Glu Thr Thr Ala Ser Gly Val Gly Asp Leu Gly Gly

1115 1120 1125

Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val Gly

1130 1135 1140

Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Val Glu Thr Ser Thr

1145 1150 1155

Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu Val

1160 1165 1170

Leu Glu Thr Ser Thr Ser Gly Val Glu Asp Ile Ser Gly Leu Pro

1175 1180 1185

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

1190 1195 1200

Ser Glu Leu Pro Ser Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly

1205 1210 1215

Val Glu Asp Leu Ser Arg Leu Pro Ser Gly Glu Val Leu Glu Thr

1220 1225 1230

Ser Ala Ser Gly Val Gly Asp Ile Ser Gly Leu Pro Ser Gly Gly

1235 1240 1245

Glu Val Leu Glu Ile Ser Ala Ser Gly Val Gly Asp Leu Ser Gly

1250 1255 1260

Leu Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val

1265 1270 1275

Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu

1280 1285 1290

Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser Gly Leu Pro Ser Gly

1295 1300 1305

Lys Glu Asp Leu Val Gly Pro Ala Ser Gly Asp Leu Asp Leu Gly

1310 1315 1320

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

1325 1330 1335

Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly Val Asp

1340 1345 1350

Leu Gly Ser Gly Pro Pro Ser Gly Leu Pro Asp Phe Ser Gly Leu

1355 1360 1365

Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser Thr Leu Val

1370 1375 1380

Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu Glu Gly Arg Gly

1385 1390 1395

Thr Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser

1400 1405 1410

Ser Glu Leu Asp Ile Ser Gly Glu Ala Ser Gly Leu Pro Ser Gly

1415 1420 1425

Thr Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro Asp Val Ser Arg

1430 1435 1440

Glu Thr Pro Gly Leu Phe Asp Val Ser Gly Gln Pro Ser Gly Phe

1445 1450 1455

Pro Asp Ile Ser Gly Gly Thr Ser Gly Ile Ser Glu Val Ser Gly

1460 1465 1470

Gln Pro Ser Gly Phe Pro Asp Thr Ser Gly Glu Thr Ser Gly Val

1475 1480 1485

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

1490 1495 1500

Glu Ala Ser Gly Val Pro Tyr Gly Ser Ser Gln Pro Phe Gly Ile

1505 1510 1515

Thr Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp Leu Ser Gly

1520 1525 1530

Gln Pro Ser Gly Leu Pro Gly Phe Ser Gly Ala Thr Ser Gly Val

1535 1540 1545

Pro Asp Leu Val Ser Gly Ala Thr Ser Gly Ser Gly Glu Ser Ser

1550 1555 1560

Gly Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Thr Pro Thr

1565 1570 1575

Thr Phe Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly

1580 1585 1590

Leu Pro Ser Gly Glu Ala Asp Leu Ser Gly Arg Ser Gly Met Val

1595 1600 1605

Asp Val Ser Gly Glyn Phe Ser Gly Thr Val Asp Ser Ser Gly Phe

1610 1615 1620

Thr Ser Gln Thr Pro Glu Phe Ser Gly Leu Pro Ile Gly Ile Ala

1625 1630 1635

Glu Val Ser Gly Glu Ser Ser Gly Ala Glu Thr Gly Ser Ser Leu

1640 1645 1650

Pro Ser Gly Ala Tyr Tyr Gly Ser Gly Leu Pro Ser Gly Phe Pro

1655 1660 1665

Thr Val Ser Leu Val Asp Arg Thr Leu Val Glu Ser Val Thr Gln

1670 1675 1680

Ala Pro Thr Ala Gln Glu Ala Gly Glu Gly Pro Pro Gly Ile Leu

1685 1690 1695

Glu Leu Ser Gly Thr His Ser Gly Ala Pro Asp Met Ser Gly Asp

1700 1705 1710

His Ser Gly Phe Leu Asp Val Ser Gly Leu Gln Phe Gly Leu Val

1715 1720 1725

Glu Pro Ser Gly Glu Pro Pro Ser Thr Pro Tyr Phe Ser Gly Asp

1730 1735 1740

Phe Ala Ser Thr Thr Asp Val Ser Gly Glu Ser Ser Ala Ala Met

1745 1750 1755

Gly Thr Ser Gly Glu Ala Ser Gly Leu Pro Gly Val Thr Leu Ile

1760 1765 1770

Thr Ser Glu Phe Met Glu Gly Val Thr Glu Pro Thr Val Ser Gln

1775 1780 1785

Glu Leu Gly Gln Arg Pro Val Thr His Thr Pro Gln Leu Phe

1790 1795 1800

Glu Ser Ser Gly Glu Ala Ser Ala Ala Gly Asp Ile Ser Gly Ala

1805 1810 1815

Thr Pro Val Leu Pro Gly Ser Gly Val Glu Val Ser Ser Val Pro

1820 1825 1830

Glu Ser Ser Ser Glu Thr Ser Ala Tyr Pro Glu Ala Gly Val Gly

1835 1840 1845

Ala Ser Ala Ala Pro Glu Thr Ser Gly Glu Asp Ser Gly Ser Pro

1850 1855 1860

Asp Leu Ser Glu Thr Thr Ser Ala Phe His Glu Ala Asp Leu Glu

1865 1870 1875

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

1880 1885 1890

Glu Gly Glu Pro Ser Ala Ser Pro Glu Val Ser Gly Glu Ser Thr

1895 1900 1905

Thr Thr Gly Asp Val Gly Thr Glu Ala Pro Gly Leu Pro Ser Ala

1910 1915 1920

Thr Pro Thr Ala Ser Gly Asp Arg Thr Glu Ile Ser Gly Asp Leu

1925 1930 1935

Ser Gly His Thr Ser Gly Leu Gly Val Val Ile Ser Thr Ser Ile

1940 1945 1950

Pro Glu Ser Glu Trp Thr Gln Gln Thr Gln Arg Pro Ala Glu Ala

1955 1960 1965

His Leu Glu Thr Glu Ser Ser Ser Leu Leu Tyr Ser Gly Glu Glu

1970 1975 1980

Thr His Thr Ala Glu Thr Ala Thr Ser Pro Thr Asp Ala Ser Ile

1985 1990 1995

Pro Ala Ser Pro Glu Trp Thr Gly Glu Ser Glu Ser Thr Val Ala

2000 2005 2010

Asp Ile Asp Glu Cys Leu Ser Ser Pro Cys Leu Asn Gly Ala Thr

2015 2020 2025

Cys Val Asp Ala Ile Asp Ser Phe Thr Cys Leu Cys Leu Pro Ser

2030 2035 2040

Tyr Gly Gly Asp Leu Cys Glu Ile Asp Gln Glu Val Cys Glu Glu

2045 2050 2055

Gly Trp Thr Lys Tyr Gln Gly His Cys Tyr Arg His Phe Pro Asp

2060 2065 2070

Arg Glu Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln Gln

2075 2080 2085

Ser His Leu Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe Val

2090 2095 2100

Asn Asn Asn Ala Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp Arg

2105 2110 2115

Thr Ile Glu Gly Asp Phe Arg Trp Ser Asp Gly His Pro Met Gln

2120 2125 2130

Phe Glu Asn Trp Arg Pro Asn Gln Pro Asp Asn Phe Phe Ala Ala

2135 2140 2145

Gly Glu Asp Cys Val Val Met Ile Trp His Glu Lys Gly Glu Trp

2150 2155 2160

Asn Asp Val Pro Cys Asn Tyr His Leu Pro Phe Thr Cys Lys Lys

2165 2170 2175

Gly Thr Val Ala Cys Gly Glu Pro Pro Met Val Gln His Ala Arg

2180 2185 2190

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

2195 2200 2205

Arg Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg His Val Pro Thr

2210 2215 2220

Ile Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro Arg Ile Thr

2225 2230 2235

Cys Thr Asp Ala Thr Ala Tyr Lys Arg Arg Leu Gln Lys Arg Ser

2240 2245 2250

Ser Arg His Pro Arg Arg Ser Arg Pro Ser Thr Ala His

2255 2260 2265

<210> 133

<211> 2167

<212> PRT

<213> Macaca mulatta

<220>

<221> VARIANT

<222> (1910)..(1915)

