RU2781182C2 - Adamts-binding immunoglobulins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, specifically to immunoglobulins binding a disintegrin and metalloproteinase with thrombospondin motifs 5 (hereinafter – ADAMTS5); it can be used in medicine for the treatment or prevention in an individual of a disease or a disorder, in which ADAMTS5 activity is involved. Polypeptide binding ADAMTS5 is proposed, containing at least 1 immunoglobulin single variable domain (ISVD) binding ADAMTS5 and containing 3 complementarity determining regions: CDR1 with SEQ ID NO: 21, CDR2 with SEQ ID NO: 37, and CDR3 with SEQ ID NO: 55.

EFFECT: invention provides polypeptides, which are ADAMTS5 antagonists relatively to inhibition of its enzyme activity with preservation of selectivity, showing improved preventive, therapeutic, and/or pharmacological properties, including a better safety profile compared to known antibodies to ADAMTS5.

26 cl, 14 dwg, 36 tbl, 16 ex

Description

1. FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to immunoglobulins that bind ADAMTS5, and more specifically, to polypeptides containing or essentially consisting of one or more of these 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, for example, immunoglobulin single variable domains (ISVDs), or polypeptides, as well as nucleic acids encoding such immunoglobulins or polypeptides (also referred to herein as "nucleic acid(s) by invention"; to methods for producing such immunoglobulins, polypeptides and constructs; to host cells expressing or capable of expressing such immunoglobulins or polypeptides; to compositions, and in particular to pharmaceutical compositions containing such immunoglobulins, polypeptides, constructs, nucleic acids and / or host cells, and to uses of immunoglobulins, polypeptides, constructs, nucleic acids, host cells and/or compositions, in particular for prophylactic and/or therapeutic purposes, such as the prophylactic and/or therapeutic purposes mentioned in the present description. Other Aspects, Embodiments, Benefits, etc. The applications of the invention will become apparent from the further description in this document.

2. BACKGROUND OF THE INVENTION

Osteoarthritis (OA) is one of the most common causes of disability worldwide. It affects 30 million Americans and is the most common joint disorder. It is expected to affect more than 20 percent of the US population by 2025. This disease is not systemic and is usually limited to a few joints. However, the disease can occur in all joints, most commonly in the joints of the knees, hips, arms, shoulders, and spine. OA is characterized by progressive erosion of the articular cartilage (the cartilage that covers bones) leading to chronic pain and disability. Ultimately, the disease leads to complete destruction of the articular cartilage, sclerosis of the underlying bone, osteophyte formation, etc., all of which lead to loss of mobility and pain. Osteoarthritis can be defined as a diverse group of conditions characterized by a combination of symptoms, signs resulting from defects in articular cartilage, and changes in adjacent tissues, including bone, tendons, and muscle. Pain is the most prominent symptom of OA and the most common reason patients seek medical attention. There is no curative treatment for OA, ie. current treatments do not inhibit structural destruction of the OA joint. Disease management is limited to treatments that are palliative at best and make little contribution to controlling the underlying cause of disease progression. Although the onset of the disease may be multifactorial, cartilage destruction appears to be the result of uncontrolled proteolytic destruction of the extracellular matrix (ECM). The most common components of the articular cartilage ECM are collagen (primarily collagen II) and proteoglycans, mainly aggrecan (Kiani et al., 2002 Cell Research 12:19-32).

Aggrecan is important for the proper functioning of articular cartilage because it provides the hydrated gel structure that gives the 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 long region between G2 and G3 for attachment of the glycosaminoglycan chain. This extended region contains two domains, one substituted with keratan sulfate chains (KS domain) and one with chondroitin sulfate chains (CS domain). The CS domain has 100-150 glycosaminoglycan (GAG) chains attached to it. Aggrecan forms large complexes with hyaluronan, in which 50-100 aggrecan molecules interact via the G1 domain and bind the protein to one hyaluronan molecule. After absorbing water (due to the GAG content), these complexes form a reversibly deformable gel resistant to compression. Structure, fluid retention, and function of articular cartilage are related to the content of aggrecan in the matrix, and to the amount of chondroitin sulfate bound to intact core protein. Structurally, OA is characterized by aggrecan degradation, progressively releasing the G3 and G2 domains (resulting in cartilage "deflation"), and ultimately, G1 domain release and collagen degradation, irreversibly destroying cartilage structure. The most significant aggrecan cleavage site for the pathogenesis of OA is located in the TEGE ³⁷³ ↓ ³⁷⁴ ARGS sequence. This cleavage site is located in the interglobular domain (IGD) of aggrecan. Antibodies recognizing neoepitope ³⁷⁴ ARGS led to the discovery of aggrecanase-1, proven to be ADAMTS4, and aggrecanase-2, proven to be ADAMTS5. Other related ADAMTS enzymes, including ADAMTS1, -8, -9, -15 and -20, were then shown to have aggrecanase activity. ADAMTS16 and 18 are also weak aggrecanases. ADAMTS and matrix metalloproteinases (MMPs) share an aggrecan binding site that is very similar in both sequence and overall shape (El Bakali et al., 2014 Future Medicinal Chemistry (Review) 6:1399).

ADAMTS enzymes (disintegrins and metalloproteinases with thrombospondin motifs) are secreted, multidomain, matrix-associated zinc metalloendopeptidases with diverse roles in tissue morphogenesis and pathophysiological remodeling, inflammation, and vascular biology (Kelwick et al., 2015 Genome Biology 16:113 ). The human family includes 19 members, which can be divided into subgroups based on their known substrates. Aggrecanases or proteoglycans include ADAMTS1, -4, -5, -8, 9, -15, and -20, which can cleave hyaluronan-binding extracellular proteins chondroitin sulfate proteoglycans (CSPGs), including aggrecan, versican, brevican, and neurocan. The two most preferred cleavage sites in bovine aggrecan are KEEE ¹⁶⁶⁷ ↓ ¹⁶⁶⁸ GLGS followed by GELE ¹⁴⁸⁰ ↓ ¹⁴⁸¹ GRGT. Cleavage then occurs at NITEGE ³⁷³ ↓ ³⁷⁴ ARGS in IGD (in which MMP is not cleaved), releasing the aforementioned neoepitope, and at TAQE ¹⁷⁷¹ ↓ ¹⁷⁷² AGEG and VSQE ¹⁸⁷¹ ↓ ¹⁸⁷² LGQR in the CS-2 region (Fosang et al., 2008 European Cells and Materials 15:11-26). These cleavage sites are highly conserved in human, bovine, mice and rats.

Various chains of evidence indicate that ADAMTS5 is a principle enzyme involved in the pathogenesis of osteoarthritis. ADAMTS5 is the main aggrecanase present in cartilage. In human cartilage explants and chondrocytes, ADAMTS5 knockdown attenuated aggrecan degradation, suggesting that this enzyme may play a role in human tissues. The expression of this enzyme is enhanced by cytokines, such as interleukin-1 and oncostatin-M, which provoke the destruction of aggrecan in tissues. ADAMTS5-formed aggrecan fragments have been detected in the synovial fluid and serum of patients with OA (Germaschewski et al., 2014 Osteoarthritis Cartilage 22:690-697).

ADAMTS5 is first synthesized as an inactive protein, including a protease domain at the N-terminus and an accessory domain at the C-terminus. The protease domain consists of a signal peptide, a prodomain with a furin recognition sequence, and a catalytic domain. The prodomain is cleaved by proprotein convertases to produce active enzymes. ADAMTS5 additionally contains accessory domains that are actively involved in substrate recognition and modulate the protease's affinity for its substrate(s) ("exosites"). The disintegrin-like domain, central thrombospondin-like (TS) type I repeat, cysteine-rich domain, spacer region, and additional TS motif of ADAMTS5 are accessory domains with potential exosite functions. The cysteine-rich domain appears to be essential for the binding and anchoring of ADAMTS5 to glycosaminoglycans. The greatest variability among members of the ADAMTS family is found in these accessory domains (Kelwick et al., 2015 Genome Biology 16:113).

Disease-modifying osteoarthritis drugs (DMOADs), which can be defined as drugs that inhibit the structural progression of a disease and ideally also improve symptoms and/or function, are in high demand. DMOADs are likely to be prescribed for long periods in this chronic disease of an aging population, thus evidence of superior safety in a target population with multiple comorbidities and potential drug interactions is needed.

Several pharmaceutical companies have developed small molecule ADAMTS5 inhibitors. Some of these compounds are claimed to be specific to ADAMTS5, while others also have an effect on other members of the ADAMTS family or even on MMPs. These cases of cross inhibition are thought to be responsible for musculoskeletal syndrome, a side effect induced by broad spectrum inhibitors and including arthralgia, myalgia, joint stiffness and tendinitis (Santamaria et al., 2015 Biochem J 471:391-401). Wyeth's aggrecanase inhibitor AGG-523 was used in 5 phase I clinical trials in healthy subjects and patients with OA, but was not used further. No other small molecule ADAMTS inhibitors have entered any further clinical development as a potential DMOAD (Bondeson et al., 2015 Drug Discovery 10:5-14). Indeed, despite a number of recent clinical trials specifically investigating DMOAD, no such drugs have been approved to date (El Bakali et al., 2014 Future Medicinal Chemistry (Review) 6:1399).

Given the success of targeted biological therapy using antibodies ("Ab"), interest has arisen in the development of similar therapeutic methods for OA. A Rottapharm study of the monoclonal antibody (mAb) CRB0017 targeting the ADAMTS5 spacer domain showed that in mice, intra-articular administration of this mAb significantly prevented disease progression in a dose-dependent manner (Chiusaroli et al., 2013 Osteoarthritis Cartilage 21:1807). There was no comparison with systemic administration, nor an assessment of the extent to which the mAb was leaking from the synovial space. Another study used systemic administration of mAb 12F4 in mice, showing both structural modification of the disease and improvement in pain-related behavior (Miller et al., 2014 Osteoarthritis Cartilage 22iii, S35). However, a single administration of the 12F4 mAb in the cynomolgus monkey caused focal bleeding, a dose-dependent increase in mean arterial pressure, and cardiac conduction abnormalities (more specifically, ST elevations and ECG ventricular arrhythmias) indicative of cardiac ischemia, which lasted up to 8 months after administration. single dose (Larkin et al., 2014 Osteoarthritis Cartilage 22iii, S483). These side effects stopped further clinical development of mAb 12F4.

WO2008/074840 from Ablynx NV describes the production of nanobodies® against members of the disintegrin and metalloproteinase (ADAM) family, including ADAMTS5.

Therapeutic interventions in the joints are further hampered by the difficulty of targeting drugs to the articular cartilage. Since articular cartilage is a vascular and lymphatic tissue, the traditional methods of drug delivery (oral, intravenous, intramuscular) ultimately depend on the transfer through the synovial space of drugs from the capillaries of the synovium to the cartilage through passive diffusion. This necessitates the development of intra-articular (IA) drug delivery.

On the other hand, IA delivery of therapeutic proteins is limited by their rapid clearance from the joint space and lack of retention within the cartilage; and limitation of large joints. The synovial retention time of the drug in the joint is often less than 24 hours (Edwards 2011 Vet J 190:15-21; Larsen et al., 2008 J Pham Sci 97:4622-4654). Due to the rapid elimination of most injected IA drugs, frequent injections are necessary to maintain effective concentrations (Owen et al., 1994 Br J Clin Pharmacol 38:349-355). In addition, IA delivery of therapeutic proteins is not practical for small joints, which precludes the treatment of, for example, fingers in OA.

The need for efficient DMOADs remains.

3. SUMMARY OF THE INVENTION

It is an object of the present invention to provide anti-OA polypeptides with improved prophylactic, therapeutic and/or pharmacological properties, in addition to other advantageous properties (eg, such as improved ease of preparation, good stability, and/or reduced cost) compared to amino acid sequences and antibodies. prior art. In particular, it is an object of the present invention to provide polypeptides that inhibit ADAMTS, and especially ADAMTS5.

ADAM, ADAMTS and MMP share aggrecan binding sites that are very similar in both sequence and overall shape. Because various other members of the ADAM (including TACE) and ADAMTS families have diverse roles in normal physiology, strongly associated with many common pathological conditions, including asthma, arthritis, malignancies, connective tissue disorders, or thrombotic thrombocytopenic purpura (El Bakali et al., supra ), the authors of the present invention recognize the importance of maintaining selectivity. For effective termination of ADAMTS5 activity alone, targeting the catalytic domain appears to be the best option. However, the catalytic domains of ADAMTS4 and ADAMTS5 share a high degree of sequence similarity (cf. El Bakali et al., supra). In addition, it turned out that the high conservation of the sequence of the catalytic domain between different species deters from a strong immune response.

Surprisingly, the ISVDs of the invention meet these seemingly mutually exclusive requirements of inhibiting ADAMTS5 (enzymatic) activity on the one hand while maintaining selectivity on the other hand.

The various monovalent ISVDs of the present invention were equally active with the conventional divalent antibody mAb 12F4 H4L0 in the AlphaLISA enzyme assay. In addition, the ISVDs of the present invention were also equally active with the conventional bivalent antibody mAb12F4 H4L0 at high or even excessive concentrations of the aggrecan substrate, resembling concentrations in the joints. On the other hand, in an ex vivo bovine explant assay even more closely resembling physiological conditions, most of the monovalent ISVDs had a better IC ₅₀ than comparative mAb 12F4 H4L0 (“12F4”) and mAb CRB0017 from Rottapharm. In the ex vivo human explant assay, the ISVDs of the present invention were also significantly better than the conventional prior art CRB0017 antibody as shown by IC ₅₀ .

After further modification of the ISVDs, taking into account various diverse and favorable features, including stability, affinity and inhibitory activity, as well as minimization of immunogenicity, these ISVDs were then evaluated in vivo.

Systemic administration of the ISVD of the present invention shows strong inhibition of aggrecanase activity in vivo as assessed in the cynomolgus monkey. In addition, ISVDs were also safe to use, unlike the prior art 12F4 antibody. Also in an in vivo medial meniscus destabilization model (DMM) in mice, the ISVD of the present invention has shown a structural advantage of up to 50% for both prophylactic and therapeutic treatment via systemic administration. Early treatment with the ISVD of the present invention produced a dose dependent, significant and significant symptomatic benefit during anterior cruciate ligament transection and medial meniscus resection (ACLT+tMx) induced OA in rats.

ISVDs are ultimately designed to inhibit ADAMTS5 in the joints and thus must withstand the conditions of synovial fluid in the joints, which contains various proteases. In addition to the favorable characteristics above, isolated ISVDs have been shown to be extremely stable in synovial fluid. This stability is believed to allow a less frequent dosing regimen.