<223> Xaa can be any naturally occurring amino acids

<400> 133

Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Ala Ala

1 5 10 15

Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

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

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

Pro Ser Asp Ala Thr Leu Glu Ile Gln Ser Leu Arg Ser Asn Asp Ser

115 120 125

Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

Cys Arg Arg Leu Gly Ala Arg Leu Ala Thr Thr Gly Gln Leu Tyr Leu

275 280 285

Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu

370 375 380

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

385 390 395 400

Val Lys Pro Ile Phe Asp Val Ser Pro Ser Pro Leu Glu Pro Glu Glu

405 410 415

Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Pro Glu Val

420 425 430

Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro

435 440 445

Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln

450 455 460

Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe

465 470 475 480

His Tyr Arg Pro Gly Ser Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala

485 490 495

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

500 505 510

Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp

515 520 525

Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro

530 535 540

Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val

545 550 555 560

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

565 570 575

Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln

580 585 590

Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Leu Ala Thr Thr

595 600 605

Gly Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala

610 615 620

Gly Trp Leu Ala Asp Gly Ser Leu Arg Tyr Pro Ile Val Thr Pro Arg

625 630 635 640

Pro Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr

645 650 655

Pro Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe

660 665 670

Cys Phe Arg Gly Val Ser Ala Val Pro Ser Pro Gly Glu Glu Glu Gly

675 680 685

Gly Thr Pro Thr Ser Pro Ser Gly Val Glu Asp Trp Ile Ala Thr Gln

690 695 700

Val Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala

705 710 715 720

Val Pro Leu Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro

725 730 735

Glu Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Met Gly Thr Ser

740 745 750

Pro Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Thr Ala Thr

755 760 765

Glu Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Leu Thr Ala Ser

770 775 780

Lys Glu Pro Ser Pro Pro Glu Val Pro Phe Pro Ser Glu Glu Pro Ser

785 790 795 800

Pro Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu

805 810 815

Pro Ser Pro Ser Glu Glu Pro Phe Pro Ser Val Glu Pro Ser Pro Ser

820 825 830

Glu Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Val

835 840 845

Pro Ser Trp Thr Glu Leu Pro Gly Ser Gly Glu Glu Ser Gly Ala Pro

850 855 860

Asp Val Ser Gly Asp Phe Ile Gly Ser Gly Asp Val Ser Gly His Leu

865 870 875 880

Asp Phe Ser Gly Gln Leu Ser Gly Asp Arg Ile Ser Gly Leu Pro Ser

885 890 895

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

900 905 910

Pro Val Asp Ser Gly Leu Ala Ser Gly Asp Glu Glu Arg Ile Glu Trp

915 920 925

Ser Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu

930 935 940

Glu Gly Ser Ala Ser Glu Val Gly Asp Leu Ser Gly Leu Pro Ser Gly

945 950 955 960

Asp Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu

965 970 975

Pro Ser Gly Glu Val Leu Glu Thr Ser Val Ser Gly Val Gly Asp Leu

980 985 990

Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val

995 1000 1005

Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ser

1010 1015 1020

Thr Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Ala Gly Glu

1025 1030 1035

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

1040 1045 1050

Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser Gly Val Glu Asp

1055 1060 1065

Ile Ser Gly Phe Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Ser

1070 1075 1080

Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu

1085 1090 1095

Thr Thr Ala Ser Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly

1100 1105 1110

Glu Val Leu Glu Thr Thr Ala Ser Gly Val Gly Asp Leu Gly Gly

1115 1120 1125

Leu Pro Ser Gly Glu Val Leu Glu Thr Ser Thr Ser Gly Val Gly

1130 1135 1140

Asp Leu Ser Gly Leu Pro Ser Gly Glu Val Val Glu Thr Ser Thr

1145 1150 1155

Ser Gly Val Glu Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu Val

1160 1165 1170

Leu Glu Thr Ser Thr Ser Gly Val Glu Asp Ile Ser Gly Leu Pro

1175 1180 1185

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

1190 1195 1200

Ser Glu Leu Pro Ser Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly

1205 1210 1215

Val Glu Asp Leu Ser Arg Leu Pro Ser Gly Glu Val Leu Glu Thr

1220 1225 1230

Ser Ala Ser Gly Val Gly Asp Ile Ser Gly Leu Pro Ser Gly Gly

1235 1240 1245

Glu Val Leu Glu Ile Ser Ala Ser Gly Val Gly Asp Leu Ser Gly

1250 1255 1260

Leu Pro Ser Gly Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val

1265 1270 1275

Gly Thr Asp Leu Ser Gly Leu Pro Ser Gly Arg Glu Gly Leu Glu

1280 1285 1290

Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser Gly Leu Pro Ser Gly

1295 1300 1305

Lys Glu Asp Leu Val Gly Pro Ala Ser Gly Asp Leu Asp Leu Gly

1310 1315 1320

Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln Ala Pro Glu Thr

1325 1330 1335

Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly Val Asp

1340 1345 1350

Leu Gly Ser Gly Pro Pro Ser Gly Leu Pro Asp Phe Ser Gly Leu

1355 1360 1365

Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser Thr Leu Val

1370 1375 1380

Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu Glu Gly Arg Gly

1385 1390 1395

Thr Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser

1400 1405 1410

Ser Glu Leu Asp Ile Ser Gly Glu Ala Ser Gly Leu Pro Ser Gly

1415 1420 1425

Thr Glu Leu Ser Gly Gln Ala Ser Gly Ser Pro Asp Val Ser Arg

1430 1435 1440

Glu Thr Ser Gly Leu Phe Asp Val Ser Gly Gln Pro Ser Gly Phe

1445 1450 1455

Pro Asp Thr Ser Gly Glu Thr Ser Gly Val Thr Glu Leu Ser Gly

1460 1465 1470

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

1475 1480 1485

Pro Tyr Gly Ser Ser Gln Pro Phe Gly Ile Thr Asp Leu Ser Gly

1490 1495 1500

Glu Thr Ser Gly Val Pro Asp Leu Ser Gly Gln Pro Ser Gly Leu

1505 1510 1515

Pro Gly Phe Ser Gly Ala Thr Ser Gly Val Pro Asp Leu Val Ser

1520 1525 1530

Gly Ala Thr Ser Gly Ser Gly Glu Ser Ser Asp Ile Thr Phe Val

1535 1540 1545

Asp Thr Ser Leu Val Glu Val Thr Pro Thr Thr Phe Lys Glu Glu

1550 1555 1560