Accordingly, the present invention provides a polypeptide comprising at least 1 immunoglobulin single variable domain (ISVD) binding disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), preferably wherein said ADAMTS is selected from the group consisting of ADAMTS1 - ADAMTS19, preferably ADAMTS5 , ADAMTS4, ADAMTS1, ADAMTS8, ADAMTS9 and ADAMTS15, and ADAMTS20, most preferably ADAMTS5. In one aspect, the invention provides a polypeptide as described herein, wherein said ADAMTS-binding ISVD, preferably ADAMTS5, does not bind to ADAMTS4, MMP1, or MMP14.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said ISVD specifically binding ADAMTS5 mainly consists of 4 framework regions (FR1-FR4, respectively) and 3 complementarity-determining regions (CDR1-CDR3, respectively). ) wherein (i) CDR1 is selected from the group consisting of SEQ ID NOs: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; and amino acid sequences having 1, 2 or 3 amino acid difference(s) from SEQ ID NOs: 21, 35, 20, 22, 25, 33, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; (ii) CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and amino acid sequences having 1, 2 or 3 amino acid difference(s) from SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and (iii) CDR3 is selected from the group consisting of SEQ ID NO: SEQ ID NO: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65 , 66, 67 and 70; and amino acid sequences having 1, 2, 3 or 4 amino acid difference(s) from SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said ISVD specifically binding ADAMTS5 is primarily composed 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: 22; and (b) an amino acid sequence having 1, 2, 3, 4, 5 or 6 amino acid difference(s) from SEQ ID NO: 22, where at position 2 S is replaced by R; in position 3 A replaced by T; in position 4 V is replaced by F; in position 6 V is replaced by S; in position 7 N is replaced by Y; and/or in position 10 A replaced by G; (ii) CDR2 is SEQ ID NO: 36; and (iii) CDR3 is SEQ ID NO: 54.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said ISVD specifically binding ADAMTS5 is primarily composed of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively) , in which (i) CDR1 is SEQ ID NO: 33; (ii) CDR2 is selected from the group consisting of (c) SEQ ID NO: 50; and (d) an amino acid sequence having 1, 2, or 3 amino acid difference(s) from SEQ ID NO: 50, where at position 8 M is replaced by I; in position 9, P is replaced by T; and/or in position 10 Y is replaced by F; and (iii) CDR3 is selected from the group consisting of (e) SEQ ID NO: 68; and (f) an amino acid sequence having 1 or 2 amino acid difference(s) from SEQ ID NO: 68, where at position 5 F is replaced by L; and/or in position 11 D is replaced by E.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said ISVD specifically binding ADAMTS5 is primarily composed of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively) in which (i) CDR1 is SEQ ID NO: 28; (ii) CDR2 is selected from the group consisting of (c) SEQ ID NO: 44; and (d) an amino acid sequence having 1, 2, or 3 amino acid difference(s) from SEQ ID NO: 44, where at position 3 S is replaced by T; in position 4 R is replaced by W; in position 8 T is replaced by I; and/or in position 9 T is replaced by L; and (iii) CDR3 is selected from the group consisting of (e) SEQ ID NO: 62; and (f) an amino acid sequence having 1 or 2 amino acid difference(s) from SEQ ID NO: 62, where at position 1 G is replaced by S; and/or in position 14 D is replaced by E.

In preferred embodiments of all aspects of the invention, the immunoglobulin single variable domain (ISVD) of the invention preferably consists of, or substantially consists of, 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions CDR1, CDR2, and CDR3, as described in the present description above and below. Preferred framework sequences are described, for example, in Table A-2 below and can be used in the ISVD of the invention. Preferably, the CDRs described in Table A-2 are combined with corresponding framework regions of the same ISVD design.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said ISVD is selected from the group ISVD, where: CDR1 is selected from the group consisting of SEQ ID NOs: 21, 35, 20, 22, 25, 33, 28 , 24, 23, 26, 27, 29, 30, 31, 32 and 34; CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and CDR3 is selected from the group consisting of SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70.

In an additional preferred aspect, the invention relates to a polypeptide as described in the present description, where the specified ISVD is selected from the group ISVD, where:

CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55;

CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 118;

CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 71;

CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54;

CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54;

CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58;

CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68;

CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69;

CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62;

CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63;

CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61;

CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57;

CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56;

CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59;

CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 119, and CDR3 is SEQ ID NO: 60;

CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60;

CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64;

CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65;

CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66;

CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; and

CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55.

In a further preferred aspect, the invention provides a polypeptide as described herein, wherein said ISVD is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide binds to ADAMTS5 with a K _D between 1E- ⁰⁷ M and 1E- ¹³ M, e.g. between 1E- ⁰⁸ M and 1E- ¹² M, preferably , not more than 1E- ⁰⁷ M, preferably lower than 1E- ⁰⁸ M or 1E- ⁰⁹ M, or even lower than 1E ¹⁰ M, for example, 5E- ¹¹ M, 4E- ¹¹ M, 3E ¹¹ M, 2E- ¹¹ M, 1.7E- ¹¹ M, 1E ¹¹ M, or even 5E- ¹² M, 4E- ¹² M, 3E ¹² M, 1E- ¹² M, for example, as determined by KinExA, or alternatively, by Gyrolab.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide inhibits ADAMTS5 activity with an IC ₅₀ between 1E- ⁰⁷ M and 1E- ¹² M, e.g. between 1E- ⁰⁸ M and 1E- ¹¹ M, e.g. , as determined by human FRET or human AlphaLISA analysis.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide inhibits the (enzymatic) activity of ^ADAMTS5 with an IC _{50 of} not more than 1E-07M, preferably 1E ^-08M , 5E- ^09M or 4E ^-9 M, 3E ^-9 M, 2E ^-9 M, e.g. 1E ^-9 M.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide modulates ^ADAMTS5 with an EC ₅₀ between 1E ^- 07M and 1E-12M, e.g. between 1E ^-08M and 1E ^- 11M, e.g. as determined by ELISA binding.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide binds to ADAMTS5 at a dissociation rate of less than 1E ^-04 s ^-1 , eg as determined by SPR.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said ADAMTS5 is human ADAMTS5 (SEQ ID NO: 149), bovine ADAMTS5 (SEQ ID NO: 150), rat ADAMTS5 (SEQ ID NO: 151) , guinea pig ADAMTS5 (SEQ ID NO: 152), mouse ADAMTS5 (SEQ ID NO: 153) or cynomolgus ADAMTS5, (SEQ ID NO: 154), preferably human ADAMTS5, most preferably SEQ ID NO: 149.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide is an antagonist of ADAMTS5 activity, e.g. ; preferably, it is an antagonist of ADAMTS5 aggrecanase activity.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide blocks the binding of ADAMTS5 to aggrecan by at least 20%, e.g., at least 30%, 40%, 50%, 60%, 70 %, 80%, 90%, 95% or even more, for example, as determined by FRET, AlplaLISA or ELISA.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide inhibits the protease activity of ADAMTS5, e.g. inhibits proteolysis of a substrate such as aggrecan, versican, brevican, neurocan, decorin and/or biglycan, preferably aggrecan.

In a further preferred aspect, the invention provides a polypeptide as described herein comprising at least 2 ISVDs, wherein at least 1 ISVD specifically binds to ADAMTS, preferably ADAMTS5, preferably selected from the group consisting of SEQ ID NO: 2 , 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18, preferably wherein said at least 2 ISVDs bind specifically to ADAMTS, preferably ADAMTS5.

In a further preferred aspect, the invention provides a polypeptide comprising two or more ISVDs that specifically bind to ADAMTS5, wherein (a) at least the "first" ISVD specifically binds to a first antigenic determinant, epitope, moiety, domain, subunit, or conformation of ADAMTS5 ; and where (b) at least the "second" ISVD specifically binds to a second antigenic determinant, epitope, moiety, domain, subunit, or conformation of ADAMTS5 different from the first antigenic determinant, epitope, moiety, domain, subunit, or conformation, respectively, preferably where said "first" ISVD specifically binding ADAMTS5 is selected from the group consisting of SEQ ID NOs: 2, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18, preferably wherein said "second" ISVD specifically binding ADAMTS5 is SEQ ID NO: 118 or 19, even more preferably said polypeptide is selected from the group consisting of SEQ ID NO: 127 (clone 130 049-093-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb) and SEQ ID NO: 128 (clone 131 9D3-093- alb).

In a further preferred aspect, the invention relates to a polypeptide as described herein, further comprising a serum albumin binding ISVD, preferably wherein said serum albumin binding ISVD mainly consists of 4 framework regions (FR1 - FR4, respectively) and 3 defining complementarity of regions (CDR1 - CDR3, respectively) in which CDR1 is SEQ ID NO: 146, CDR2 is SEQ ID NO: 147, and CDR3 is SEQ ID NO: 148, even more preferably, wherein said ISVD binding serum albumin selected from the group consisting of ALB8 (SEQ ID NO: 131), ALB23 (SEQ ID NO: 132), ALB129 (SEQ ID NO: 133), ALB132 (SEQ ID NO: 134), ALB11 (SEQ ID NO: 135), ALB11 (S112K)-A (SEQ ID NO: 136), ALB82 (SEQ ID NO: 137), ALB82-A (SEQ ID NO: 138), ALB82-AA (SEQ ID NO: 139), ALB82- AAA (SEQ ID NO: 140), ALB82-G (SEQ ID NO: 141), ALB82-GG (SEQ ID NO: 142), ALB82-GGG (SEQ ID NO: 143), ALB92 (SEQ ID N O: 144), and ALB223 (SEQ ID NO: 145), even more preferably, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 129 (clone 577 2F3 ^SO -Alb), SEQ ID NO: 130 (clone 579 2F3 ^SO -093-Alb), SEQ ID NO: 120 (clone 4 2A12-Alb), SEQ ID NO: 121 (clone 5 2D7-Alb), SEQ ID NO: 122 (clone 6 2F3-Alb), SEQ ID NO : 123 (clone 69 049-Alb), SEQ ID NO: 124 (clone 70 9D3-Alb), SEQ ID NO: 125 (clone 71 3B2-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb) , SEQ ID NO: 127 (clone 130 049-093-Alb), and SEQ ID NO: 128 (clone 131 9D3-093-Alb).

In a further preferred aspect, the invention relates to a polypeptide as described herein, further comprising at least one aggrecan-specific binding ISVD, preferably selected from the group consisting of SEQ ID NO: 156 (nanobody 00745 PEA114F08) and SEQ ID NO : 157 (nanobody 00747 PEA604F02).

In a further preferred aspect, the invention relates to a polypeptide as described herein, comprising at least 2 aggrecan-specific binding ISVDs, wherein said at least 2 aggrecan-specific binding ISVDs may be the same or different, preferably wherein said at least At least 2 aggrecan-specific ISVDs are independently selected from the group consisting of SEQ ID NOs: 156 and 157, even more preferably, wherein said aggrecan-specific ISVD specifically binds human aggrecan [SEQ ID NO: 155]. Preferably, said aggrecan-specific ISVD specifically binds dog aggrecan (see also Table 2), bovine aggrecan, rat aggrecan; pig aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and/or rhesus monkey aggrecan. Preferably, said ISVD that specifically binds aggrecan preferentially binds to cartilage tissue such as cartilage and/or meniscus.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide 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.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said at least two ISVDs are directly linked to each other or linked via a linker, preferably said linker is selected from the group consisting of SEQ ID NOs: 158 to 174 (i.e. SEQ ID NOs: 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 and 174), preferably SEQ ID NOs: 169.

In a further preferred aspect, the invention provides a polypeptide as described herein further comprising a C-terminal extension, preferably wherein said C-terminal extension is a (X)n C-terminal extension wherein n is 1-10 , preferably 1-5, eg 1, 2, 3, 4 or 5 (and preferably 1 or 2, eg 1); and each X is a (preferably natural) amino acid residue which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I ).

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide has at least 80%, 90%, 95%, or 100% sequence identity to any of SEQ ID NOs: 1-19 (i.e. SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19), 116-117 or 120- 130 (i.e. SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 and 130).

In an additional preferred aspect, the invention relates to a method for treating and/or preventing diseases or disorders in an individual, for example, those in which ADAMTS5 activity is involved, comprising administering a polypeptide according to any one of paragraphs. 1-43 to said individual in an amount effective to treat or prevent a symptom of said disease or disorder, preferably wherein said diseases or disorders are selected from the group consisting of arthropathies and chondrodystrophies, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration disease, degenerative joint disease, and recurrent polychondritis, osteochondritis dissecans, and aggrecanopathy. More preferably, said disease or disorder is an arthritic disease, and most preferably, osteoarthritis.

In an additional preferred aspect, the invention provides a polypeptide as described herein for use as a drug.

In a further preferred aspect, the invention relates to a polypeptide as described herein for use in the treatment or prevention of a symptom of an ADAMTS5 associated disease such as arthropathy and chondrodystrophy, arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration disease, degenerative joint disease, and recurrent polychondritis, osteochondritis dissecans, and aggrecanopathy. More preferably, said disease is an arthritic disease, and most preferably, osteoarthritis.

In a further preferred aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide cross-blocks the binding to ADAMTS5 of at least one of the polypeptides represented by any of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18 and/or its binding to ADAMTS5 is cross-blocked by at least one of the polypeptides represented by any of the SEQs ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

In a further preferred aspect, the invention relates to a polypeptide that cross-blocks the binding to ADAMTS5 of a polypeptide represented by any of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4 , 7, 117, 8, 12, 13, 14, 15 and 18, and/or whose binding to ADAMTS5 is cross-blocked by at least one of the polypeptides represented by any of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18, wherein said polypeptide contains at least one VH, VL, dAb, immunoglobulin single variable domain (ISVD), specifically binding ADAMTS5, where binding to ADAMTS5 modulates the activity of ADAMTS5.

Other aspects, advantages, applications and uses of the polypeptides and compositions will become apparent from the further description herein. Several documents are cited throughout the text of this description. The entire contents of each of the documents cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), either above or below, are hereby incorporated by reference. Nothing in the present description should be construed as an admission that this invention has no retroactive rights to such a description of the prior invention.

4. DESCRIPTION OF THE DRAWINGS

Figure 1: ADAMTS5 binding ISVDs do not inhibit MMP1 (A) or MMP14 (B) activity.

Figure 2: ADAMTS5 binding ISVDs do not inhibit ADAMTS4 activity

Figure 3: Preexisting antibody (preAb) binding levels to nanobody construct 581 (A) and nanobody construct 579 (B) from 3 sets of donor samples obtained from: healthy subjects, subjects with osteoarthritis, and donors with residual preAb binding.

Figure 4: Nanobody construct 581 ("C011400581") does not bind human ADAMTS1 (A) and does not bind human ADAMTS4 or human ADAMTS15 (B).

Figure 5: Activity in the human explant system. Shows the accumulation of GAG in the supernatant after 7 days.

Figure 6: A) Time dependence of GAG release in the supernatant in a bovine co-culture system.

B) Calculation of the area under the curve (AUC) for GAG release over time (7-28 days). The number of repetitions n=4. The data is shown in box-and-whisker format from min. up to max.

C) Data show GAG content [ng/mg cartilage] after papain digestion. The number of repetitions n=4. The data are shown in box-and-whisker format from min. up to max.

Figure 7: Calculation of the area under the curve (AUC) for the release of exAGNx1 over time (7-28 days). The number of repetitions n=4. The data is shown in box-and-whisker format from min. up to max.

Figure 8: Calculation of area under the curve (AUC) for C2M release over time (7-28 days). The number of repetitions n=4. The data is shown in box-and-whisker format from min. up to max.

Figure 9: Calculation of area under the curve (AUC) for C3M release over time (7-28 days). The number of repetitions n=4. The data is shown in box-and-whisker format from min. up to max.

Figure 10: Inhibition of aggrecanase activity by NHP.

Figure 11: Inhibition of cartilage degeneration. The median amount of cartilage degeneration is shown for prophylactic (A) and therapeutic (B) treatments in the DMM mouse model.

Figure 12: Symptomatic behavior in a model of surgical OA in rats.

Figure 13: Width of medial tibial cartilage degeneration.

Figure 14: Effect of anti-ADAMTS-5 nanobodies on ex vivo aggrecanase-induced degradation of aggrecan in cartilage cultures (A, B, C) and cartilage and synovial membrane co-cultures (D, E). The concentrations listed are the concentration of the nanobody. Statistical analysis was performed using conventional one-way ANOVA or two-way ANOVA. Statistical significance was recognized when p<0.05.

5. DETAILED DESCRIPTION

There remains a need for safe and effective drugs against OA, in particular DMOAD. These medicinal products must satisfy different and often conflicting requirements, especially when a widely applicable format is envisaged. Thus, the format should preferably be applicable to a wide range of patients. The format should preferably be safe and should not induce infections due to frequent administration. In addition, the format should preferably be patient friendly, eg, the format should allow for a convenient dosing regimen and route of administration, eg, systemic administration. For example, it is preferred that the format is not removed from the circulation immediately after administration. However, the half-life extension should preferably not result in non-specific activity and side effects or limit efficacy.

The present invention implements at least one of these requirements.

Based on non-conventional screening, characterization and combination methods, the present inventors have surprisingly found that immunoglobulin single variable domains (ISVDs) performed remarkably well in in vitro and in vivo experiments.

In addition, the present inventors have succeeded in redesigning ISVDs further superior to comparative drugs in alleviating OA. In addition, the ISVDs of the invention have also been shown to be significantly safer than prior art compounds.

The aim of the present invention is to provide polypeptides that are antagonists of ADAMTS, in particular ADAMTS5, with improved prophylactic, therapeutic and/or pharmacological properties, including a better safety profile, compared to the amino acid sequences and antibodies of the prior art.

Accordingly, the present invention relates to ISVDs and polypeptides that are targeted against and/or that can specifically bind (as defined herein) ADAMTS5.

Accordingly, the present invention relates to ISVDs and polypeptides that are targeted against and/or that can specifically bind (as defined herein) ADAMTS and modulate their activity, in particular, a polypeptide containing at least one ISVD that specifically binds ADAMTS5, where binding to ADAMTS5 modulates ADAMTS5 activity.

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

Unless otherwise indicated, all methods, steps, methods and manipulations not specifically described in detail can be carried out and carried out in a manner known per se, as understood by a person skilled in the art. Again, reference is made to, for example, the standard manuals and the general art mentioned in the present description, and additional references cited in these documents; as well as, for example, the following reviews Presta (Adv. Drug Deliv. Rev. 58 (5-6): 640-56, 2006), Levin and Weiss (Mol. Biosyst. 2(1): 49-57, 2006 ), Irving et al. (J. Immunol. Methods 248(1-2): 31-45, 2001), Schmitz et al. (Placenta 21 Suppl. A: S106-12, 2000), Gonzales et al. (Tumour Biol. 26(1): 31-43, 2005), which describe protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desirable properties of proteins such as immunoglobulins.

It should be noted that, within the scope of the invention, the singular forms include references to the plural, unless the context clearly indicates otherwise. Thus, for example, reference to "reagent" includes one or more of these various reagents, and reference to "method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or replace the methods described herein. .

Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. A person skilled in the art knows, or can determine, using no more than conventional experimentation, many equivalents of the specific embodiments of the invention described in the present description. Such equivalents are intended to be included in the present invention.

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

The term "about" or "approximately", as used herein, means within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range.

Throughout this description and the following claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a specified integer or step, or group of integers or stages, but not to the exclusion of any other integer or stage, or group of integers or stages. Within the scope of the invention, the term "comprising" can be replaced by the term "comprising" or "concluding", or sometimes, when used in the present description, the term "having".

The term "sequence", within the meaning of the invention (for example, in terms like the terms "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V _HH sequence", or "protein sequence"), should be generally understood to include the corresponding amino acid sequence, as well as the nucleic acid or nucleotide sequences encoding it, unless the context requires more limited interpretation.

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

Amino acids are L-amino acids commonly found in natural proteins. Amino acid residues are indicated according to the standard three-letter or one-letter amino acid code. Reference is made to, for example, Table A-2 on page 48 of WO 08/020079. Amino acid sequences containing D-amino acids are not intended to be included in this definition. Any amino acid sequence containing post-translationally modified amino acids can be described as the amino acid sequence originally translated using the symbols shown in this Table A-2, with the positions of the modifications; for example, hydroxylation or glycosylation, however, these modifications should not be explicitly indicated in the amino acid sequence. Any peptide or protein that can be expressed in the form of a sequence with modified bonds, cross-links and end caps, non-peptidyl bonds, etc., is included in this definition, all as known in the art.

The terms "protein", "peptide", "protein/peptide" and "polypeptide" are used interchangeably throughout this description, and each has the same meaning for the purposes of this description. Each term refers to an organic compound made up of a linear chain of two or more amino acids. A compound may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. One skilled in the art will appreciate that polypeptides generally contain fewer amino acids than proteins, although there is no art-accepted cut-off point for the number of amino acids that differentiates polypeptides from proteins; 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, as is known in the art. Traditionally, the amide bond in the primary structure of polypeptides is in the order in which the amino acids are written, in which the amino-terminus (N-terminus) of the polypeptide is always on the left, while the acidic-terminus (C-terminus) is on the right.

A nucleic acid sequence or amino acid sequence is considered to be "(in) (substantially) isolated (form)"—for example, compared to the reaction medium or culture medium from which it is derived—when it is separated from at least one other component, with with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or minor component. In particular, a nucleic acid sequence or amino acid sequence is considered to be "(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 times or more. A nucleic acid or amino acid that is "in (substantially) isolated form" is preferably substantially homogeneous, as determined using a suitable method, such as a suitable chromatographic method, for example, polyacrylamide gel electrophoresis.

When a nucleotide sequence or amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or "primarily consists of" another nucleotide sequence or amino acid sequence, this may mean that the last nucleotide sequence or amino acid sequence is introduced into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually, this generally means that the first mentioned nucleotide sequence or amino acid sequence contains within its sequence a fragment of nucleotides or amino acid residues, respectively, having the same nucleotide sequence or amino acid sequence, respectively, as the last sequence, regardless of how the first mentioned sequence was actually formed or obtained (which may, for example, be carried out by any suitable method described in the present description). As a non-limiting example, when a polypeptide of the invention is said to contain an immunoglobulin single variable domain ("ISVD"), this may mean that said immunoglobulin single variable domain sequence is introduced into the sequence of the polypeptide of the invention, but more usually, it generally means, that the polypeptide of the invention contains within its sequence the sequence of single variable domains of immunoglobulins, regardless of how the specified polypeptide of the invention is formed or obtained. Also, when a nucleic acid or nucleotide sequence is said to contain another nucleotide sequence, the first nucleic acid or nucleotide sequence mentioned is preferably such that when it is expressed prior to an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence, forms part of said expression product (in other words, that the last nucleotide sequence is in the same reading frame as the first mentioned longer nucleic acid or nucleotide sequence). Also, when a construct of the invention is said to contain a polypeptide or ISVD, this may mean that said construct at least includes said polypeptide or ISVD, respectively, but more usually, it means that said construct includes groups, residues (e.g., amino acid residues), fragments, and/or linking units, in addition to said polypeptide or ISVD, regardless of how said polypeptide or ISVD is linked to said groups, residues (e.g., amino acid residues), fragments, and/or linking units, and regardless of how said construct is formed or obtained.

By "essentially consists of" is meant that the ISVD used according to the invention is either exactly the same as the ISVD according to the invention, or corresponds to the ISVD according to the invention, having a limited number of amino acid residues, for example, 1-20 amino acid residues, for example, 1 -10 amino acid residues and preferably 1-6 amino acid residues, e.g. 1, 2, 3, 4, 5 or 6 amino acid residues added at the amino terminus, at the carboxy terminus, or both at the amino terminus and at the carboxy -end of ISVD.

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 [total nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference in one nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using a known computer sequence alignment algorithm such as NCBI Blast v2.0 using standard settings. Some other methods, computer algorithms and conditions for determining the degree of sequence identity are, for example, described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768. Typically, for for the purpose of determining the percentage of "sequence identity" between two nucleotide sequences, according to the calculation method described herein above, the nucleotide sequence with the highest number of nucleotides is taken as the "first" nucleotide sequence, and the other nucleotide sequence is taken as the "second" nucleotide sequence.

For purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between the first amino acid sequence and the second amino acid sequence (also referred to herein as "amino acid identity") can be calculated by dividing the [number of amino acid residues in the first amino acid sequence that are identical amino acid residues at the corresponding positions in the second amino acid sequence] by [total number of amino acid residues in the first amino acid sequence] and multiplying by [100%] where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence is 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 herein above, the amino acid sequence with the most amino acid residues is taken as the "first" amino acid sequence, and the other amino acid sequence is taken as the "second » amino acid sequence.

Also, when determining the degree of sequence identity between two amino acid sequences, one skilled in the art can take into account so-called "conservative" amino acid substitutions, which can be generally described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue of similar chemical structure, and which have 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 can be selected based on the relevant explanations from WO 04/037999 as well as WO 98/49185 and from additional references cited in these documents.

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

Any amino acid substitutions applicable to the polypeptides described herein may also be based on the frequency analysis of amino acid variants between homologous proteins from different species developed by Schulz et al. ("Principles of Protein Structure", Springer-Verlag, 1978), on structure-forming potential analyzes developed by Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45-149, 1978), and on the analysis hydrophobicity patterns in proteins developed by Eisenberg et al. (Proc. Natl. Acad Sci. USA 81: 140-144, 1984), Kyte and Doolittle (J. Molec. Biol. 157: 105-132, 1981), and Goldman et al. (Ann. Rev. Biophys. Chem. 15: 321-353, 1986), all of which are incorporated herein by reference in their entirety. Information about the primary, secondary and tertiary structure of nanobodies is given in the description in this document and in the general level of technology cited above. Also, for this purpose, the crystal structure of the llama V _HH domain is given, for example, in Desmyter et al. (Nature Structural Biology, 3: 803, 1996), Spinelli et al. (Natural Structural Biology, 3: 752-757, 1996) and Decanniere et al. (Structure, 7 (4): 361, 1999). Additional information about some of the amino acid residues that form the V _H /V _L contact surface in conventional V _H domains and potential camellization at these positions can be found in the prior art cited above.

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

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

An "amino acid(s) difference" can be any one, two, three, four, or up to five substitutions, deletions, or insertions, or any combination thereof, that either enhances the properties of an ADAMTS5 binding molecule of the invention, such as a polypeptide of the invention, or that at least not impair too much the desired properties, or the balance or combination of desirable properties of an ADAMTS5 binding molecule of the invention, such as a polypeptide of the invention. In this regard, the resulting ADAMTS5 binding molecule of the invention, such as a polypeptide of the invention, should at least bind ADAMTS5 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. Affinity can be measured by any suitable method known in the art, but is preferably measured by the method described in the examples section.

In this regard, the amino acid sequence of the CDR according to b), d) and/or f), as indicated below, 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 affinity maturation methods as known per se, or as described in the examples. For example, and depending on the host organism used to express the polypeptide of the invention, such deletions and/or substitutions can be engineered in such a way that one or more sites of post-translational modification (such as one or more glycosylation sites) are removed as is within the purview of specialist in this field (cf. examples).

"Nanobody family", "V _HH family", or "family", as used herein, refers to a group of nanobody and/or V _HH sequences that are identical in length (i.e., they have the same number of amino acids in the sequence) and in which the amino acid the 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, recognized by antigen-binding molecules such as immunoglobulins, conventional antibodies, immunoglobulin single variable domains, and/or polypeptides of the invention, and more. specifically, the antigen-binding site of these molecules. Epitopes define the minimum binding site for an immunoglobulin, and thus represent a target for immunoglobulin specificity.

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

An amino acid sequence (such as an immunoglobulin single variable domain, an antibody, a polypeptide of the invention, or in general an antigen-binding protein or polypeptide, or fragment thereof) that can "bind to" or "specifically bind to" that "has affinity for" and/ or which "has specificity for" a particular epitope, antigen or protein (or at least one part, fragment or epitope thereof), is said to be "against" or "targeted against" the specified epitope, antigen or protein, or is "binding" a molecule with respect to such an epitope, antigen, or protein, or is referred to as an "anti" epitope, "anti" antigen, or "anti" protein (eg, "anti"-ADAMTS5).

Affinity refers to the strength or stability of a molecular interaction. Affinity is usually given as K _D , or the dissociation constant, which has the dimension of mol/liter (or M). Affinity can also be expressed as a binding constant, K _A , which is equal to 1/K _D and has the unit of (mol/liter) - ¹ (or M - ¹ ). In the present description, the stability of an interaction between two molecules can be basically expressed in terms of the K _D value of their interaction; one of skill 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 refers to the change in the free energy (DG) of the binding through the well-known relationship DG=RT.ln(K _D ) (equivalently, DG=-RT.ln(K _A )) , where R is equal to the gas constant, T is equal to 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 rate of dissociation of the complex, denoted k _off , to the rate of its binding, denoted k _on (so that K _D =k _off /k _on and K _A =k _on /k _off ). The dissociation rate k _off has the dimension c ^-1 (where c is the SI notation for second). k _on for the speed of binding has the dimension of M ^-1 s ^-1 . The binding rate can vary between 10 ² M ^-1 s ^-1 and approximately 10 ⁷ M ^-1 s ^-1 , approaching the rate constant of diffusion-limited binding for bimolecular interaction. The dissociation rate is related to the half-life of this molecular interaction by the ratio t _1/2 =ln(2)/k _off . The dissociation rate can vary between 10 ^-6 s ^-1 (almost irreversible complex with t _1/2 for several days) and 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 competitive sandwich assays, and various variations thereof known per se in the art; as well as through other methods mentioned in the present description.

The affinity of a molecular interaction between two molecules can be measured by various methods known per se, such as the well-known surface plasmon resonance (SPR) 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 to obtain measurements of k _on , k _off , and thus K _D (or K _A ) values. This can, for example, be carried out using well-known BIACORE® devices (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 assays can also be performed using the GYROLAB® Immunoassay System, which provides an automated bioassay platform and rapid sample turnover (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 method of measurement affects the binding affinity inherent in the intended molecules in any way, for example due to artifacts associated with coating the biosensor with a single molecule. Also apparent K _D can be measured if one molecule contains more than one recognition site for another molecule. In such a situation, the measured affinity can be affected by the avidity of the interaction between the two molecules. In particular, accurate measurement of K _D can be quite cumbersome, and as a result, often apparent K _D values are determined to estimate the binding strength of two molecules. It should be noted that, provided that all measurements are made in a consistent manner (e.g., keeping the analysis conditions unchanged), apparent K _D measurements can be used as an approximation of the true K _D and thus, in the present description, K _D and apparent K _D should be considered with equal importance or authority.

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 (the contents of which are incorporated herein by reference), which also describes preferred methods for measuring binding between an antigen-binding molecule. (such as a polypeptide or ISVD of the invention) and the corresponding antigen. Typically, antigen-binding proteins (such as ISVDs and/or polypeptides of the invention) bind to their antigen with a dissociation constant (K _D ) of 10 ^-5 -10 ^-12 mol/liter or less and preferably 10 ^-7 -10 ¹² mol/ liter or less, and more preferably 10 ^-8 -10 ^-12 mol/liter (i.e. with a binding constant (K _A ) 10 ⁵ -10 ¹² liters/mol or more and preferably 10 ⁷ -10 ¹² liters/ mol or more, and more preferably 10 ⁸ -10 ¹² liters/mol). Any K _D value greater than 10 ^-4 mol/liter (or any K _A value lower than 10 ⁴ liters/mol) is generally considered to indicate non-specific binding. Preferably, the monovalent ISVD of the invention binds to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 nM, such as between 10 and 5 nM or less. . Reference is also made to section n) on pages 53-56 of WO 08/020079.

An ISVD and/or polypeptide is considered 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 appropriately expressed as the value K _D , 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 the ISVD and/or polypeptide binds to the second target or antigen. For example, the ISVD and/or polypeptide may bind to the first target or antigen with a K _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 less. this than the K _D with which said ISVD and/or polypeptide binds to the second target or antigen. Preferably, when the ISVD and/or polypeptide is "specific for" the first target or antigen, as compared to the second target or antigen, it is directed against (as defined 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 radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and various variants thereof. known in the art; the same as other methods mentioned in the present description.

A preferred method that can be used to assess affinity is the 2-step ELISA method from Friguet et al., 1985 (J. Immunol. Methods 77: 305-19). This method provides a measurement of the binding equilibrium in the solution phase and eliminates possible artifacts associated with the adsorption of one of the molecules on the substrate, such as plastic. As one skilled in the art would appreciate, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining the dissociation constant are clear to the person skilled in the art, and include, for example, the methods mentioned on pages 53-56 of WO 08/020079.

Finally, it should be noted that in many situations, one of skill in the art may find it convenient to determine the binding affinity for the same reference molecule. For example, to assess the strength of binding between molecules A and B, one can, for example, use a reference molecule C, which is known to bind to B, and which is suitably labeled using a fluorophore or chromophore moiety, or another chemical moiety such as biotin, for ease of detection in ELISA or FACS (fluorescence-activated cell sorting) or another format (fluorophore for fluorescent detection, chromophore for light absorption detection, biotin for streptavidin-mediated detection in ELISA). Typically, the reference molecule C is maintained at a fixed concentration and the concentration of A is varied 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 measured for C in the absence of A is halved. Provided that the K _{D of the reference ..,} the K _D of the reference molecule is known, as well as the total concentration with the _reference. reference molecule, the apparent K _D for the interaction AB can be obtained from the following formula: K _D = IC ₅₀ /(1+s _reference /K _Dreference ). It should be noted that if with a _standard. << K _Dreference. , K _D ≈ IC ₅₀ . Provided that the IC ₅₀ measurement is carried out in a constant manner (for example, keeping the _standard fixed) for the compared binding molecules, differences in the strength or stability of the molecular interaction can be assessed by comparing the IC ₅₀ and this measurement is considered equivalent to K _D or apparent K _D by throughout this text.