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

1565 1570 1575

Ala Asp Leu Ser Gly Arg Ser Gly Met Val Asp Val Ser Gly Gln

1580 1585 1590

Phe Ser Gly Thr Val Asp Ser Ser Gly Phe Thr Ser Gln Thr Pro

1595 1600 1605

Glu Phe Ser Gly Leu Pro Ile Gly Ile Ala Glu Val Ser Gly Glu

1610 1615 1620

Ser Ser Gly Ala Glu Thr Gly Ser Ser Leu Pro Ser Gly Ala Tyr

1625 1630 1635

Tyr Gly Ser Glu Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val

1640 1645 1650

Asp Arg Thr Leu Val Glu Ser Val Thr Gln Ala Pro Thr Ala Gln

1655 1660 1665

Glu Ala Gly Glu Gly Pro Pro Gly Ile Leu Glu Leu Ser Gly Thr

1670 1675 1680

His Ser Gly Ala Pro Asp Met Ser Gly Asp His Ser Gly Phe Leu

1685 1690 1695

Asp Val Ser Gly Leu Gln Phe Gly Leu Val Glu Pro Ser Gly Glu

1700 1705 1710

Pro Pro Ser Thr Pro Tyr Phe Ser Gly Asp Phe Ala Ser Thr Thr

1715 1720 1725

Asp Val Ser Gly Glu Ser Ser Ala Ala Met Gly Thr Asn Gly Glu

1730 1735 1740

Ala Ser Gly Leu Pro Glu Val Thr Leu Ile Thr Ser Glu Phe Met

1745 1750 1755

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

1760 1765 1770

Pro Pro Val Thr His Thr Pro Gln Leu Phe Glu Ser Ser Gly Glu

1775 1780 1785

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

1790 1795 1800

Gly Ser Gly Val Glu Val Ser Ser Val Pro Glu Ser Ser Ser Glu

1805 1810 1815

Thr Ser Ala Tyr Pro Glu Ala Gly Val Gly Ala Ser Ala Ala Pro

1820 1825 1830

Glu Thr Ser Gly Glu Asp Ser Gly Ser Pro Asp Leu Ser Glu Thr

1835 1840 1845

Thr Ser Ala Phe His Glu Ala Asp Leu Glu Arg Ser Ser Gly Leu

1850 1855 1860

Gly Val Ser Gly Ser Thr Leu Thr Phe Gln Glu Gly Glu Pro Ser

1865 1870 1875

Ala Ser Pro Glu Val Ser Gly Glu Ser Thr Thr Thr Gly Asp Val

1880 1885 1890

Gly Thr Glu Ala Pro Gly Leu Pro Ser Ala Thr Pro Thr Ala Ser

1895 1900 1905

Gly Xaa Xaa Xaa Xaa Xaa Xaa Pro Thr Arg Ser Cys Ala Glu Glu

1910 1915 1920

Pro Cys Gly Ala Gly Thr Cys Lys Glu Thr Glu Gly His Val Ile

1925 1930 1935

Cys Leu Cys Pro Pro Gly Tyr Thr Gly Glu His Cys Asn Ile Asp

1940 1945 1950

Gln Glu Val Cys Glu Glu Gly Trp Thr Lys Tyr Gln Gly His Cys

1955 1960 1965

Tyr Arg His Phe Pro Asp Arg Glu Thr Trp Val Asp Ala Glu Arg

1970 1975 1980

Arg Cys Arg Glu Gln Gln Ser His Leu Ser Ser Ile Val Thr Pro

1985 1990 1995

Glu Glu Gln Glu Phe Val Asn Asn Asn Ala Gln Asp Tyr Gln Trp

2000 2005 2010

Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp Phe Arg Trp Ser

2015 2020 2025

Asp Gly His Pro Met Gln Phe Glu Asn Trp Arg Pro Asn Gln Pro

2030 2035 2040

Asp Asn Phe Phe Ala Ala Gly Glu Asp Cys Val Val Met Ile Trp

2045 2050 2055

His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr His Leu

2060 2065 2070

Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu Pro Pro

2075 2080 2085

Met Val Gln His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg Tyr

2090 2095 2100

Glu Ile Asn Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly Phe Val

2105 2110 2115

Gln Arg His Val Pro Thr Ile Arg Cys Gln Pro Ser Gly His Trp

2120 2125 2130

Glu Glu Pro Arg Ile Thr Cys Thr Asp Ala Thr Ala Tyr Lys Arg

2135 2140 2145

Arg Leu Gln Lys Arg Ser Ser Arg His Pro Arg Arg Ser Arg Pro

2150 2155 2160

Ser Thr Ala His

2165

<210> 134

<211> 1321

<212> PRT

<213> Homo sapiens

<400> 134

Met Gly Ala Pro Phe Val Trp Ala Leu Gly Leu Leu Met Leu Gln Met

1 5 10 15

Leu Leu Phe Val Ala Gly Glu Gln Gly Thr Gln Asp Ile Thr Asp Ala

20 25 30

Ser Glu Arg Gly Leu His Met Gln Lys Leu Gly Ser Gly Ser Val Gln

35 40 45

Ala Ala Leu Ala Glu Leu Val Ala Leu Pro Cys Leu Phe Thr Leu Gln

50 55 60

Pro Arg Pro Ser Ala Ala Arg Asp Ala Pro Arg Ile Lys Trp Thr Lys

65 70 75 80

Val Arg Thr Ala Ser Gly Gln Arg Gln Asp Leu Pro Ile Leu Val Ala

85 90 95

Lys Asp Asn Val Val Arg Val Ala Lys Ser Trp Gln Gly Arg Val Ser

100 105 110

Leu Pro Ser Tyr Pro Arg Arg Arg Ala Asn Ala Thr Leu Leu Leu Gly

115 120 125

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

130 135 140

Gly Ile Glu Asp Glu Gln Asp Leu Val Pro Leu Glu Val Thr Gly Val

145 150 155 160

Val Phe His Tyr Arg Ser Ala Arg Asp Arg Tyr Ala Leu Thr Phe Ala

165 170 175

Glu Ala Gln Glu Ala Cys Arg Leu Ser Ser Ala Ile Ile Ala Ala Pro

180 185 190

Arg His Leu Gln Ala Ala Phe Glu Asp Gly Phe Asp Asn Cys Asp Ala

195 200 205

Gly Trp Leu Ser Asp Arg Thr Val Arg Tyr Pro Ile Thr Gln Ser Arg

210 215 220

Pro Gly Cys Tyr Gly Asp Arg Ser Ser Leu Pro Gly Val Arg Ser Tyr

225 230 235 240

Gly Arg Arg Asn Pro Gln Glu Leu Tyr Asp Val Tyr Cys Phe Ala Arg

245 250 255

Glu Leu Gly Gly Glu Val Phe Tyr Val Gly Pro Ala Arg Arg Leu Thr

260 265 270

Leu Ala Gly Ala Arg Ala Gln Cys Arg Arg Gln Gly Ala Ala Leu Ala

275 280 285

Ser Val Gly Gln Leu His Leu Ala Trp His Glu Gly Leu Asp Gln Cys

290 295 300

Asp Pro Gly Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Gln Thr

305 310 315 320

Pro Arg Arg Arg Cys Gly Gly Pro Ala Pro Gly Val Arg Thr Val Tyr

325 330 335

Arg Phe Ala Asn Arg Thr Gly Phe Pro Ser Pro Ala Glu Arg Phe Asp

340 345 350

Ala Tyr Cys Phe Arg Ala His His Pro Thr Ser Gln His Gly Asp Leu

355 360 365

Glu Thr Pro Ser Ser Gly Asp Glu Gly Glu Ile Leu Ser Ala Glu Gly

370 375 380

Pro Pro Val Arg Glu Leu Glu Pro Thr Leu Glu Glu Glu Glu Glu Val Val

385 390 395 400

Thr Pro Asp Phe Gln Glu Pro Leu Val Ser Ser Gly Glu Glu Glu Thr

405 410 415

Leu Ile Leu Glu Glu Lys Gln Glu Ser Gln Gln Thr Leu Ser Pro Thr

420 425 430

Pro Gly Asp Pro Met Leu Ala Ser Trp Pro Thr Gly Glu Val Trp Leu

435 440 445

Ser Thr Val Ala Pro Ser Pro Ser Asp Met Gly Ala Gly Thr Ala Ala

450 455 460

Ser Ser His Thr Glu Val Ala Pro Thr Asp Pro Met Pro Arg Arg Arg

465 470 475 480

Gly Arg Phe Lys Gly Leu Asn Gly Arg Tyr Phe Gln Gln Gln Glu Pro

485 490 495

Glu Pro Gly Leu Gln Gly Gly Met Glu Ala Ser Ala Gln Pro Pro Thr

500 505 510

Ser Glu Ala Ala Val Asn Gln Met Glu Pro Pro Leu Ala Met Ala Val

515 520 525

Thr Glu Met Leu Gly Ser Gly Gln Ser Arg Ser Pro Trp Ala Asp Leu

530 535 540

Thr Asn Glu Val Asp Met Pro Gly Ala Gly Ser Ala Gly Gly Lys Ser

545 550 555 560

Ser Pro Glu Pro Trp Leu Trp Pro Pro Thr Met Val Pro Pro Ser Ile

565 570 575

Ser Gly His Ser Arg Ala Pro Val Leu Glu Leu Glu Lys Ala Glu Gly

580 585 590

Pro Ser Ala Arg Pro Ala Thr Pro Asp Leu Phe Trp Ser Pro Leu Glu

595 600 605

Ala Thr Val Ser Ala Pro Ser Pro Ala Pro Trp Glu Ala Phe Pro Val

610 615 620

Ala Thr Ser Pro Asp Leu Pro Met Met Ala Met Leu Arg Gly Pro Lys

625 630 635 640

Glu Trp Met Leu Pro His Pro Thr Pro Ile Ser Thr Glu Ala Asn Arg

645 650 655

Val Glu Ala His Gly Glu Ala Thr Ala Thr Ala Pro Pro Ser Pro Ala

660 665 670