The half maximum inhibitory concentration (IC ₅₀ ) may also be an indication of the effectiveness of a compound in inhibiting a biological or biochemical function, such as a pharmacological effect. This score indicates how much of a polypeptide or ISVD (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.) 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 ISVD (eg, nanobody) 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 testing the effect of different concentrations of an antagonist, such as a polypeptide or ISVD (eg, nanobody) of the invention, on reversing agonist activity.

The term "half maximum effective concentration" (EC ₅₀ ) refers to the concentration of a compound that induces half the response between background and maximum after a specified exposure time. In the present context, it is used as an indicator of the activity of a polypeptide, ISVD (eg, nanobodies). The EC ₅₀ in the graduated dose response curve represents the concentration of a compound when 50% of its maximum effect is observed. The concentration is preferably expressed in molar units.

In biological systems, small changes in ligand concentration typically result in rapid response changes following a sigmoidal function. The inflection point at which the increase in response with increasing ligand concentration begins to slow down is the EC ₅₀ . This can be determined mathematically by deriving a best fit curve. Based on a graph for evaluation is convenient in most cases. In the case where the EC ₅₀ is presented in the examples section, the experiments were designed to reflect the 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 sum of the data divided by the number of data values).

This also applies to the IC ₅₀ , which is an indication of the compound in relation to its inhibition (50% inhibition). For competition binding assays and functional antagonist assays, IC ₅₀ is the most common summary measure of the dose-response curve. For agonist/stimulant assays, the most common summary measure is EC ₅₀ .

The inhibition constant (Ki) is a measure of how active an inhibitor is; it represents the concentration required to obtain half of the maximum inhibition. Unlike IC ₅₀ , which can vary with experimental conditions, Ki is an absolute value and is often referred to as the drug's inhibition constant. The inhibition constant K _i can be calculated using the Cheng Prusov equation:

,

,

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

The term "activity" of the polypeptide and/or ISVD according to the invention, within the scope of the invention, is a function of the amount of the polypeptide and/or ISVD according to the invention, necessary for the occurrence of its specific effect. It refers to the ability of said polypeptide and/or ISVD of the invention to modulate and/or partially or completely inhibit the activity of ADAMTS5. More specifically, it may refer to the ability of the specified polypeptide and/or ISVD to reduce or even completely inhibit the activity of ADAMTS5, as defined in the present description. Thus, it may refer to the ability of said polypeptide and/or ISVD to inhibit ADAMTS5 activity, e.g., enzymatic activity such as proteolysis, e.g., protease activity and/or endopeptidase activity, as well as substrate binding, including, but not limited to, aggrecan, versican, brevican, neurocan, decorin and/or biglycan, preferably aggrecan cleavage. Said polypeptide and/or ISVD is preferably an ADAMTS5 aggrecanase activity antagonist. Activity can be measured by any suitable assay known in the art or described herein. Within the scope of the invention, "aggrecanase activity" is defined as the proteolytic cleavage of aggrecan.

The "potency" of a polypeptide of the invention measures the maximum strength of the effect itself, at saturating concentrations of the polypeptide. Efficacy indicates the maximum response achievable for a polypeptide of the invention. It refers to the ability of a polypeptide to exert a desired (therapeutic) effect.

In one aspect, the invention provides a polypeptide as described herein, wherein said polypeptide binds to ^ADAMTS5 with a K _D between 1E ^- 07M and 1E-13M, e.g. between 1E ^-08M and 1E ^- 12M, preferably not more than 1E ^-07 M, preferably lower than 1E ^-08 M or 1E ^-09 M, or even lower than 1E ¹⁰ M, e.g. 5E- ¹¹ M, 4E- ¹¹ M, 3E ¹¹ M, 2E- ¹¹ M , 1.7E- ¹¹ M, 1E ¹¹ M, or even 5E ^-12 M, 4E ^-12 M, 3E ¹² M, 1E ^-12 M, for example, as determined by Gyrolab or KinExA.

In one aspect, the invention provides a polypeptide as described herein, wherein said polypeptide modulates ADAMTS5 with an EC ₅₀ between 1E- ⁰⁷ M and 1E ^-12 M, such as between 1E ^-08 M and 1E ^-11 M, such as determined by ELISA binding.

In one aspect, the invention provides a polypeptide as described herein, wherein said polypeptide binds to ADAMTS5 at a dissociation rate of less than 5E ^-04 s ^-1 , such as less than 1E ^-04 s ^-1 or 5E ^-05 s ^-1 , or even less than 1E ^-05 s ^{- 1} , for example, as determined by SPR.

In one aspect, the invention provides a polypeptide as described herein, wherein said polypeptide inhibits ADAMTS5 activity with an IC ₅₀ between 1E- ⁰⁷ M and 1E ^-12 M, e.g. between 1E ^-08 M and 1E ^-11 M, e.g. as determined by human FRET or human AlphaLISA assay.

In one aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide inhibits ADAMTS5 enzyme activity with an IC _{50 of} not more than 1E ^-07 M, preferably 1E ^-08 M, 5E ^-09 M, or 4E ^-9 M, 3E ^-9 M, 2E ^-9 M, e.g. 1E ^-9 M.

An amino acid sequence, such as an ISVD or a polypeptide, is said to have "cross-reactivity" for two different antigens or antigenic determinants (e.g., such as ADAMTS5 from different mammalian species, e.g., such as human ADAMTS5, bovine ADAMTS5, rat ADAMTS5, ADAMTS5 guinea pig, mouse ADAMTS5, or cynomolgus ADAMTS5) if it is specific for (as defined herein) these various antigens or antigenic determinants. It is understood that an ISVD or polypeptide can be considered to be cross-reactive, although the binding affinity for two different antigens may differ, for example, by factors of 2, 5, 10, 50, 100 or even more, provided that it is specific for (as defined herein) of these various antigens or antigenic determinants.

ADAMTS5 is also known as ADAMTS11, ADMP-2 or aggrecanase-2.

The relevant structural information for ADAMTS5 can be found, for example, in access numbers in UniProt, as described in Table 1 below (cf. Table B).

Table 1

Protein access number Gene organism SEQID NO: Q9UNA0 ADAMTS5 H. sapiens 149 Q9TT92 ADAMTS5 B. taurus 150 Q6TY19 ADAMTS5 R.norvegicus 151 H0VFP0 ADAMTS5 Cavia Porcellus 152 Q9R001 ADAMTS5 M.musculus 153 F6Z3S6 ADAMTS5 M. mulatta 154

"Human ADAMTS5" refers to ADAMTS5 comprising the amino acid sequence of SEQ ID NO: 149. In one aspect, a polypeptide of the invention specifically binds to ADAMTS5 from Human sapiens, Mus musculus, Cavia Porcellus, Bos taurus, Macaca mulatta and/or Rattus norvegicus, preferably human ADAMTS5, preferably SEQ ID NO: 149.

The terms "(cross)block", "(cross)block", "(cross)block", "competitive binding", "(cross)compete", "(cross)compete" and "(cross)compete" are used herein. description is used interchangeably to refer to the ability of an immunoglobulin, antibody, ISVD, polypeptide, or other binding molecule to interfere with the binding of other immunoglobulins, antibodies, ISVDs, polypeptides, or binding molecules to a given target. The degree to which an immunoglobulin, antibody, ISVD, polypeptide, or other binding molecule is capable of interfering with the binding of another to a target, and thus whether they can be called cross-blocking according to the invention, can be determined using competitive binding assays generally accepted in the art, for example, such as screening purified ISVDs against phage-exposed ISVDs in a competitive ELISA. Particularly suitable quantitative cross-blocking assays include ELISA.

Other methods for determining whether an immunoglobulin, antibody, ISVD, polypeptide, or other binding molecule targeted against a target is (cross) blocking, (cross) blocking, competitively binding, or (cross) competitive as defined herein, can be evaluate in a "sandwich analysis" based on the SPR, for example, such as described in the examples section. Other suitable methods are described, for example, in Xiao-Chi Jia et al. (Journal of Immunological Methods 288: 91-98, 2004), Miller et al. (Journal of Immunological Methods 365: 118-125, 2011).

Accordingly, the present invention relates to a polypeptide as described herein, such as represented by SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7 , 117, 8, 12, 13, 14, 15, or 18 (cf. Table A-1), wherein said polypeptide competes with a cross-blocking polypeptide, eg, as determined by competitive ELISA.

The present invention relates to a method for determining competitors, such as polypeptides competing with a polypeptide as described herein, such as represented by any of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10 , 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18, wherein the polypeptide as described herein competes or cross-blocks with a competitor, such as the polypeptide, for binding to ADAMTS5, such as, for example, human ADAMTS5 (SEQ ID NO: 149), wherein binding to competitor ADAMTS5 is reduced by at least 5%, such as 10%, 20%, 30%, 40%, 50%, or even more, for example, 80%, 90%, or even 100% (ie, virtually undetectable in this assay) in the presence of a polypeptide of the invention, compared to binding to competitor ADAMTS5 in the absence of a polypeptide of the invention. Competition and cross-blocking can be determined by any means known in the art, such as, for example, competitive ELISA or FACS analysis. In one aspect, the present invention relates to a polypeptide according to the invention, where the specified polypeptide cross-blocks the binding to ADAMTS5 of at least one of the polypeptides represented by SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18 and/or its binding to ADAMTS5 is cross-blocked by at least one of the polypeptides represented by SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18.

The present invention also relates to competitors competing with a polypeptide as described herein, such as SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7 , 117, 8, 12, 13, 14, 15 or 18, where the competitor competes or cross-blocks for the polypeptide as described herein in relation to binding to ADAMTS5, where the binding to ADAMTS5 of the polypeptide of the invention is reduced by at least 5 %, such as 10%, 20%, 30%, 40%, 50%, or even more, such as 80%, or even more, such as at least 90% or even 100% (i.e. virtually undetectable in this assay) in the presence of said competitor, as compared to ADAMTS5 binding of a polypeptide of the invention in the absence of said competitor. In one aspect, the present invention relates to a polypeptide that cross-blocks the binding to ADAMTS5 of a polypeptide of the invention, such as one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5 , 4, 7, 117, 8, 12, 13, 14, 15, or 18, and/or its binding to ADAMTS5 is cross-blocked by at least one of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16 , 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18, preferably wherein said polypeptide contains at least one VH, VL, dAb, or ISVD that specifically binds ADAMTS5 , where binding to ADAMTS5 modulates ADAMTS5 activity.

"ADAMTS5 activities" and "ADAMTS5 activities" (these terms are used interchangeably herein) include, but are not limited to, enzyme activities such as proteolysis, for example, protease activity (also referred to as protease or peptidase activity), and activities endopeptidases, on the one hand, and exosite activities, such as recognition and/or binding of a substrate, for example, via a disintegrin-like domain, a central thrombospondin (TS) type I repeat, a cysteine-rich domain, a spacer region and/or additional motifs TS. ADAMTS5 activities include binding and/or proteolysis of substrates such as hyaluronan-binding extracellular proteins chondroitin sulfate proteoglycans (CSPG) such as aggrecan, versican, brevican, neurocan, decorin and biglycan. Within the scope of the invention, proteolysis is the breakdown of proteins into smaller polypeptides or amino acids through the hydrolysis of peptide bonds that link amino acids together in a polypeptide chain.

In the context of the present invention, "modulate" or "modulate" generally means to alter the activity of ADAMTS5 as measured using an appropriate in vitro assay, cellular assay, or in vivo assay (such as the assays mentioned herein). In particular, "modulation" or "modulate" can mean either a reduction or inhibition of activity, or alternatively, an increase in ADAMTS5 activity as measured using an appropriate in vitro assay, cellular assay, or in vivo assay (e.g., such as those mentioned in present description), at least 1%, preferably at least 5%, for example, at least 10% or at least 25%, for example, at least 50%, at least 60 %, at least 70%, at least 80%, or 90% or more, compared to ADAMTS5 activity in the same assay under the same conditions, but in the absence of ISVD or a polypeptide of the invention.

Accordingly, the present invention provides a polypeptide as described herein, wherein said polypeptide modulates ADAMTS5 activity, preferably inhibits ADAMTS5 activity.

Accordingly, the present invention relates to a polypeptide as described herein, wherein said polypeptide inhibits the protease activity of ADAMTS5, eg inhibits proteolysis of a substrate such as aggrecan, versican, brevican, neurocan, decorin and/or biglycan.

Accordingly, the present invention relates to a polypeptide as described herein, wherein said polypeptide blocks the binding of ADAMTS5 to a substrate such as aggrecan, versican, brevican, neurocan, decorin, and/or biglycan, wherein said substrate is preferably aggrecan.

In one aspect, the invention provides a polypeptide as described herein, wherein said polypeptide blocks ADAMTS5 binding to aggrecan by at least 20%, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, for example, as determined by ELISA.

In one aspect, the invention provides a polypeptide as described herein, wherein said polypeptide antagonizes or inhibits ADAMTS5 activity, such as (i) protease activity, preferably cleavage of aggrecan, versican, brevican, neurocan, decorin and/or biglycan, preferably cleavage of aggrecan; preferably, is an antagonist of ADAMTS5 aggrecanase activity; (ii) binding the substrate to ADAMTS5, eg, an ADAMTS5 exosite, eg, a disintegrin-like domain, a type I central thrombospondin (TS)-like repeat, a cysteine-rich domain, a spacer region, or an additional TS motif.

Accordingly, the present invention provides a polypeptide as described herein, wherein said polypeptide inhibits ADAMTS5 protease activity, preferably by at least 5%, such as 10%, 20%, 30%, 40%, 50%, or even greater, such as at least 60%, 70%, 80%, 90%, 95%, or even greater, as determined by any suitable method known in the art, such as, for example, enzyme inhibition assays, or as described in examples section.

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

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

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

The term "domain" (polypeptide or protein), within the meaning of the invention, refers to a folded protein structure having the ability to maintain its tertiary structure independently of the rest of the protein. As a rule, domains are responsible for individual functional properties of proteins, and in many cases, can be added, removed or transferred to other proteins without losing the function of the rest of the protein and/or other domains.

The term "immunoglobulin domain", as used herein, refers to the globular region of an antibody chain (eg, such as a conventional 4-chain or heavy chain antibody chain), or to a polypeptide that primarily consists of such a globular region. Immunoglobulin domains are distinct in that they retain the characteristics of immunoglobulin folding of antibody molecules, which consists of a bilayer sandwich of approximately seven antiparallel beta chains arranged into two beta sheets, optionally stabilized by a conservative disulfide bond.

The term "immunoglobulin variable domain", as used herein, refers to an immunoglobulin domain essentially consisting of four "framework regions" referred to in the art and hereinafter as "framework 1" or "FR1"; as "wireframe 2" or "FR2"; as "wireframe 3" or "FR3"; and as "frame region 4" or "FR4", respectively; where the framework regions are interrupted by three "complementarity-determining regions" or "CDRs", designated in this area and in the present description below as "complementarity-determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be specified as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The variable domain(s) of an immunoglobulin imparts specificity to an antibody for an antigen because it carries an antigen-binding site.

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

With 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, e.g. binding of an Fv or scFv fragment or a diabody (all known in the art) derived from such a conventional 4-chain antibody is not usually considered a single immunoglobulin variable domain because, in these cases, binding to the corresponding antigen epitope does not normally occur with one (single) immunoglobulin domain, but occurs with a pair of (associated) immunoglobulin domains, such as light and heavy chain variable domains, ie. with a pair of V _H -V _L immunoglobulin domains, which together bind to the epitope of the corresponding antigen.