Ala Glu Thr Lys Val Tyr Ser Leu Pro Leu Ser Leu Thr Pro Thr Gly

675 680 685

Gln Gly Gly Glu Ala Met Pro Thr Thr Pro Glu Ser Pro Arg Ala Asp

690 695 700

Phe Arg Glu Thr Gly Glu Thr Ser Pro Ala Gln Val Asn Lys Ala Glu

705 710 715 720

His Ser Ser Ser Ser Pro Trp Pro Ser Val Asn Arg Asn Val Ala Val

725 730 735

Gly Phe Val Pro Thr Glu Thr Ala Thr Glu Pro Thr Gly Leu Arg Gly

740 745 750

Ile Pro Gly Ser Glu Ser Gly Val Phe Asp Thr Ala Glu Ser Pro Thr

755 760 765

Ser Gly Leu Gln Ala Thr Val Asp Glu Val Gln Asp Pro Trp Pro Ser

770 775 780

Val Tyr Ser Lys Gly Leu Asp Ala Ser Ser Pro Ser Ala Pro Leu Gly

785 790 795 800

Ser Pro Gly Val Phe Leu Val Pro Lys Val Thr Pro Asn Leu Glu Pro

805 810 815

Trp Val Ala Thr Asp Glu Gly Pro Thr Val Asn Pro Met Asp Ser Thr

820 825 830

Val Thr Pro Ala Pro Ser Asp Ala Ser Gly Ile Trp Glu Pro Gly Ser

835 840 845

Gln Val Phe Glu Glu Ala Glu Ser Thr Thr Leu Ser Pro Gln Val Ala

850 855 860

Leu Asp Thr Ser Ile Val Thr Pro Leu Thr Thr Leu Glu Gln Gly Asp

865 870 875 880

Lys Val Gly Val Pro Ala Met Ser Thr Leu Gly Ser Ser Ser Ser Ser Gln

885 890 895

Pro His Pro Glu Pro Glu Asp Gln Val Glu Thr Gln Gly Thr Ser Gly

900 905 910

Ala Ser Val Pro Pro His Gln Ser Ser Pro Leu Gly Lys Pro Ala Val

915 920 925

Pro Pro Gly Thr Pro Thr Ala Ala Ser Val Gly Glu Ser Ala Ser Val

930 935 940

Ser Ser Gly Glu Pro Thr Val Pro Trp Asp Pro Ser Ser Thr Leu Leu

945 950 955 960

Pro Val Thr Leu Gly Ile Glu Asp Phe Glu Leu Glu Val Leu Ala Gly

965 970 975

Ser Pro Gly Val Glu Ser Phe Trp Glu Glu Val Ala Ser Gly Glu Glu

980 985 990

Pro Ala Leu Pro Gly Thr Pro Met Asn Ala Gly Ala Glu Glu Val His

995 1000 1005

Ser Asp Pro Cys Glu Asn Asn Pro Cys Leu His Gly Gly Thr Cys

1010 1015 1020

Asn Ala Asn Gly Thr Met Tyr Gly Cys Ser Cys Asp Gln Gly Phe

1025 1030 1035

Ala Gly Glu Asn Cys Glu Ile Asp Ile Asp Asp Cys Leu Cys Ser

1040 1045 1050

Pro Cys Glu Asn Gly Gly Thr Cys Ile Asp Glu Val Asn Gly Phe

1055 1060 1065

Val Cys Leu Cys Leu Pro Ser Tyr Gly Gly Ser Phe Cys Glu Lys

1070 1075 1080

Asp Thr Glu Gly Cys Asp Arg Gly Trp His Lys Phe Gln Gly His

1085 1090 1095

Cys Tyr Arg Tyr Phe Ala His Arg Arg Ala Trp Glu Asp Ala Glu

1100 1105 1110

Lys Asp Cys Arg Arg Arg Ser Gly His Leu Thr Ser Val His Ser

1115 1120 1125

Pro Glu Glu His Ser Phe Ile Asn Ser Phe Gly His Glu Asn Thr

1130 1135 1140

Trp Ile Gly Leu Asn Asp Arg Ile Val Glu Arg Asp Phe Gln Trp

1145 1150 1155

Thr Asp Asn Thr Gly Leu Gln Phe Glu Asn Trp Arg Glu Asn Gln

1160 1165 1170

Pro Asp Asn Phe Phe Ala Gly Gly Glu Asp Cys Val Val Met Val

1175 1180 1185

Ala His Glu Ser Gly Arg Trp Asn Asp Val Pro Cys Asn Tyr Asn

1190 1195 1200

Leu Pro Tyr Val Cys Lys Lys Gly Thr Val Leu Cys Gly Pro Pro

1205 1210 1215

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

1220 1225 1230

Tyr Asn Val His Ala Thr Val Arg Tyr Gln Cys Asn Glu Gly Phe

1235 1240 1245

Ala Gln His His Val Ala Thr Ile Arg Cys Arg Ser Asn Gly Lys

1250 1255 1260

Trp Asp Arg Pro Gln Ile Val Cys Thr Lys Pro Arg Arg Ser His

1265 1270 1275

Arg Met Arg Arg His His His His His Gln His His His Gln His

1280 1285 1290

His His His Lys Ser Arg Lys Glu Arg Arg Lys His Lys Lys His

1295 1300 1305

Pro Thr Glu Asp Trp Glu Lys Asp Glu Gly Asn Phe Cys

1310 1315 1320

<210> 135

<211> 911

<212> PRT

<213> Homo sapiens

<400> 135

Met Ala Gln Leu Phe Leu Pro Leu Leu Ala Ala Leu Val Leu Ala Gln

1 5 10 15

Ala Pro Ala Ala Leu Ala Asp Val Leu Glu Gly Asp Ser Ser Glu Asp

20 25 30

Arg Ala Phe Arg Val Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val

35 40 45

Leu Gly Gly Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro

50 55 60

Pro Pro Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr

65 70 75 80

Phe Leu Ser Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly Val

85 90 95

Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro Ala

100 105 110

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

115 120 125

Pro Asn Asp Ser Gly Ile Tyr Arg Cys Glu Val Gln His Gly Ile Asp

130 135 140

Asp Ser Ser Asp Ala Val Glu Val Lys Val Lys Gly Val Val Phe Leu

145 150 155 160

Tyr Arg Glu Gly Ser Ala Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln

165 170 175

Glu Ala Cys Ala Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu

180 185 190

Tyr Ala Ala Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu

195 200 205

Ser Asp Gln Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala Cys

210 215 220

Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr Gly Val Val

225 230 235 240

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

245 250 255

Gly Glu Leu Phe Leu Gly Asp Pro Glu Lys Leu Thr Leu Glu Glu

260 265 270

Ala Arg Ala Tyr Cys Gln Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly

275 280 285

Gln Leu Tyr Ala Ala Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly

290 295 300

Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln

305 310 315 320

Arg Cys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro

325 330 335

Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val Tyr Cys

340 345 350

Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu Ala Ser Asn Pro

355 360 365

Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu Ala Ile Val Thr Val Thr

370 375 380

Glu Thr Leu Glu Glu Leu Gln Leu Pro Gln Glu Ala Thr Glu Ser Glu

385 390 395 400

Ser Arg Gly Ala Ile Tyr Ser Ile Pro Ile Met Glu Asp Gly Gly Gly

405 410 415

Gly Ser Ser Thr Pro Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu

420 425 430

Glu Phe Glu Thr Gln Ser Met Val Pro Thr Gly Phe Ser Glu Glu

435 440 445

Glu Gly Lys Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu Glu

450 455 460

Lys Glu Glu Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala Leu Trp

465 470 475 480

Ala Trp Pro Ser Glu Leu Ser Ser Pro Gly Pro Glu Ala Ser Leu Pro

485 490 495

Thr Glu Pro Ala Ala Gln Glu Glu Ser Leu Ser Gln Ala Pro Ala Arg

500 505 510

Ala Val Leu Gln Pro Gly Ala Ser Pro Leu Pro Asp Gly Glu Ser Glu

515 520 525

Ala Ser Arg Pro Pro Arg Val His Gly Pro Thr Glu Thr Leu Pro

530 535 540

Thr Pro Arg Glu Arg Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val

545 550 555 560

Glu Ala Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly

565 570 575

Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly Ala Pro

580 585 590

Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg Glu Leu Glu

595 600 605

Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr Ala Pro Ala Gly Thr Ser

610 615 620

Val Gln Ala Gln Pro Val Leu Pro Thr Asp Ser Ala Ser Arg Gly Gly