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

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

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

For example, an ISVD can be a (single) domain antibody (or an amino acid suitable for use with a (single) domain antibody), a "dAb" or a dAb (or an amino acid suitable for use with a dAb), or a nanobody (as defined herein). description, including but not limited to VHH); other single variable domains, or any suitable fragment of any one of them.

In particular, the ISVD may be a nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobody®, and Nanoclone® are registered trade names from Ablynx N.V.] For a general description of nanobody, reference is made to the additional description below, as well as to the prior art cited herein, for example, as described in WO 08/020079 (page 16).

" _VHH domains", also known as VHH, _VHH domains, VHH antibody fragments, and VHH antibodies, were originally described as the antigen-binding (variable) domain of an immunoglobulin from "heavy chain antibodies" (i.e., from "antibodies lacking lungs chains"; Hamers-Casterman et al. 1993 Nature 363: 446-448). The term " _VHH domain" was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as " _VH domains" or "VH domains") and from the light chain variable domains, present in conventional 4-chain antibodies (referred to herein as "VL _domains " or "VL domains"). For a further description of VHH and nanobodies, reference is made to a review article from Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications cited as general 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 Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06 /122787 and WO 06/122825, from Ablynx NV and further published patent applications from Ablynx NV Reference is also made to additional prior art references mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153, where this list and references are given in the present description by reference. As described in these references, nanobodies (in particular, VHH sequences and partially humanized nanobodies) can, in particular, be characterized by the presence of one or more "characteristic residues" in one or more of the framework sequences. Additional description of nanobodies, including humanization and/or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or "fused nanobodies", multivalent constructs (including some non-limiting examples of linker sequences), and various modifications to increase the half-life of nanobodies and their preparations can be found, for example, in WO 08/101985 and WO 08/142164. For a further general description of nanobodies, reference is made to the prior art cited herein, such as, for example, as described in WO 08/020079 (page 16).

In particular, the framework sequences present in ADAMTS5 binding molecules of the invention, such as ISVDs and/or polypeptides of the invention, may contain one or more characteristic residues (for example, as described in WO 08/020079 (Tables A-3 - A-8 )), so that the ADAMTS5 binding molecule of the invention is a nanobody. Some preferred but non-limiting examples (suitable combinations) of such framework sequences will become apparent from the further description herein (see, for example, Table A-2). In general, nanobodies (particularly V _HH sequences and partially humanized nanobodies) can in particular be characterized by the presence of one or more "characteristic residues" in one or more of the framework sequences (as, for example, further described in WO 08/020079 , page 61, line 24 - page 98, line 3).

More specifically, the invention relates to ADAMTS5 binding molecules containing at least one immunoglobulin single variable domain, which is an amino acid sequence with the (general) structure

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

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

i) has 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: 2, 116, 19, 1, 3, 6, 16, 17 , 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15, or 18 (see Table A-1), in which, for purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences , are not taken into account. In this regard, reference is also made to Table A-2, which lists framework 1 sequences (SEQ ID NOs: 72-84), framework 2 sequences (SEQ ID NOs: 85-94), framework 3 sequences (SEQ ID NOs: 95-113) and framework sequence 4 (SEQ ID NOs: 114-115) of immunoglobulin single variable domains from SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18; or

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

and in which:

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

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

In particular, ADAMTS5 binding molecules of the invention, such as ISVDs 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 Patent Applications, when an ADAMTS5 binding molecule of the invention, such as an ISVD and/or a polypeptide of the invention, contains mutations according to one of (i) to (v) above (or a suitable combination thereof) :

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

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

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

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

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

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

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

As mentioned in the co-pending US Provisional Patent Applications, these mutations are effective in preventing or reducing the binding of so-called "preexisting antibodies" to the immunoglobulins and compounds of the invention. For this purpose, ADAMTS5 binding molecules of the invention, such as ISVDs and/or polypeptides of the invention, may also contain (optionally in combination with the indicated mutations) a C-terminal extension (X)n (in which n is 1-10, preferably 1 -5, such as 1, 2, 3, 4, or 5 (and preferably 1 or 2, such as 1); and each X is a (preferably natural) amino acid residue which is independently selected, and preferably independently selected from a group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)), see for example US Provisional Patent Applications as well as WO 12/175741. In particular, an ADAMTS5 binding molecule of the invention, such as an ISVD and/or a polypeptide of the invention, may contain such a C-terminal extension when it forms the C-terminus of its containing protein, polypeptide, or other compound or construct (see, for example, those U.S. provisional patent applications, as well as WO 12/175741).

The ADAMTS5 binding molecule of the invention may be an immunoglobulin, such as an immunoglobulin single variable domain, obtained by any suitable method and from any suitable source, and may, for example, be natural V _HH sequences (i.e., from a suitable camelid species) or synthetic or semi-synthetic amino acid sequences, including, but not limited to, “humanized” (as defined herein) nanobodies or VHH sequences, “camelized” (as defined herein) immunoglobulin sequences (and in particular, camelized heavy chain variable domain sequences) , as well as nanobodies produced by methods such as affinity maturation (for example, starting from synthetic, random, or natural immunoglobulin sequences), CDR grafting, masking of surface residues, combining fragments derived from different sequences, and immunoglobulins, assembly by PCR using overlapping primers, and similar methods for constructing immunoglobulin sequences, well known to the person skilled in the art; or any suitable combination of any of the above, as further described in the present description. Also, when an immunoglobulin contains the V _HH sequence, said immunoglobulin can be appropriately humanized, as described hereinafter, so as to obtain one or more further (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 can optionally be further humanized in an appropriate manner, again as described herein, again so as to obtain one or more additionally (partially or fully) humanized immunoglobulins according to the invention.

"Domain antibodies", also known as "Dab", "Domain Antibodies" and "dAb" (the terms "domain antibodies" and "dAb" are used as trade names in the GlaxoSmithKline group of companies), are described, for example, in EP 0368684, Ward et al. (Nature 341: 544-546, 1989), Holt et al. (Tends in Biotechnology 21: 484-490, 2003) and WO 03/002609 as well as, for example, WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications from Domantis Ltd. Domain antibodies generally correspond to VH or VL domains from non-camelid mammals, in particular from human 4-chain antibodies. For binding to an epitope in the form of a single antigen-binding domain, ie. without mating to a VL or VH domain, respectively, specific selection for such antigen binding properties is required, eg using a human VH or VL single domain sequence library. 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 human therapeutic use.

It should also be noted that, although least preferred in the context of the present invention because they are not derived from mammals, single variable domains can be derived from specific shark species (e.g. so-called "IgNAR domains", see e.g. WO 05/18629 ).

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

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

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

With regard to CDRs, as is well known in the art, there are many conventions for defining and describing a CDR from a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is the most commonly accepted) and the Chothia definition (which is based on the localization of structural regions). loops). A link is provided to, for example, the website http://www.bioinf.org.uk/abs/. For the purposes of this specification and claims, CDR is most preferably determined based on the Abm definition (which is based on the AbM antibody modeling software from Oxford Molecular) as it is considered to be the best compromise between the Kabat and Chothia definitions (cf. http:// www.bioinf.org.uk/abs/) within the scope of the invention, FR1 contains amino acid residues at positions 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acids at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113.

As used herein, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from a non-human source, preferably a camelid, preferably a camelid heavy chain antibody. They may be humanized as described herein. In addition, the term includes polypeptides derived from non-camelid sources, such as mouse or human, that have been "camelized" as described herein.

Thus, ISVDs such as domain antibodies and nanobodies (including VHH domains) can be humanized. In particular, humanized ISVDs such as nanobodies (including VHH domains) may be ISVDs that are as generally defined herein, but in which at least one amino acid residue (and in particular, at least in one of the framework residues) which is and/or which corresponds to a humanizing substitution (as defined herein). Humanizing substitutions that can potentially be used can be determined by comparing the framework sequence of the naturally occurring V _HH sequence with the corresponding framework sequence from one or more closely related human V _H sequences, followed by one or more humanizing substitutions (or combinations thereof) that can potentially be used, thus defined, can be introduced into the indicated V _HH sequence (by any method known per se, as further described in the present description) and the resulting humanized V _HH sequences can be tested for target affinity, stability, simplicity and level of expression, and/or other desirable properties. Thus, through a limited amount of trial and error, one skilled in the art can determine other suitable humanizing substitutions (or suitable combinations thereof) based on the disclosures herein. Also, based on the foregoing, the (framework regions) of an ISVD such as a nanobody (including VHH domains) can be partially humanized or fully humanized.

Another particularly preferred class of ISVDs of the invention comprises an ISVD with an amino acid sequence that corresponds to the amino acid sequence of the natural V _H domain, but which has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of the native V _H domain of a conventional 4 chain antibody with one or more of the amino acid residues occurring at the corresponding position(s) in the heavy chain antibody _HH V domain. This can be done in a manner known per se, which is clear to a person skilled in the art, for example, based on the description in this document. Such "camelizing" substitutions are preferably introduced at amino acid positions forming the contact surface and/or present at the V _H -V _L contact surface and/or in the so-called camelid characteristic residues as defined herein (see also, for example, WO 94/04678 and Davies and Riechmann (1994 and 1996)). Preferably, the V _H sequence that is used as a starting material or starting point for the production or construction of camelized immunoglobulin single variable domains is preferably a mammalian V _H sequence, more preferably a human V _H sequence, e.g., a V _H 3 sequence. However, , it should be noted that such camelized immunoglobulin single variable domains of the invention may be prepared by any suitable method known per se, and thus are not strictly limited to polypeptides prepared using a natural V _H domain-containing polypeptide as a starting material. Reference is made to Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13: 475-479, 1995; Prot. Eng. 9: 531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999)

For example, again as further described herein, both "humanization" and "camelization" can be performed by obtaining a nucleotide sequence encoding a natural V _HH domain or a V _H 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 "camelized" ISVD of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to obtain the desired ISVDs of the invention. Alternatively, based on the amino acid sequence of the natural _HH V domain or the V _H domain, respectively, the amino acid sequence of the desired humanized or camelized ISVDs of the invention, respectively, can be designed and then synthesized de novo using peptide synthesis methods known per se. Also based on the amino acid sequence or nucleotide sequence of the natural V _HH domain or the V _H domain, respectively, a nucleotide sequence encoding the desired humanized or camelized ISVDs of the invention, respectively, can be designed and then synthesized de novo using nucleic acid synthesis methods known per se. , after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to obtain the desired ISVDs of the invention.

ISVDs such as domain antibodies and nanobodies (including VHH domains and humanized VHH domains) can also be affinity matured by introducing one or more changes to the amino acid sequence of one or more CDRs, where the changes result in improved affinity of the resulting ISVD for the corresponding antigen, compared to the corresponding parent molecule. Affinity matured ISVD molecules of the invention can be prepared by methods known in the art, for example as described in 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 method of constructing/selecting and/or obtaining a polypeptide, starting with an ISVD, such as V _H , V _L , V _HH , domain antibody or nanobody, is also referred to in the present description as "formatting" the specified ISVD; and an ISVD that is part of a polypeptide is referred to as being "formatted" or being "in the format" of said polypeptide. Examples of ways in which an ISVD can be formatted, and examples of such formats, will be understood by one of ordinary skill in the art based on the descriptions herein; and such a formatted single immunoglobulin variable domain constitutes a further aspect of the invention.

Preferred CDRs are shown in Table A-2.

In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binding ADAMTS5 mainly consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which

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

amino acid sequences having 1, 2 or 3 amino acid difference(s) from SEQ ID NOs: 21, 35, 20, 22, 25, 33, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34;

(ii) CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and

amino acid sequences having 1, 2 or 3 amino acid difference(s) from SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47 , 48, 49 and 52; and

(iii) CDR3 is selected from the group consisting of SEQ ID NO: SEQ ID NO: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70; and

amino acid sequences having 1, 2, 3 or 4 amino acid difference(s) from SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64 , 65, 66, 67 and 70.

In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binding ADAMTS5 mainly consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which

(i) CDR1 is selected from the group consisting of

(a) SEQ ID NO: 22; and

(b) an amino acid sequence having 1, 2, 3, 4, 5, or 6 amino acid difference(s) from SEQ ID NO: 22, wherein

- in position 2 S replaced by R;

- in position 3 A replaced by T;

- in position 4 V is replaced by F;

- in position 6 V is replaced by S;

- in position 7 N is replaced by Y; and/or

- in position 10 A replaced by G;

(ii) CDR2 is SEQ ID NO: 36; and

(iii) CDR3 is SEQ ID NO: 54.

In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binding ADAMTS5 mainly consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which

(i) CDR1 is SEQ ID NO: 33;

(ii) CDR2 is selected from the group consisting of

(c) SEQ ID NO: 50; and

(d) an amino acid sequence having 1, 2, or 3 amino acid difference(s) from SEQ ID NO: 50, wherein

- in position 8 M is replaced by I;

- in position 9 P is replaced by T; and/or

- in position 10 Y is replaced by F; and

(iii) CDR3 is selected from the group consisting of

(e) SEQ ID NO: 68; and

(f) an amino acid sequence having 1 or 2 amino acid difference(s) from SEQ ID NO: 68, wherein

- in position 5 F is replaced by L; and/or

- in position 11 D replaced by E.

In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binding ADAMTS5 mainly consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which

(i) CDR1 is SEQ ID NO: 28;

(ii) CDR2 is selected from the group consisting of

(c) SEQ ID NO: 44; and

(d) an amino acid sequence having 1, 2, or 3 amino acid difference(s) from SEQ ID NO: 44, wherein

- in position 3 S replaced by T;

- in position 4 R is replaced by W;

- in position 8 T is replaced by I; and/or

- in position 9 T is replaced by L; and

(iii) CDR3 is selected from the group consisting of

(e) SEQ ID NO: 62; and

(f) an amino acid sequence having 1 or 2 amino acid difference(s) from SEQ ID NO: 62, wherein

- in position 1 G is replaced by S; and/or

- in position 14 D replaced by E.

In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and is mainly composed 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: 21, 35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34;

- CDR2 is selected from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and

- CDR3 is selected from the group consisting of SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70.

In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and is mainly composed of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively), in which the specified ISVD is selected from a group of ISVDs where:

CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55;

CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 118;

CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53, and CDR3 is SEQ ID NO: 71;

CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54;

CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 36, and CDR3 is SEQ ID NO: 54;

CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 40, and CDR3 is SEQ ID NO: 58;

CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 50, and CDR3 is SEQ ID NO: 68;

CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 51, and CDR3 is SEQ ID NO: 69;

CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 44, and CDR3 is SEQ ID NO: 62;

CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 45, and CDR3 is SEQ ID NO: 63;

CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 43, and CDR3 is SEQ ID NO: 61;

CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 39, and CDR3 is SEQ ID NO: 57;

CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 38, and CDR3 is SEQ ID NO: 56;

CDR1 is SEQ ID NO: 26, CDR2 is SEQ ID NO: 41, and CDR3 is SEQ ID NO: 59;

CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 119, and CDR3 is SEQ ID NO: 60;

CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 42, and CDR3 is SEQ ID NO: 60;

CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 46, and CDR3 is SEQ ID NO: 64;

CDR1 is SEQ ID NO: 30, CDR2 is SEQ ID NO: 47, and CDR3 is SEQ ID NO: 65;

CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 48, and CDR3 is SEQ ID NO: 66;

CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 49, and CDR3 is SEQ ID NO: 67; and

CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 52, and CDR3 is SEQ ID NO: 70.

In a specific preferred aspect, the present invention provides an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and is primarily composed of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively). ), wherein CDR1 is or contains SEQ ID NO: 21, CDR2 is SEQ ID NO: 37, and CDR3 is SEQ ID NO: 55.

In particular, the present invention relates to an ISVD as described herein, wherein said ISVD specifically binds to ADAMTS5 and is mainly composed of 4 framework regions (FR1 - FR4, respectively) and 3 complementarity determining regions (CDR1 - CDR3, respectively), wherein said ISVD is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18.