625 630 635 640

Val Ala Val Val Pro Ala Ser Gly Asp Cys Val Pro Ser Pro Cys His

645 650 655

Asn Gly Gly Thr Cys Leu Glu Glu Glu Glu Gly Val Arg Cys Leu Cys

660 665 670

Leu Pro Gly Tyr Gly Gly Asp Leu Cys Asp Val Gly Leu Arg Phe Cys

675 680 685

Asn Pro Gly Trp Asp Ala Phe Gln Gly Ala Cys Tyr Lys His Phe Ser

690 695 700

Thr Arg Arg Ser Trp Glu Glu Ala Glu Thr Gln Cys Arg Met Tyr Gly

705 710 715 720

Ala His Leu Ala Ser Ile Ser Thr Pro Glu Glu Gln Asp Phe Ile Asn

725 730 735

Asn Arg Tyr Arg Glu Tyr Gln Trp Ile Gly Leu Asn Asp Arg Thr Ile

740 745 750

Glu Gly Asp Phe Leu Trp Ser Asp Gly Val Pro Leu Leu Tyr Glu Asn

755 760 765

Trp Asn Pro Gly Gln Pro Asp Ser Tyr Phe Leu Ser Gly Glu Asn Cys

770 775 780

Val Val Met Val Trp His Asp Gln Gly Gln Trp Ser Asp Val Pro Cys

785 790 795 800

Asn Tyr His Leu Ser Tyr Thr Cys Lys Met Gly Leu Val Ser Cys Gly

805 810 815

Pro Pro Pro Glu Leu Pro Leu Ala Gln Val Phe Gly Arg Pro Arg Leu

820 825 830

Arg Tyr Glu Val Asp Thr Val Leu Arg Tyr Arg Cys Arg Glu Gly Leu

835 840 845

Ala Gln Arg Asn Leu Pro Leu Ile Arg Cys Gln Glu Asn Gly Arg Trp

850 855 860

Glu Ala Pro Gln Ile Ser Cys Val Pro Arg Arg Pro Ala Arg Ala Leu

865 870 875 880

His Pro Glu Glu Asp Pro Glu Gly Arg Gln Gly Arg Leu Leu Gly Arg

885 890 895

Trp Lys Ala Leu Leu Ile Pro Ser Ser Pro Met Pro Gly Pro

900 905 910

<210> 136

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 136

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 137

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 137

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 138

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 138

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Ala

115

<210> 139

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 139

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Ala

115

<210> 140

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 140

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 141

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 141

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Ala

115

<210> 142

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 142

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 143

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 143

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Ala

115

<210> 144

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 144

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Ala Ala

115

<210> 145

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 145

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Ala Ala Ala

115

<210> 146

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 146

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Gly

115

<210> 147

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 147

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Gly Gly

115

<210> 148

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 148

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Gly Gly Gly

115

<210> 149

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 149

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 150

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 150

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Ala

115

<210> 151

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 151

Ser Phe Gly Met Ser

fifteen

<210> 152

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 152

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

1 5 10 15

gly

<210> 153

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 153

Gly Gly Ser Leu Ser Arg

fifteen

<210> 154

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 154

Ala Ala Ala

one

<210> 155

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 155

Gly Gly Gly Gly Ser

fifteen

<210> 156

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 156

Ser Gly Gly Ser Gly Gly Ser

fifteen

<210> 157

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 157

Gly Gly Gly Gly Gly Gly Gly Ser

fifteen

<210> 158

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 158

Gly Gly Gly Gly Ser Gly Gly Gly Ser

fifteen

<210> 159

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 159

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 160

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 160

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

1 5 10 15

<210> 161

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 161

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

1 5 10 15

Gly Ser

<210> 162

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 162

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

1 5 10 15

Gly Gly Gly Ser

20

<210> 163

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 163

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

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 164

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 164

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

1 5 10 15

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

20 25 30

<210> 165

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 165

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

1 5 10 15

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

20 25 30

Gly Gly Ser

35

<210> 166

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 166

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

1 5 10 15

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

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser

35 40

<210> 167

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 167

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

1 5 10 15

<210> 168

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 168

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

1 5 10 15

Thr His Thr Cys Pro Pro Cys Pro

20

<210> 169

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 169

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

1 5 10

<210> 170

<211> 62

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker Sequence

<400> 170

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

<210> 171

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Nanobody Sequence

<400> 171

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Lys Ser Ala

115

<210> 172

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> myc tag

<400> 172

Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala

1 5 10 15

Ala

<---

Claims (375)