It is understood that, without limitation, the immunoglobulin single variable domains of the present invention can be used as "building blocks" to produce a polypeptide, which may optionally contain one or more additional immunoglobulin single variable domains that can serve as building blocks (i.e., i.e. against the same or a different epitope on ADAMTS5 and/or against one or more antigens, proteins or targets other than ADAMTS5).

The polypeptide of the invention (also referred to herein as "nanobody construct") contains at least one ADAMTS-binding ISVD, preferably ADAMTS5, for example, two ADAMTS5-binding ISVDs, and preferably also an albumin-binding ISVD. In a polypeptide of the invention, ISVDs may be linked directly or linked via a linker. Even more preferably, the polypeptide of the invention contains a C-terminal extension. As detailed herein, C-terminal extension basically prevents/stops binding of pre-existing antibodies/factors in most samples from human subjects/patients. The C-terminal extension is present at the C-terminus of the last amino acid residue (usually a serine residue) of the last (localized closest to the C-terminus) ISVD.

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

In general, polypeptides or constructs containing or essentially consisting of a single building block, a single ISVD, or a single nanobody are referred to herein as "monovalent" polypeptides and "monovalent constructs", respectively. Polypeptides or constructs containing two or more building blocks (such as ISVDs) are also referred to herein as "multivalent" polypeptides or constructs, and building blocks/ISVDs present in such polypeptides or constructs are also referred to herein as being in a "multivalent format". For example, a "bivalent" polypeptide may contain two ISVDs optionally linked via a linker sequence, while a "trivalent" polypeptide may contain three ISVDs optionally linked via two linker sequences; while a "tetravalent" polypeptide may contain four ISVDs, optionally linked via three linker sequences, and so on.

In a multivalent polypeptide, two or more ISVDs may be the same or different, and may be directed against the same antigen or antigenic determinant (e.g., against the same part(s) or epitope(s), or against different parts or epitopes), or may alternatively be directed against different antigens or antigenic determinants; or may be any suitable combination thereof. Polypeptides and constructs containing at least two building blocks (such as ISVD) in which at least one building block is directed against the first antigen (i.e. ADAMTS5) and at least one building block is directed against the second antigen ( i.e., other than ADAMTS5) may also be referred to as "multispecific" polypeptides and constructs, and the building blocks (e.g., such as ISVD) present in such polypeptides and constructs are also referred to herein as being in a "multispecific format" . Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide containing at least one ISVD directed against a first antigen (i.e., ADAMTS5) and at least one additional ISVD directed against a second antigen (i.e., ADAMTS5). other than ADAMTS5), while a "trispecific" polypeptide of the invention is a polypeptide containing at least one ISVD directed against the first antigen (i.e. ADAMTS5), at least one additional ISVD directed against the second antigen ( ie, different from ADAMTS5) and at least one additional ISVD directed against the third antigen (ie, different from both ADAMTS5 and the second antigen); etc.

In one aspect, the present invention relates to a polypeptide containing at least 2 ISVD, where at least 1 ISVD specifically binds to ADAMTS, preferably ADAMTS5, more preferably selected from the group consisting of SEQ ID NOS: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

In one aspect, the present invention provides a polypeptide comprising at least 2 ISVDs, wherein said at least 2 ISVDs specifically bind to ADAMTS, preferably ADAMTS5, more preferably, each ISVD of said 2 ISVDs is independently selected from the group consisting of SEQ ID NO: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

"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 building blocks, each of which has a different paratope.

Accordingly, ADAMTS5-binding ISVDs of the invention may be in substantially isolated form (as defined herein), or they may form part of a construct or polypeptide, which may contain or primarily consist of one or more ADAMTS5-binding ISVDs and which may optionally further comprise one or more additional amino acid sequences (all optionally linked via one or more suitable linkers). The present invention relates to a polypeptide or construct that comprises or essentially consists of at least one ISVD of the invention, such as one or more ISVDs of the invention (or suitable fragments thereof) that bind ADAMTS5.

One or more ISVDs of the invention may be used as building blocks in such a polypeptide or construct, so as to produce a monovalent, multivalent, or multiparatopic polypeptide or construct of the invention, respectively, all as described herein. The present invention thus also relates to a polypeptide which is a monovalent construct comprising or essentially consisting of a single monovalent polypeptide or ISVD of the invention.

The present invention thus also relates to a polypeptide or construct that is a multivalent polypeptide or a multivalent construct, respectively, such as, for example, a divalent or trivalent polypeptide or construct containing or essentially consisting of two or more ISVDs of the invention (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. WO 99/23221 and WO 2010/115998).

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 ISVD, such as a nanobody directed against ADAMTS5, and an identical second ISVD, such as a nanobody directed against ADAMTS5, where said first and said second ISVDs, such as nanobodies, may optionally be linked via a linker sequence (as defined herein). In another form, the multivalent polypeptide or construct of the invention may be a trivalent polypeptide or construct of the invention comprising a first ISVD, such as a nanobody directed against ADAMTS5, identical to a second ISVD, such as a nanobody directed against ADAMTS5, and an identical third ISVD, such as as a nanobody directed against ADAMTS5, wherein said first, second and third ISVDs, such as nanobodies, may optionally be linked via one or more, and in particular two, linker sequences. In one aspect, the invention relates to a polypeptide or construct containing or essentially consisting of at least two ISVDs of the invention, for example, 2, 3 or 4 ISVDs (or suitable fragments thereof) that bind ADAMTS5. Two or more ISVDs may optionally be linked via one or more peptide linkers.

In another aspect, the multivalent polypeptide or construct of the invention may be a bispecific polypeptide or construct of the invention comprising a first ISVD such as a nanobody directed against ADAMTS5 and a second ISVD such as a nanobody directed against a second antigen such as aggrecan. in which said first and second ISVDs, such as nanobodies, may optionally be linked via a linker sequence (as defined herein); while a multivalent polypeptide or construct of the invention may also be a trispecific polypeptide or construct of the invention comprising a first ISVD such as a nanobody directed against ADAMTS5, a second ISVD such as a nanobody directed against a second antigen such as aggrecan , and a third ISVD, such as a nanobody directed against a third antigen, wherein said first, second, and third ISVDs, such as nanobodies, may optionally be linked via one or more, and in particular two, linker sequences.

The invention further provides a multivalent polypeptide comprising or (substantially) consisting of at least one ISVD (or suitable fragments thereof) that binds ADAMTS5, preferably human ADAMTS5, and one additional ISVD, such as an aggrecan-binding ISVD.

Particularly preferred bivalent, bispecific polypeptides or constructs of the invention are those shown in the Examples described herein and in Table A-1 (cf. SEQ ID NO: 120-130 (i.e. SEQ ID NO: 120 , 121, 122, 123, 124, 125, 126, 127, 128, 129 and 130), most preferably SEQ ID NOS: 129 and 130).

In a preferred aspect, the polypeptide or construct of the invention comprises or essentially consists of at least two ISVDs, wherein said at least two ISVDs may be the same or different, but of which at least one ISVD is directed against ADAMTS5, preferably said ISVD binding ADAMTS5 is selected from the group consisting of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

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

In one aspect of the invention, the first ISVD and the second ISVD present in the multiparatopic (preferably biparatopic or triparatopic) polypeptide or construct of the invention do not (cross)compete with each other for binding to ADAMTS5 and thus belong to different families. Accordingly, the present invention provides a multiparatopic (preferably biparatopic) polypeptide or construct containing two or more ISVDs, where each ISVD belongs to a different family. In one aspect, the first ISVD of the multiparatopic (preferably biparatopic) polypeptide or construct of the invention does not cross-block the binding to ADAMTS5 of the second ISVD of the multiparatopic (preferably biparatopic) polypeptide or construct of the invention, and/or the binding of the first ISVD to ADAMTS5 does not is cross-blocked by the second ISVD. In another aspect, the first ISVD of the multiparatopic (preferably biparatopic) polypeptide or construct of the invention cross-blocks the binding to ADAMTS5 of the second ISVD of the multiparatopic (preferably biparatopic) polypeptide or construct of the invention, and/or the binding of the first ISVD thereof to ADAMTS5 is cross-blocked second ISVD.

In a particularly preferred aspect, the polypeptide or construct of the invention contains or essentially consists of three or more ISVDs, of which at least two ISVDs are directed against ADAMTS5. It should be understood that the at least two ISVDs directed against ADAMTS5 may be the same or different, may be directed against the same epitope or different ADAMTS5 epitopes, may belong to the same epitope group or to different epitope groups, and/or may bind to the same or different ADAMTS5 domains.

The relative affinity may depend on the location of the ISVD in the polypeptide. It should be understood that the order of the ISVD in the polypeptide of the invention (orientation) can be chosen, as appropriate, by one 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 ISVD (eg, ISVD 1) and second ISVD (eg, ISVD 2) in a polypeptide of the invention may be (N-terminus to C-terminus): (i) ISVD 1 (eg, nanobody 1) - [linker] - ISVD 2 (eg nanobody 2) - [C-terminal extension]; or (ii) ISVD 2 (eg nanobody 2) - [linker] - ISVD 1 (eg nanobody 1) - [C-terminal extension]; (where the groups in square brackets, i.e. linker and C-terminal extension, are optional). All orientations are included in the invention. Polypeptides containing an ISVD orientation that provides the desired binding characteristics can be easily identified by conventional screening, for example, as illustrated in the Examples section. The preferred order is from N-terminus to C-terminus: ISVD binding ADAMTS5 - [linker] - ISVD binding Albumin or aggrecan - [C-terminal extension], where groups in square brackets are optional.

In one aspect, the present invention relates to a polypeptide containing two or more ISVDs that specifically bind to ADAMTS5, where

a) at least the "first" ISVD specifically binds to a first antigenic determinant, epitope, moiety, domain, subunit or conformation of ADAMTS5, preferably wherein said "first" ISVD specifically binding ADAMTS5 is selected from the group consisting of SEQ ID NO: 2, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18; and

b) at least a "second" ISVD specifically binds to a second antigenic determinant, epitope, moiety, domain, subunit, or conformation of ADAMTS5 different from the first antigenic determinant, epitope, moiety, domain, subunit, or conformation, respectively, preferably, wherein said " the second" ISVD specifically binding ADAMTS5 is SEQ ID NO: 116 or 19.

In a preferred aspect, the polypeptide or construct of the invention comprises or essentially consists of at least two ADAMTS5 binding ISVDs, wherein said at least two ISVDs may be the same or different, which are independently selected from the group consisting of SEQ ID NO: 2 , 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

In a further aspect, the invention relates to a multiparatopic (preferably biparatopic) polypeptide or construct comprising two or more ISVDs directed against ADAMTS5 binding the same epitope(s) that bind to any of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

In a further aspect, the invention relates to a polypeptide as described herein, wherein said polypeptide has at least 80%, 90%, 95%, or 100% sequence identity with any of SEQ ID NOs: 1-19 (i.e. SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19), 116-117 or 120-130 (i.e., SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 and 130).

In one aspect, the present invention relates to a polypeptide as described herein, which is selected from the group consisting of SEQ ID NO: 127 (clone 130 049-093-Alb), SEQ ID NO: 126 (clone 129 2F3-093- Alb), SEQ ID NO: 127 (clone 130 049-093-Alb) and SEQ ID NO: 128 (clone 131 9D3-093-Alb).

There is a need in the art for more effective therapies against disorders that affect cartilage in the joints, such as osteoarthritis. Especially when administered systemically, the retention time of most drugs is insufficient. The present inventors hypothesized that the efficacy of a therapeutic drug, such as the construct, polypeptide, and ISVD of the invention, can be significantly increased by associating the therapeutic drug with a moiety that increases drug half-life and therefore increases drug retention, but which does not must interfere with the efficacy of said therapeutic medicinal product.

In a particular aspect of the invention, a construct or polypeptide of the invention may have a group conferring an increased half-life compared to a corresponding construct or polypeptide of the invention without said group. 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 the further description herein, and for example, include ISVDs or polypeptides of the invention chemically modified to increase half-life (eg, by pegylation); ADAMTS5 binding molecules of the invention, such as ISVDs and/or polypeptides of the invention, containing at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention comprising at least one ISVD of the invention linked to at least one group (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, containing such half-life prolonging groups or ISVDs will become apparent to those skilled in the art based on the further description herein; and, for example, include, without limitation, polypeptides in which one or more ISVDs of the invention are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or one or more binding units that can bind to serum proteins (e.g., such as domain antibodies, immunoglobulin single variable domains suitable for use as a domain antibody, single domain antibodies, immunoglobulin single variable domains suitable for use as a single domain antibody, dAbs, immunoglobulin single variable domains suitable for use as a dAb or nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin; reference is made to additional description and cc labels mentioned in the present description); polypeptides in which the amino acid sequence of the invention is linked to an Fc fragment (such as a human Fc) or a corresponding portion or fragment thereof; or polypeptides in which one or more single variable domains of the immunoglobulins of the invention are suitably linked to one or more small proteins or peptides that can bind to serum proteins, such as, without limitation, 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, or a polypeptide, wherein said construct or said polypeptide further comprises a whey protein binding group or whey protein. Preferably, said serum protein binding group binds serum albumin, such as human serum albumin.

In one aspect, the present invention provides a polypeptide as described herein comprising an ISVD that binds serum albumin.

In general, constructs or polypeptides of the invention with increased half-life preferably have a half-life of at least 1.5 times, preferably at least 2 times, for example at least 5 times, for example at least 10 times or more than 20 times the intrinsic half-life of the respective constructs or polypeptides of the invention, i. e. without a group conferring an extended half-life. For example, constructs or polypeptides of the invention with extended half-life may have a half-life, for example, in humans, increased by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example, more than 12 hours, or even more than 24, 48 or 72 hours, compared to the actual corresponding constructs or polypeptides of the invention, ie. without a group conferring an extended half-life.

In a preferred but non-limiting aspect of the invention, the constructs of the invention and the polypeptides of the invention have a serum half-life, for example in humans, 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, in comparison with the actual corresponding constructs or polypeptides according to the invention, i. without a group conferring an extended half-life.

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

In a particularly preferred but non-limiting aspect of the invention, the invention relates to a construct of the invention and a polypeptide of the invention comprising, in addition to one or more building blocks that bind ADAMTS5, at least one building block that binds serum albumin, such as ISVD that binds serum albumin. albumin, such as human serum albumin, as described herein. Preferably, said serum albumin binding ISVD 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 ISVD is selected from the group consisting of Alb8, Alb23, Alb129, Alb132, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G , Alb82-GG, Alb82-GGG, Alb92 or Alb223 (cf. table D).

In one aspect, the present invention provides a polypeptide as described herein, comprising at least one ADAMTS5 binding ISVD and a serum albumin binding ISVD, preferably selected from the group consisting of SEQ ID NO: 129 (clone 577 2F3 *-Alb), SEQ ID NO: 130 (clone 579 2F3*-093-Alb), SEQ ID NO: 120 (clone 4 2A12-Alb), SEQ ID NO: 121 (clone 5 2D7-Alb), SEQ ID NO : 122 (clone 6 2F3-Alb), SEQ ID NO: 123 (clone 69 049-Alb), SEQ ID NO: 124 (clone 70 9D3-Alb), SEQ ID NO: 125 (clone 71 3B2-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb) and SEQ ID NO: 128 (clone 131 9D3-093-Alb) (cf. Table A-1 ).

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

There is a need in the art for more effective therapies against disorders that affect cartilage in the joints, such as osteoarthritis. Even with intra-articular administration, the retention time of most drugs for the treatment of affected cartilage is insufficient. The present inventors hypothesized that the efficacy of a therapeutic drug, such as a construct, polypeptide, and ISVD of the invention, can be modulated by associating the therapeutic drug with a moiety that can anchor the drug in the joint and therefore increase drug retention, but which should not interfere with the effectiveness of the specified therapeutic drug (this group is also referred to in the present description as "cartilage anchoring protein" or "CAP"). This concept of anchoring can modulate not only the efficacy of a drug, but also the specificity of action for the diseased joint, by reducing toxicity and side effects, thus expanding the number of possible useful drugs.

The format of the molecule for clinical use is envisaged to contain one or two building blocks, such as ISVDs that bind ADAMTS5, and one or more building blocks, such as ISVDs, with such a retention mechanism, and optionally additional groups. The co-pending application shows that such formats retain both ADAMTS5 binding and therapeutic effect, eg, inhibitory activity, as well as retention properties. One or more building blocks, such as ISVD, with a retaining mechanism of action, can be any building block that has a retaining effect ("CAP building block") in diseases in which ADAMTS5 is involved, such as arthritic disease, osteoarthritis, spondyloepimetaphyseal dysplasia, lumbar disc degeneration disease, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, aggrecanopathy.

The “CAP building block” is used to guide, anchor and/or retain other, e.g. therapeutic, building blocks, such as ADAMTS5 binding ISVDs, at a desired site, such as, for example, a joint where said other, e.g., therapeutic, building block block should have its effect, for example, binding and/or inhibition of ADAMTS5.

The present inventors further hypothesize that aggrecan-binding molecules, such as aggrecan-binding ISVDs, could potentially function as such an anchor, although aggrecan is highly glycosylated and degraded in various disorders affecting cartilage in the joints. In addition, given the cost and extensive testing in various animal models required before a drug can enter the clinic, such aggrecan-binding molecules should preferably have broad cross-reactivity, e.g., aggrecan-binding molecules should bind to aggrecan. from various types.

Using various inventive methods of immunization, screening and characterization, the authors of the present invention were able to identify various aggrecan-binding molecules with excellent features of selectivity, stability and specificity, providing prolonged retention and activity in the joint (cf. concurrent application).

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

In the present invention, the term "reducing and/or inhibiting outflow" means reducing and/or inhibiting the outflow 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 100%, compared to the efflux of the above composition, polypeptide or construct in the joint under the same conditions but in the absence of an aggrecan-binding molecule of the invention, for example, aggrecan-binding ISVDs.

It is envisaged that, in addition to the diseases in which ADAMTS5 is involved, such as arthritic disease, osteoarthritis, spondyloepimetaphyseal dysplasia, lumbar disc degeneration disease, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, and aggrecanopathy, the aggrecan binding molecules of the invention can also be used in various other diseases affecting cartilage such as arthropathies and chondrodystrophies, arthritic disease (such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment), achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration disease, degenerative joint disease and relapsing polychondritis (collectively referred to herein as "aggrecan-associated diseases").

Said CAP building block, eg ISVDs, that bind aggrecan preferably binds to cartilage tissue such as cartilage and/or meniscus. In a preferred aspect, the CAP building block is cross-reactive with other species and specifically binds one or more of human aggrecan (SEQ ID NO: 155), dog aggrecan, bovine aggrecan, rat aggrecan; pig aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and/or rhesus monkey aggrecan. Relevant structural information for aggrecan can be found, for example, in access numbers (UniProt) as shown in Table 2 below.

The preferred building block of CAP is an ISVD that binds aggrecan, preferably human aggrecan, preferably represented by SEQ ID NO: 155, as shown in Table B.

table 2

Name access number human aggrecan (SEQ ID NO: 155) P16112 aggrecan dog Q28343 aggrecan bull P13608 rat aggrecan P07897 pig aggrecan (kor) Q29011 mouse aggrecan Q61282 rabbit aggrecan G1U677-1 aggrecan cynomolgus macaque XP_002804990.1 aggrecan rhesus monkey XP_002804990.1

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

The present invention thus relates to a polypeptide or construct of the invention further comprising at least one aggrecan-specific binding ISVD, preferably selected from the ISVDs shown in SEQ ID NOs: 156 and 157.

In one aspect, the present invention relates to a polypeptide, as described in the present description, containing at least 2 ISVDs that specifically bind aggrecan.

In one aspect, the present invention provides a polypeptide as described herein comprising at least 2 aggrecan-specific binding ISVDs, wherein said at least 2 aggrecan-specific binding ISVDs may be the same or different.

In one aspect, the present invention provides a polypeptide as described herein comprising at least 2 aggrecan-specific binding ISVDs, wherein said at least 2 aggrecan-specific binding ISVDs are independently selected from the group consisting of SEQ ID NO: 156 -157.

In one aspect, the present invention provides a polypeptide as described herein comprising at least 2 aggrecan-specific binding ISVDs, wherein said at least 2 aggrecan-specific binding ISVDs are represented by SEQ ID NOs: 156-157.

In one aspect, the present invention provides a polypeptide as described herein comprising an aggrecan-specific binding ISVD, wherein said aggrecan-specific binding ISVD specifically binds human aggrecan [SEQ ID NO: 155].

In one aspect, the present invention relates to a polypeptide as described herein, wherein said aggrecan-specific ISVD specifically binds human aggrecan (SEQ ID NO: 155), dog aggrecan, bovine aggrecan, rat aggrecan; pig aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and/or rhesus monkey aggrecan.

In one aspect, the present invention relates to a polypeptide as described herein, wherein said ISVD specifically binding aggrecan preferably binds to cartilage tissue such as cartilage and/or meniscus.

It should be understood that the ISVD, polypeptide and construct of the invention are preferably stable. The stability of a polypeptide, construct, or ISVD of the invention can be measured by conventional assays known to one of skill in the art. Exemplary assays include, but are not limited to, assays that determine the activity of the indicated polypeptide, construct, or ISVD, followed by incubation in synovial fluid for the desired period of time, after which the activity is determined again.

In one aspect, the present invention provides an ISVD, 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.

The desired activity of a therapeutic building block, eg, ADAMTS5 binding ISVD, in a multivalent polypeptide or construct of the invention can be measured by conventional assays known to one of skill in the art.

In one aspect, the present invention provides a construct as described herein comprising at least one ISVD or polypeptide and one or more other groups, residues, motifs, or linking units. One or more other groups, residues, motifs or binding units are preferably selected from the group consisting of a polyethylene glycol molecule, whey proteins or fragments thereof, binding units that can bind to whey proteins, an Fc fragment, and small proteins or peptides that can bind to serum proteins, additional amino acid residues, labels or other functional groups, such as toxins, labels, radioactive chemicals, etc.

In one embodiment, as mentioned below, the present invention relates to a construct of the invention, such as a polypeptide containing a group that provides an extension of half-life, where the specified group is a PEG. Thus, the present invention also 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:

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

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

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

d) may be one or more amino acid residues that have been functionalized and/or that may serve as a site for the attachment of functional groups. Suitable amino acid residues and functional groups are known to those skilled in the art and include, but are not limited to, the amino acid residues and functional groups mentioned herein for the polypeptide derivatives of the invention.

The present invention also relates to constructs containing a polypeptide and/or ISVD according to the invention, which additionally contain other functional groups, for example, toxins, labels, radioactive chemicals, etc.

Other groups, residues, motifs or linking units may, for example, be chemical groups, residues, motifs, which themselves may or may not be biologically and/or pharmacologically active. For example, and without limitation, such groups can be linked to one or more ISVDs or polypeptides of the invention in such a way as to produce a "derivative" of the polypeptide or construct of the invention.

Accordingly, the invention, in its broadest sense, also relates to constructs and/or polypeptides that are derivatives of the constructs and/or polypeptides of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (eg, enzymatic) modification, of the constructs and/or polypeptides of the invention and/or of one or more amino acid residues forming a polypeptide of the invention.

Examples of such modifications, as well as examples of amino acid residues in polypeptide sequences that can be modified in this way (i.e. either in the protein backbone, but preferably on the side chain), methods and methods that can be used to introduce such modifications, and the potential applications and benefits of such modifications are known 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 bonding or any other suitable method) of one or more of the (functional) groups, residues or motifs in or on the polypeptide of the invention, and in particular, one or more of the functional groups, residues or motifs conferring one or more desirable properties or functionalities on a construct and/or polypeptide of the invention. Examples of such functional groups are known to the 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 motifs that increase the half-life, solubility and/or absorption of the construct or polypeptide of the invention, which reduce the immunogenicity and/or toxicity of the construct, or a polypeptide of the invention that eliminates or reduces any undesirable side effects of the construct or polypeptide of the invention and/or that provides other advantageous properties and/or reduces undesirable properties of the construct or polypeptide of the invention; or any combination of two or more of the above. Examples of such functional motifs and methods of introducing them are known to those skilled in the art, and may generally include the functional motifs and methods mentioned in the general art cited herein above, as well as the functional motifs and methods known per se for modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ^ScFv and single domain antibodies), for which reference is made to, for example, Remington (Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA, 1980 ). Such functional motifs can, for example, be linked directly (eg, covalently) to the polypeptide of the invention, or optionally via a suitable linker or spacer, again as known to one of skill in the art.

One specific example is a derivative of a polypeptide or construct of the invention, wherein the polypeptide or construct of the invention is 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 decreasing the immunogenicity of pharmaceutical proteins and involves attaching a suitable pharmacologically acceptable polymer such as poly(ethylene glycol) (PEG) or derivatives thereof (such as methoxypoly(ethylene glycol) or mPEG). In general, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including, but not limited to, (single)domain antibodies and ScFvs); reference is made to, for example, Chapman (Nat. Biotechnol. 54: 531-545, 2002), Veronese and Harris (Adv. Drug Deliv. Rev. 54: 453-456, 2003), Harris and Chess (Nat. Rev. Drug Discov 2: 214-221, 2003) and WO 04/060965. Various protein pegylation reagents are also commercially available, for example from Nektar Therapeutics, USA.

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

Preferably, for the constructs or 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 20000-80000.

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-emitting groups or motifs, depending on the intended use of the polypeptide or construct of the invention. Suitable labels and methods for attaching, using and detecting them are known to those skilled in the art, and for example include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phtaldehyde and fluorescamine, and fluorescent metals such as ¹⁵² Eu or other metals from the lanthanide group), 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) , radioactive isotopes (such as ³ H, ¹²⁵ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe and ⁷⁵ Se), metals, metal chelates or metal cations (e.g., such as metal cations ^99m Tc, ¹²³ I, ¹¹¹ In, ¹³¹ I, ⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga and ⁶⁸ Ga or other metals or metal cations particularly suitable for use in in vivo, in vitro or in situ diagnostic and imaging applications , such as to ( ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr and ⁵⁶ Fe)), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcus nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels are known to the person skilled in the art and include, for example, motifs that can be detected using NMR or ESR spectroscopy.

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

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

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

Other potential chemical and enzymatic modifications are known to those skilled in the art. Such modifications may also be introduced for research purposes (eg, to investigate function-activity relationships). Reference is made to, for example, Lundblad and Bradshaw (Biotechnol. Appl. Biochem. 26: 143-151, 1997).

Preferably, the constructs, polypeptides and/or derivatives are such that they bind ADAMTS5, with an affinity (appropriately measured and/or expressed as a K _D value (true or apparent), a K _A value (true or apparent), k _on rate, or binding rate and/or k _off or dissociation rate, or alternatively as an IC ₅₀ value, as further described herein), i.e., as defined herein (eg, as defined for the polypeptides of the invention).

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

In one aspect, the present invention relates to a construct of the invention which contains or essentially consists of an ISVD of the invention or a polypeptide of the invention, and which further comprises one or more other groups, residues, motifs, or linking units, which are optionally linked via one or more peptide linkers.

In one aspect, the present invention provides a construct of the invention wherein one or more of the other groups, residues, motifs, 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 serum proteins, an Fc fragment, and small proteins or peptides that can bind to serum proteins.

In the constructs of the invention, such as the polypeptides of the invention, two or more building blocks, such as the ISVD, and optionally, one or more other groups, drugs, agents, residues, motifs, or binding units may be directly linked to each other. the other (eg, as described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof. Suitable spacers or linkers for use in multivalent and multispecific polypeptides are known to those skilled in the art, and 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 for pharmaceutical use.

For example, a polypeptide of the invention may, for example, be a trivalent, trispecific polypeptide containing one building block, such as an ADAMTS5-binding ISVD, an albumin-binding ISVD, and potentially another building block, such as a third ISVD, wherein said first, the second and third building blocks, such as ISVD, may optionally be linked via one or more, and in particular 2, linker sequences. The present invention also relates to a construct or polypeptide of the invention comprising a first ADAMTS5-binding ISVD and possibly a second albumin-binding ISVD and/or possibly a third ISVD and/or possibly a fourth ISVD, wherein said first ISVD and /or said second ISVD and/or possibly said third ISVD and/or possibly said fourth ISVD are connected via linkers, in particular 3 linkers.

Some particularly preferred linkers include those 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 used in the art to construct diabodies or ScFv fragments (in this regard, however, it should be noted that while in diabodies and ScFv fragments, the linker sequence used must have a length, degree of flexibility, and other properties that allow convergence of the respective V _H and V _L domains to form a complete antigen-binding site, there is no particular limitation on the length or flexibility of the linker used in the polypeptide of the invention, since each ISVD , such as a nanobody, itself forms a complete antigen-binding site).

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

Some other particularly preferred linkers are poly-alanine (such as AAA), as are the linkers GS30 (see also SEQ ID NO: 85 in WO 06/122825) and GS9 (see also SEQ ID NO: 84 in WO 06 /122825).

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

It is included 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 effect on the properties of the final construct of the invention, such as a polypeptide of the invention, including, but not limited to, affinity, specificity, or avidity for a chemokine, or for one or more other antigens. Based on the description herein, one of skill in the art is able to determine the optimal linker(s) for use in a particular construct of the invention, such as a polypeptide of the invention, optionally after some limited routine experimentation.

For example, in multivalent polypeptides of the invention containing building blocks, ISVDs or nanobodies directed against ADAMTS5 and another target, the length and flexibility of the linker is preferably such that each building block, such as an ISVD of the invention, present in the polypeptide, bind to a related target, for example, an antigenic determinant on each of the targets. Again, based on the description herein, one skilled in the art can determine the optimal linker(s) for use in a construct of the invention, such as a polypeptide of the invention, optionally after some limited routine experimentation.

It is also included within the scope of the invention that the linker(s) used confer one or more other advantageous properties or functionalities to the constructs of the invention, such as the polypeptides of the invention, and/or provide one or more sites for derivatization and/or attachment of functional groups (eg, as described herein for the ISVD 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 description herein, one of skill in the art is able to determine the optimal linkers for use in a particular polypeptide of the invention, optionally 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 description herein, one of skill in the art is able to determine the optimal linkers for use in a particular construct or polypeptide of the invention, optionally after some limited routine experimentation.

Typically, for ease of expression and production, a construct of the invention, such as a polypeptide of the invention, is 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, ISVDs or nanobodies, one can link them using a linker with three or more "arms", where each "arm" is associated with a building block, ISVD or nanobody, so as to obtain a "star-shaped" design. It is also possible, although generally 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 ISVDs are directly linked to each other or linked via a linker.

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

Accordingly, the present invention provides a construct of the invention, such as a polypeptide of the invention, wherein said linker is selected from the group consisting of linkers from 3A, 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, 35GS, poly -A, 8GS, 40GS, G1 hinge, 9GS-G1 hinge, lama upper long hinge region, and G3 hinge, such as those shown in Table C (SEQ ID NO: 158-174).

Accordingly, the present invention relates to a construct of the invention, such as a polypeptide of the invention, wherein said polypeptide is selected from the group consisting of SEQ ID NOs: 120-130.

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

The multivalent polypeptides of the invention can generally be prepared by a process that includes at least the step of linking, in an appropriate manner, an ISVD and/or a monovalent polypeptide of the invention to one or more additional ISVDs, optionally via one or more suitable linkers, so as to obtain multivalent polypeptide according to the invention. The polypeptides of the invention can also be produced by a method that generally includes at least the steps of obtaining a nucleic acid encoding a polypeptide of the invention, expressing said nucleic acid in an appropriate manner, and isolating the expressed polypeptide of the invention. Such methods can be carried out in a manner known per se, known to a person skilled in the art, for example, based on the methods and methods further described in the present description.