1. An immunoglobulin single variable domain (ISV) that specifically binds to human aggrecan, preferably human aggrecan having the sequence shown in SEQ ID NO: 125 and optionally dog aggrecan having the sequence shown in SEQ ID NO: 126, bovine aggrecan having the sequence shown in SEQ ID NO: 127; rat aggrecan having the sequence shown in SEQ ID NO: 128; porcine aggrecan having the sequence shown in SEQ ID NO: 129; mouse aggrecan having the sequence shown in SEQ ID NO: 130, rabbit aggrecan having the sequence shown in SEQ ID NO: 131; cynomolgus monkey aggrecan having the sequence shown in SEQ ID NO: 132 and/or rhesus monkey aggrecan having the sequence shown in SEQ ID NO: 133, wherein (A) i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 24, 20, 21, 109; and b) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 24, where - in position 2, S is replaced by R, F, I or T; - in position 3 T is replaced by I; - in position 5 I replaced by S; - in position 6 I replaced by S, T or M; - in position 7 N is replaced by Y or R; - in position 8 V is replaced by A, Y, T or G; - in position 9 V replaced by M; and/or - in position 10 R replaced by G, K or A; and ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 42, 38, 39 and 110; and d) amino acid sequences that differ by 5, 4, 3, 2 or 1 amino acids compared to the amino acid sequence of SEQ ID NO: 42, where - in position 1, T is replaced by A or G; - S or N inserted between position 3 and position 4; - in position 3 S replaced by R, W, N or T; - in position 4 S is replaced by T or G; - in position 5 G is replaced by S; - in position 6 G is replaced by S or R; - in position 7, N is replaced by S, T or R; - in position 8 A replaced by T; and/or - in position 9, N is replaced by D or Y; and iii) CDR3 is selected from the group consisting of: e) SEQ ID NOS: 60, 56, 57 and 111; and f) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 60, where - in position 1, P is replaced by G, R, D or E or absent; - in position 2 T replaced by R, L, P or V or missing; - in position 3 T replaced by M, S or R or missing; - in position 4 H is replaced by D, Y, G or T; - in position 5 Y is replaced by F, V, T or G; - in position 6 G is replaced by L, D, S, Y or W; - R, T, Y or V is inserted between positions 6 and 7; - in position 7 G is replaced by P or S; - in position 8, V is replaced by G, T, H, R, L or Y; - in position 9 Y is replaced by R, A, S, D or G; - in position 10 Y is replaced by N, E, G, W or S; - W inserted between positions 10 and 11; - in position 11 G is replaced by S, K or Y; - in position 12 P replaced by E or D or missing; and/or - in position 13 Y is replaced by L or absent; (B) (i) CDR1 is selected from the group consisting of: a) SEQ ID NOs: 32, 30 and 23; and b) amino acid sequences that differ by 3, 2 or 1 amino acids compared to the amino acid sequence of SEQ ID NO: 32, where - in position 2 R is replaced by L; - in position 6 S replaced by T; and/or - in position 8 T replaced by A; and ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 50, 41, 48 and 51; and d) amino acid sequences in which there are 2 or 1 amino acid differences compared to the amino acid sequence of SEQ ID NO: 50, where - in position 7 G is replaced by S or R; and/or - in position 8 R is replaced by T; and iii) CDR3 is selected from the group consisting of: e) SEQ ID NOS: 68, 59, 66 and 69; and f) amino acid sequences that differ by 5, 4, 3, 2 or 1 amino acids compared to the amino acid sequence of SEQ ID NO: 68, where - in position 4 R is replaced by V or P; - in position 6 A is replaced by Y; - in position 7 S replaced by T; - in position 8 S is absent; - in position 9, N is replaced by P; - in position 10 R is replaced by T or L; - in position 11 G is replaced by E; and/or - in position 12 L is replaced by T or V; or (C) (i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 28; and b) amino acid sequences that differ by 5, 4, 3, 2 or 1 amino acids compared to the amino acid sequence of SEQ ID NO: 28, where - in position 1 G is replaced by R; - in position 2 P is replaced by S or R; - in position 3 T is replaced by I; - in position 5 S replaced by N; - in position 6, R is replaced by N, M or S; - in position 7, Y is replaced by R or is missing; - in position 8 A replaced by F or missing; and/or - in position 10 G is replaced by Y; and ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 46; and d) amino acid sequences that differ by 5, 4, 3, 2 or 1 amino acids compared to the amino acid sequence of SEQ ID NO: 46, where - in position 1 A is replaced by S or Y; - in position 4 W is replaced by L; - in position 5 S replaced by N; - in position 6 S is absent; - in position 7 G is absent; - in position 8 G is replaced by A; - in position 9, R is replaced by S, D or T; and/or - in position 11 Y is replaced by N or R; and iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 64; and f) amino acid sequences that differ by 5, 4, 3, 2 or 1 amino acids compared to the amino acid sequence of SEQ ID NO: 64, where - in position 1 A is replaced by R or F; - in position 2, R is replaced by I or L; - in position 3 I replaced by H or Q; - in position 4 P is replaced by G or N; - in position 5 V is replaced by S; - in position 6, R is replaced by G, N or F; - in position 7 T is replaced by R, W or Y; - in position 8, Y is replaced by R or S or is missing; - in position 9 T replaced by S or missing; - in position 10 S is replaced by E, K or absent; - in position 11 E replaced by N, A or missing; - in position 12 W is replaced by D or absent; - in position 13 N replaced by D or missing; - in position 14 Y is absent; and/or - D and N added after position 14 in SEQ ID NO: 64. 2. ISV according to claim 1, characterized in that it essentially consists of 4 framework regions FR1-FR4, respectively, and 3 complementarity-determining regions CDR1-CDR3, respectively, in which: - CDR1 is selected from the group consisting of SEQ ID NOs: 24, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 and 109, preferably selected from the group consisting of SEQ ID NOs: 24, 20, 21, 25, 27, 29, 31, 34, 35, 36, 37 and 109; - CDR2 is selected from the group consisting of SEQ ID NOs: 42, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 110, preferably selected from the group consisting of SEQ ID NOs: 42, 38, 39, 43, 45, 47, 49, 50, 53, 54, 55 and 110; and - CDR3 is selected from the group consisting of SEQ ID NOs: 60, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 111 is preferably selected from the group consisting of SEQ ID NOs: 60, 56, 57, 61, 63, 65, 67, 71, 72, 73, 74 and 111. 3. An ISV according to claim 1 or 2, which essentially consists of 4 framework regions FR1-FR4, respectively, and 3 complementarity-determining regions CDR1-CDR3, respectively, in which: i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 24, 20, 21, 109; b) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 24, where - in position 2 S changed to R, F, I or T; - in position 3 T changed to I; - in position 5 I was changed to S; - in position 6 I was changed to S, T or M; - in position 7 N changed to Y or R; - in position 8 V changed to A, Y, T or G; - in position 9 V changed to M; and/or - in position 10 R changed to G, K or A; and/or ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 42, 38, 39 and 110; and d) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 42, where - in position 1, T is changed to A or G; - S or N inserted between position 3 and position 4; - in position 3 S changed to R, W, N or T; - in position 4 S changed to T or G; - in position 5 G changed to S; - in position 6 G is changed to S or R; - in position 7 N changed to S, T or R; - in position 8 A changed to T; and/or - in position 9, N is changed to D or Y; and/or iii) CDR3 is selected from the group consisting of: e) SEQ ID NOS: 60, 56, 57 and 111; and f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 60, where - in position 1 P changed to G, R, D or E or missing; - in position 2, T is changed to R, L, P or V or absent; - in position 3, T is changed to M, S or R or absent; - in position 4 H changed to D, Y, G or T; - in position 5 Y changed to F, V, T or G; - in position 6 G is changed to L, D, S, Y or W; - R, T, Y or V is inserted between position 6 and position 7; - in position 7 G is changed to P or S; - in position 8 V is changed to G, T, H, R, L or Y; - in position 9, Y is changed to R, A, S, D or G; - in position 10 Y changed to N, E, G, W or S; - W inserted between position 10 and position 11; - in position 11 G is changed to S, K or Y; - in position 12 P is changed to E or D or missing; and/or - in position 13 Y changed to L or missing. 4. ISV according to claim 1 or 2, characterized in that it essentially consists of 4 framework regions FR1-FR4, respectively, and 3 complementarity-determining regions CDR1-CDR3, respectively, in which: i) CDR1 is selected from the group consisting of: a) SEQ ID NOs: 32, 30 and 23; and b) amino acid sequences that have a 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 32, where - in position 2 R changed to L; - in position 6 S changed to T; and/or - in position 8 T changed to A; and/or ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 50, 41, 48 and 51; and d) amino acid sequences that have a difference of 2 or 1 amino acid from the amino acid sequence of SEQ ID NO: 50, where - in position 7 G changed to S or R; and/or - in position 8 R changed to T; and/or iii) CDR3 is selected from the group consisting of: e) SEQ ID NOS: 68, 59, 66 and 69; and f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 68, where - in position 4 R changed to V or P; - in position 6 A changed to Y; - in position 7 S changed to T; - in position 8 S is absent; - in position 9 N changed to P; - in position 10 R changed to T or L; - in position 11 G changed to E; and/or - in position 12 L changed to T or V. 5. ISV according to claim 1 or 2, which essentially consists of 4 framework regions FR1-FR4, respectively, and 3 complementarity-determining regions CDR1-CDR3, respectively, in which: i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 28; and b) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 28, where - in position 1 G changed to R; - in position 2 P changed to S or R; - in position 3 T changed to I; - in position 5 S changed to N; - in position 6, R is changed to N, M or S; - in position 7 Y changed to R or missing; - in position 8 A changed to F or missing; and/or - in position 10 G changed to Y; and/or ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 46; and d) amino acid sequences that have a 5, 4, 3, 2, or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 46, where - in position 1 A changed to S or Y; - in position 4 W changed to L; - in position 5 S changed to N; - in position 6 S is absent; - in position 7 G is absent; - in position 8 G changed to A; - in position 9 R changed to S, D or T; and/or - in position 11 Y changed to N or R; and/or iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 64; and f) amino acid sequences that have a 5, 4, 3, 2 or 1 amino acid difference from the amino acid sequence of SEQ ID NO: 64, where - in position 1 A changed to R or F; - in position 2 R changed to I or L; - in position 3, I was changed to H or Q; - in position 4 P changed to G or N; - in position 5 V changed to S; - in position 6 R changed to G, N or F; - in position 7, T is changed to R, W or Y; - in position 8 Y changed to R or S or missing; - in position 9, T is changed to S or absent; - in position 10 S changed to E, K or absent; - in position 11 E changed to N, A or missing; - in position 12 W changed to D or absent; - in position 13 N changed to D or missing; - in position 14 Y is absent; and/or - D and N are added after position 14 of SEQ ID NO: 64. 6. ISV according to any one of paragraphs. 2-5, characterized in that the specified ISV is selected from the group of ISVs in which: - CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 42 and CDR3 is SEQ ID NO: 60; - CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 38 and CDR3 is SEQ ID NO: 56; - CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 39 and CDR3 is SEQ ID NO: 57; - CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 43 and CDR3 is SEQ ID NO: 61; - CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 45 and CDR3 is SEQ ID NO: 63; - CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 47 and CDR3 is SEQ ID NO: 65; - CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 49 and CDR3 is SEQ ID NO: 67; - CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 50 and CDR3 is SEQ ID NO: 71; - CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53 and CDR3 is SEQ ID NO: 72; - CDR1 is SEQ ID NO: 36, CDR2 is SEQ ID NO: 54 and CDR3 is SEQ ID NO: 73; - CDR1 is SEQ ID NO: 37, CDR2 is SEQ ID NO: 55 and CDR3 is SEQ ID NO: 74; - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 50 and CDR3 is SEQ ID NO: 68; - CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 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; - CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 41 and CDR3 is SEQ ID NO: 59; - CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 46 and CDR3 is SEQ ID NO: 64; CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58; - CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 44 and CDR3 is SEQ ID NO: 62; and - CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 52; and CDR3 is SEQ ID NO: 70. 7. ISV according to any one of paragraphs. 1-6, characterized in that said ISV is selected from the group consisting of ISVs consisting of SEQ ID NOs: 1-19 and 114-118, preferably SEQ ID NOs: 5, 1, 2, 6, 8, 10, 12 , 16, 17, 18 and 19, and ISVs that have greater than 80%, e.g. 90% or 95% sequence identity with any of SEQ ID NOs: 1-19 and 114-118, preferably SEQ ID NOs: 5, 1 , 2, 6, 8, 10, 12, 16, 17, 18 and 19. 8. An anti-aggrecan polypeptide that binds to the G2 domain of aggrecan, in which (A) i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 24, 20, 21, 109; and b) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 24, where - in position 2, S is replaced by R, F, I or T; - in position 3 T is replaced by I; - in position 5 I replaced by S; - in position 6 I replaced by S, T or M; - in position 7 N is replaced by Y or R; - in position 8 V is replaced by A, Y, T or G; - in position 9 V replaced by M; and/or - in position 10 R replaced by G, K or A; and ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 42, 38, 39 and 110; and d) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 42, where - in position 1, T is replaced by A or G; - S or N inserted between positions 3 and 4; - in position 3 S replaced by R, W, N or T; - in position 4 S is replaced by T or G; - in position 5 G is replaced by S; - in position 6 G is replaced by S or R; - in position 7, N is replaced by S, T or R; - in position 8 A replaced by T; and/or - in position 9, N is replaced by D or Y; and iii) CDR3 is selected from the group consisting of: e) SEQ ID NOS: 60, 56, 57 and 111; and f) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 60, where - in position 1, P is replaced by G, R, D or E or is absent; - in position 2 T replaced by R, L, P or V or missing; - in position 3 T replaced by M, S or R or missing; - in position 4 H is replaced by D, Y, G or T; - in position 5 Y is replaced by F, V, T or G; - in position 6 G is replaced by L, D, S, Y or W; - R, T, Y or V is inserted between positions 6 and 7; - in position 7 G is replaced by P or S; - in position 8, V is replaced by G, T, H, R, L or Y; - in position 9 Y is replaced by R, A, S, D or G; - in position 10 Y is replaced by N, E, G, W or S; - a W is inserted between positions 10 and 11; - in position 11 G is replaced by S, K or Y; - in position 12 P replaced by E or D or missing; and/or - in position 13 Y is replaced by L or absent; (B) (i) CDR1 is selected from the group consisting of: a) SEQ ID NOs: 32, 30 and 23; and b) amino acid sequences that differ by 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 32, where - in position 2 R is replaced by L; - in position 6 S replaced by T; and/or - in position 8 T replaced by A; and ii) CDR2 is selected from the group consisting of: c) SEQ ID NOs: 50, 41, 48 and 51; and d) amino acid sequences that differ by 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 50, where - in position 7 G is replaced by S or R; and/or - in position 8 R is replaced by T; and iii) CDR3 is selected from the group consisting of: e) SEQ ID NOS: 68, 59, 66 and 69; and f) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 68, where - in position 4 R is replaced by V or P; - in position 6 A is replaced by Y; - in position 7 S replaced by T; - in position 8 S is absent; - in position 9, N is replaced by P; - in position 10 R is replaced by T or L; - in position 11 G is replaced by E; and/or - in position 12 L is replaced by T or V; or (C) (i) CDR1 is selected from the group consisting of: a) SEQ ID NO: 28; and b) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 28, where - in position 1 G is replaced by R; - in position 2 P is replaced by S or R; - in position 3 T is replaced by I; - in position 5 S replaced by N; - in position 6, R is replaced by N, M or S; - in position 7, Y is replaced by R or is absent; - in position 8 A replaced by F or missing; and/or - in position 10 G is replaced by Y; and ii) CDR2 is selected from the group consisting of: c) SEQ ID NO: 46; and d) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 46, where - in position 1 A is replaced by S or Y; - in position 4 W is replaced by L; - in position 5 S replaced by N; - in position 6 S is absent; - in position 7 G is absent; - in position 8 G is replaced by A; - in position 9, R is replaced by S, D or T; and/or - in position 11 Y is replaced by N or R; and iii) CDR3 is selected from the group consisting of: e) SEQ ID NO: 64; and f) amino acid sequences that differ by 5, 4, 3, 2 amino acids or 1 amino acid compared to the amino acid sequence of SEQ ID NO: 64, where - in position 1 A is replaced by R or F; - in position 2, R is replaced by I or L; - in position 3 I replaced by H or Q; - in position 4 P is replaced by G or N; - in position 5 V is replaced by S; - in position 6, R is replaced by G, N or F; - in position 7 T is replaced by R, W or Y; - in position 8, Y is replaced by R or S or is missing; - in position 9 T replaced by S or missing; - in position 10 S is replaced by E, K or absent; - in position 11 E replaced by N, A or missing; - in position 12 W is replaced by D or absent; - in position 13 N replaced by D or missing; - in position 14 Y is absent; and/or - D and N added after position 14 in SEQ ID NO: 64, and which competes for aggrecan binding to the ISV of claim 6, wherein said aggrecan-directed polypeptide is selected from 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, dAb, an immunoglobulin that is suitable for use as a dAb, a nanobody, a VHH sequence, a humanized VHH sequence, an overblown VH sequence, or a VHH sequence that has been obtained by affinity maturation that binds to aggrecan G1 domain epitope group 1, a VHH sequence that has been obtained by affinity maturation and which binds to the G1-IGD-G2 domain of aggrecan, or a VHH sequence which has been obtained by affinity maturation. 9. A polypeptide that specifically binds to human aggrecan, preferably human aggrecan having the sequence shown in SEQ ID NO: 125, and optionally dog aggrecan having the sequence shown in SEQ ID NO: 126, bovine aggrecan having the sequence presented in SEQ ID NO: 127, rat aggrecan having the sequence shown in SEQ ID NO: 128; porcine aggrecan having the sequence shown in SEQ ID NO: 129; mouse aggrecan having the sequence shown in SEQ ID NO: 130, rabbit aggrecan having the sequence shown in SEQ ID NO: 131; cynomolgus monkey aggrecan having the sequence shown in SEQ ID NO: 132, and/or rhesus monkey aggrecan having the sequence shown in SEQ ID NO: 133, containing at least one ISV according to any one of paragraphs. 1-7 and possibly a second ISV, possibly a third ISV and possibly a fourth ISV, or containing a polypeptide according to claim 8. 10. A polypeptide that specifically binds to human aggrecan, preferably human aggrecan having the sequence shown in SEQ ID NO: 125, and optionally dog aggrecan having the sequence shown in SEQ ID NO: 126, bovine aggrecan having the sequence presented in SEQ ID NO: 127, rat aggrecan having the sequence shown in SEQ ID NO: 128; porcine aggrecan having the sequence shown in SEQ ID NO: 129; mouse aggrecan having the sequence shown in SEQ ID NO: 130, rabbit aggrecan having the sequence shown in SEQ ID NO: 131; cynomolgus monkey aggrecan having the sequence shown in SEQ ID NO: 132 and/or rhesus monkey aggrecan having the sequence shown in SEQ ID NO: 133, which contains at least two ISV according to any one of paragraphs. 1-7 and possibly a third ISV and possibly a fourth ISV. 11. A polypeptide according to claim 10, wherein said at least two ISVs may be the same or different, preferably said at least two ISVs are independently selected from the group consisting of SEQ ID NOs: 1-19 and 114-118. 12. The polypeptide according to any one of paragraphs. 9-11, characterized in that said at least one ISV according to any one of paragraphs. 1-8 or said at least two ISVs according to any one of paragraphs. 1-8 bind to a member of the serine protease family, cathepsins, (MMP)/matrixin matrix metalloproteinases or disintegrin A and metalloproteinase with thrombospondin motifs (ADAMTS), preferably MMP8, MMP13, MMP19, MMP20, ADAMTS5 (aggrecanase-2), ADAMTS4 ( aggrecanase-1) and/or ADAMTS11. 13. The polypeptide according to any one of paragraphs. 9-12, characterized in that said polypeptide further comprises a whey protein-binding fragment or whey protein. 14. A construct that specifically binds to human aggrecan, preferably human aggrecan having the sequence shown in SEQ ID NO: 125, and optionally dog aggrecan having the sequence shown in SEQ ID NO: 126, bovine aggrecan having the sequence presented in SEQ ID NO: 127, rat aggrecan having the sequence shown in SEQ ID NO: 128; porcine aggrecan having the sequence shown in SEQ ID NO: 129; mouse aggrecan having the sequence shown in SEQ ID NO: 130, rabbit aggrecan having the sequence shown in SEQ ID NO: 131; cynomolgus monkey aggrecan having the sequence shown in SEQ ID NO: 132 and/or rhesus monkey aggrecan having the sequence shown in SEQ ID NO: 133 and which contains or essentially consists of an ISV according to any one of paragraphs. 1-7 or a polypeptide according to any one of paragraphs. 8-13 and which further includes one or more other groups, residues, fragments or linking units, optionally linked through one or more peptide linkers. 15. A composition that specifically binds to human aggrecan, preferably human aggrecan having the sequence shown in SEQ ID NO: 125, and optionally dog aggrecan having the sequence shown in SEQ ID NO: 126, bovine aggrecan having the sequence presented in SEQ ID NO: 127, rat aggrecan having the sequence shown in SEQ ID NO: 128; porcine aggrecan having the sequence shown in SEQ ID NO: 129; mouse aggrecan having the sequence shown in SEQ ID NO: 130, rabbit aggrecan having the sequence shown in SEQ ID NO: 131; cynomolgus monkey aggrecan having the sequence shown in SEQ ID NO: 132 and/or rhesus monkey aggrecan having the sequence shown in SEQ ID NO: 133, containing in a pharmaceutically effective amount at least one ISV according to any one of paragraphs. 1-7, a polypeptide according to any one of paragraphs. 8-13, or the construct of claim 14, wherein said composition is preferably a pharmaceutical composition optionally further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant and optionally containing one or more other pharmaceutically active polypeptides and /or connections. 16. The use of ISV according to any one of paragraphs. 1-7 or a product containing an ISV according to any one of paragraphs. 1-7, for fixing drugs in the joint in the treatment of various diseases of the joint, where the specified product containing ISV according to any one of paragraphs. 1-7, selected from the composition according to p. 15, the polypeptide according to any one of paragraphs. 8-13, or designs according to item 14. 17. The use of ISV according to any one of paragraphs. 1-7 or a product containing an ISV according to any one of paragraphs. 1-7 in the prevention or treatment of 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, degenerative diseases of the lumbar disc, degenerative diseases joints and recurrent polychondritis, where the specified product containing ISV according to any one of paragraphs. 1-7, selected from the composition according to p. 15, the polypeptide according to any one of paragraphs. 8-13, or designs according to item 14.

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