The process for producing multivalent polypeptides of the invention may include at least the steps of linking two or more ISVDs of the invention and, for example, one or more linkers, in an appropriate manner. The ISVDs of the invention (and linkers) can be linked by any method known in the art and as further described herein. Preferred methods include joining nucleic acid sequences encoding the ISVDs of the invention (and linkers) to obtain a genetic construct expressing a multivalent polypeptide. Methods for joining amino acids or nucleic acids are known to those skilled in the art, and reference is again 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 ISVD of the invention to produce a multivalent polypeptide of the invention. A method for producing a multivalent polypeptide may include linking an ISVD of the invention to at least one additional ISVD of the invention, optionally via one or more linkers. The ISVD of the invention is then used as a binding domain or building block to provide and/or obtain a multivalent polypeptide containing 2 (e.g., in a divalent polypeptide), 3 (e.g., in a trivalent polypeptide), 4 (e.g., in a tetravalent polypeptide) or more ( for example, in a multivalent polypeptide) building blocks. In this regard, an ISVD of the invention can be used as a binding domain or binding unit to provide and/or obtain 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 ISVD polypeptide of the invention (as described herein) to obtain a multivalent polypeptide. A method for producing a multivalent polypeptide may include linking an ISVD of the invention to at least one additional ISVD of the invention, optionally via one or more linkers.

The polypeptides and nucleic acids of the invention can be obtained in a manner known per se, as will become clear to a person skilled in the art from the further description in this document. For example, the polypeptides of the invention can be prepared by any method known per se for making antibodies, and in particular for making antibody fragments (including, but not limited to, (single)domain antibodies and ScFv fragments). Some preferred, but non-limiting, methods for producing polypeptides and nucleic acids include those described herein.

A method for producing a polypeptide of the invention may include the following steps: expressing, in a suitable host cell or host organism (also referred to herein as "the host of the invention"), or in another suitable expression system, a nucleic acid encoding said polypeptide of the invention (also referred to herein as "nucleic acid of the invention"); optionally, followed by isolation and/or purification of the polypeptide of the invention thus obtained.

In particular, such a method may include the 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 of the invention thus obtained.

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

The nucleic acid of the invention may be in the form of single or double stranded DNA or RNA. According to one embodiment of the invention, the nucleic acid of the invention is in a substantially isolated form as defined herein. The nucleic acid of the invention may also be in the form of, present in, and/or be part of a vector, eg an expression vector such as, for example, a plasmid, cosmid, or YAC, which again may be in a 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 may be prepared or prepared in a manner known per se based on the disclosure of the polypeptides of the invention provided herein and/or isolated from a suitable natural source, as well as one skilled in the art to obtain a nucleic acid. acids of the invention, also multiple nucleotide sequences, such as at least two nucleic acids encoding an ISVD of the invention and, for example, nucleic acids encoding one or more linkers, can be joined together in an appropriate manner. Methods for producing nucleic acids of the invention are known to those skilled in the art and may, for example, include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more natural and/or synthetic sequences (or two or more parts thereof), introducing mutations resulting in the expression of a truncated expression product; introducing one or more restriction sites (for example, to obtain cassettes and/or regions that can be easily cleaved and/or ligated using suitable restriction enzymes), and/or introducing mutations via a PCR reaction using one or more "mismatched" primers. These and other methods are known to the person skilled in the art, and reference is again made to standard manuals such as Sambrook et al. and Ausubel et al., mentioned above, as well as the examples below.

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

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

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

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

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

The genetic constructs of the invention can generally be obtained by linking, in an appropriate manner, the nucleotide sequence(s) of the invention to one or more of the additional elements described above, for example, using the methods 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, i. for expression and/or production of a polypeptide of the invention. Suitable hosts or host cells are known to 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 other host cells or (non-human) hosts known per se to express and produce antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) known to one of skill in the art. Reference is also made to the general prior art cited herein above, as well as, for example, to WO 94/29457; W096/34103; WO 99/42077; Franken et al. (Res Immunol. 149: 589-99, 1998); Riechmann and Muyldermans (1999), supra; van der Linden (J. Biotechnol. 80: 261-70, 2000); Joosten et al. (Microb. Cell Fact. 2: 1, 2003); Joosten et al. (Appl. Microbiol. Biotechnol. 66: 384-92, 2005); and additional references cited in the present description. 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 are known to the person skilled in the art. Suitable methods for transforming a host or host cell of the invention are known to those skilled in the art and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is made again to the manuals and patent applications mentioned above. A transformed host cell (which may be in the form of a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) constitute further aspects of the present invention. Accordingly, the present invention relates to a host or host cell 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 are (at least) capable of expressing (e.g., under suitable conditions) a polypeptide of the invention (and, in the case of a host organism: in at least one of its 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, for example, be obtained by cell division or by sexual or asexual reproduction.

In order to produce/obtain expression of the polypeptides of the invention, the transformed host cell or transformed host organism can generally be maintained, maintained and/or cultured under conditions such that the (desired) polypeptide of the invention is expressed/produced. Suitable conditions are known to the person skilled in the art and generally may depend on the host cell/host organism used, as well as on the regulatory elements controlling the expression of the (corresponding) nucleotide sequence of the invention. Reference is again made to the manuals and patent applications mentioned above in the sections 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 methods of protein isolation and/or purification known per se, such as methods ( preparative) chromatography and/or electrophoresis, differential precipitation methods, affinity methods (e.g., using a specific, cleavable amino acid sequence fused to a polypeptide of the invention) and/or preparative immunological methods (i.e., using antibodies against the polypeptide to be isolated ).

In one aspect, the invention provides a method for producing a construct, polypeptide, or ISVD 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, an ISVD polypeptide of the invention.

In one aspect, the invention provides a composition comprising a construct, polypeptide, ISVD, or nucleic acid of the invention.

As mentioned above, there remains a need for safe and effective anti-OA drugs. Based on non-conventional screening, characterization and combination methods, the present inventors have identified ISVDs that bind and inhibit ADAMTS5. These ADAMTS5 binding molecules performed exceptionally well in both in vitro and in vivo experiments. In addition, the ISVDs of the invention have also been shown to be significantly more effective than prior art compounds. The present invention thus relates to ISVDs and ADAMTS antagonist polypeptides, in particular ADAMTS5, with improved prophylactic, therapeutic and/or pharmacological properties, including a better safety profile, compared to prior art amino acid sequences and antibodies. In addition, these ADAMTS5-binding molecules, when bound to albumin-binding ISVDs, had increased retention in the subject and could be administered systemically while maintaining activity.

In one aspect, the present invention relates to a method for treating or preventing a disease or disorder in an individual, such as those involving ADAMTS5 activity, comprising administering an ISVD or a polypeptide of the invention to said individual in an amount effective to treat or prevent a symptom of said disease or disorder.

In one aspect, the present invention provides a composition of the invention, an ISVD of the invention, a polypeptide of the invention, and/or a construct of the invention for use as a drug.

In another aspect, the invention relates to the use of an ISVD, a polypeptide and/or a construct of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one ADAMTS5 associated disease; and/or for use in one or more of the treatments mentioned herein.

The invention also relates to the use of an ISVD, a polypeptide 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, which can be prevented and/or treated by modulating the activity of ADAMTS, preferably ADAMTS5, for example, inhibition of aggrecan degradation.

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

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

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

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

In one aspect, the present invention provides a method for preventing or treating arthropathies and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar degeneration disease. disc, degenerative joint disease, and relapsing polychondritis and NASH, wherein said method comprises administering to a subject in need thereof a pharmaceutically active amount of at least a composition, immunoglobulin, polypeptide or construct of the invention to an individual in need thereof.

In one aspect, the present invention relates to the use of an ISVD, a polypeptide, composition or construct of the invention, in the preparation of a pharmaceutical composition for the treatment or prevention of, for example, arthropathies and chondrodystrophies, an arthritic disease such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture or detachment, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration disease, degenerative joint disease and recurrent polychondritis, osteochondritis dissecans and aggrecanopathies, and NASH.

It is also expected that, through binding to aggrecan, the constructs and/or polypeptides of the invention can reduce or inhibit the activity of a member of the serine protease family, cathepsins, matrix metalloproteinases (MMPs), such as, for example, MMP20, but also ADAMTS4 (aggrecanase-1) and /or ADAMTS11, for aggrecan degradation.

In the context of the present invention, the term "prevention and/or treatment" not only includes preventing and/or treating a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated disease, reducing and/or alleviating one or more of the symptoms associated with the disease, reducing the severity and/or duration of the disease and/or any symptoms associated with it, and/or preventing further increase in the severity of the disease and/or any symptoms associated with him, the prevention, reduction or reversal of any physiological damage caused by the disease, and, in general, any pharmacological action that provides an advantage to the patient being treated.

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

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

Typically, a treatment regimen may include administering one or more ISVDs, polypeptides and/or constructs of the invention, or one or more compositions containing them, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to be administered may be determined by the clinician, again based on the factors cited above. Applicable doses of the constructs, polypeptides, and/or ISVDs of the invention can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art; for example, see US 4938949.

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

The number of constructs, polypeptides and/or ISVDs of the invention required for use in treatment may vary depending not only on the particular immunoglobulin, polypeptide, compound and/or construct chosen, but also on the route of administration, the nature of the condition being treated, and age. and the condition of the patient, and is ultimately determined by the judgment of the treating physician or clinician. Also, the dosage of the constructs, polypeptides and/or ISVDs of the invention varies depending on the cell, tumor, tissue, graft, or target organ.

The desired dose may conveniently be presented as a single dose or as divided doses administered at appropriate intervals, for example in the form of two, three, four or more dose portions per day. Actually part of the dose can be further divided, for example, into several discrete injections with free intervals.

The mode of administration may include long-term, daily treatment. By "long" is meant a duration of at least two weeks and preferably several weeks, months or years. Necessary modifications in this dosage range can be determined by a person skilled in the art using only conventional experiments, taking into account the explanations in the present description. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dose may also be adjusted by the individual's physician in the event of any complication.

Generally, in the above method, an ISVD, a polypeptide and/or a construct of the invention can be used. However, the scope of the invention includes the use of two or more ISVDs, polypeptides and/or constructs of the invention in combination.

The ISVDs, polypeptides and/or constructs of the invention may be used in combination with one or more additional pharmaceutically active compounds or components, i. in the form of a combination treatment regimen, which may or may not result in a synergistic effect.

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

Again, the clinician is able to select such additional compounds or components, as well as an appropriate combination treatment regimen, based on the factors cited above and their own judgment.

In particular, the ISVDs, polypeptides, and/or constructs of the invention may be used in combination with other pharmaceutically active compounds or components that are or may be used to prevent and/or treat the diseases, disorders, and conditions cited herein, whereby receive or may not receive a synergistic effect. Examples of such compounds and components, as well as methods, methods, and pharmaceutical compositions or compositions for their administration are clear to the clinician.

When two or more substances or components are intended to be used as part of a combination treatment regimen, they may be administered via the same route of administration or via different routes of administration, substantially at the same time or at different times (e.g., essentially simultaneously, sequentially, or according to an alternative mode). When the substances or components are intended to be administered simultaneously, via the same route of administration, they may be administered in the form of different pharmaceutical formulations or compositions, or as part of a combined pharmaceutical formulation or composition, as known to those skilled in the art.

Also, when two or more active substances or methods are intended to be used as part of a combination treatment regimen, each of the substances or components can be administered in the same amount and according to the same regimen as used when the compound or component is used alone, and such combined use may or may not result in a synergistic effect. However, when the combined use of two or more active substances or components results in a synergistic effect, it may also be possible to reduce the amount of one, more than one or all of the substances or components to be administered while still achieving the desired therapeutic effect. This, for example, can be used to eliminate, limit or reduce any unwanted side effects associated with the use of one or more of the substances or components, when used in normal amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to the invention can be determined and/or monitored by any method known per se for the disease, disorder or condition in question, as understood by the clinician. The clinician is also able, when appropriate and on a case-by-case basis, to change or modify a particular treatment regimen so as to achieve the desired therapeutic effect, to eliminate, limit or reduce unwanted side effects, and/or to achieve an appropriate balance between achieving a therapeutic effect. effect on the one hand and the exclusion, limitation or reduction of unwanted side effects on the other hand.

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

Thus, in a further aspect, the invention relates to a pharmaceutical composition comprising at least one construct of the invention, at least one polypeptide of the invention, at least one ISVD 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 additional active substances. In a specific aspect, the invention relates to a pharmaceutical composition comprising a construct, polypeptide, ISVD or nucleic acid according to the invention, preferably at least one of SEQ ID NO: OOO, and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally, one or more additional active substances.

The subject to be treated may be any warm-blooded animal, but in particular is a mammal, and more particularly a human. In veterinary applications, the subject to be treated includes any animal that has been commercially bred or kept as a pet. As is known to the person skilled in the art, the subject to be treated may, in particular, be an individual suffering from or at risk of diseases, disorders and conditions mentioned in the present description. Thus, in a preferred embodiment of the invention, pharmaceutical compositions containing a polypeptide of the invention are intended for use in medicine or diagnostics. Preferably, the pharmaceutical compositions are for use in human medicine, but they can also be used for veterinary purposes.

Again, in such a pharmaceutical composition, one or more of the immunoglobulins, polypeptides, compounds and/or constructs of the invention, or the nucleotides encoding them, and/or the pharmaceutical composition containing them, can also be appropriately combined with one or more other active ingredients, such as mentioned in the present description.

The invention also relates to a composition (for example, 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 cell assay) or in vivo (for example, in in a single cell or multicellular organism, and in particular in a mammal, and more specifically in a human, for example, a human at risk for or suffering from the disease, disorder or condition of the invention).

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

In general, for pharmaceutical use, the constructs, polypeptides and/or ISVDs of the invention may be formulated into 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 excipient and/or adjuvant; and optionally, one or more pharmaceutically active polypeptides and/or compounds. As non-limiting examples, such a composition may be in a form suitable for oral administration, for parenteral administration (for example, via intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, via a skin patch, via an implant. , via a suppository, etc., where parenteral administration is preferred. Such suitable administration forms—which may be solid, semi-solid, or liquid, depending on the mode of administration—as well as the methods and carriers for use in their preparation, are known to those skilled in the art, and are further described herein. Such a pharmaceutical preparation or composition is generally referred to herein as a "pharmaceutical composition".

Mention may be made of exemplary excipients, disintegrants, binders, fillers, and lubricants. Examples of disintegrants include agar-agar, algins, calcium carbonate, cellulose, colloidal silicon dioxide, gums, magnesium aluminum silicate, methylcellulose and starch. Examples of binders include microcrystalline cellulose, hydroxymethylcellulose, hydroxypropylcellulose, and polyvinylpyrrolidone. Examples of fillers include calcium carbonate, calcium phosphate, tribasic calcium sulfate, calcium carboxymethylcellulose, cellulose, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrins, maltose, sorbitol, starch, sucrose, sugar, and xylitol. Examples of lubricants include agar, ethyl oleate, ethyl laurate, glycerin, glyceryl palmitostearate, hydrogenated vegetable oil, magnesium oxide, stearates, mannitol, poloxamer, glycols, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc. Usual stabilizers, preservatives, wetting agents and emulsifiers, texture improvers, taste enhancers, osmotic salts, buffers, solubilizers, diluents, emollients, colorants and masking agents, and antioxidants should be considered as pharmaceutical auxiliaries. .

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

In general, the constructs, polypeptides and/or ISVDs of the invention may be formulated and administered by any suitable method known per se. Reference is made, for example, to the general prior art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079) as well as to standard 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 Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see e.g. pages 252-255).

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

The constructs, polypeptides, and/or ISVDs of the invention can be formulated and administered by any method known per se for conventional antibodies and antibody fragments (including ScFvs and diabodies) and other pharmaceutically active proteins. Such formulations and methods for their preparation are known to the person skilled in the art and, for example, include formulations preferred as being suitable for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial, intrathecal, intranasal, or intrabronchial administration), as well as for topical (ie transdermal or intradermal) administration.

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

The constructs, polypeptides, and/or ISVDs of the invention may also be administered using delivery methods known in gene therapy, see, for example, US Patent No. 5399346, delivery methods for gene therapy of which are given as a reference. Using a gene therapy delivery method, primary cells transfected with a gene encoding a construct, polypeptide and/or ISVD of the invention can furthermore be transfected using tissue-specific promoters to target specific organs, tissues, transplants, tumors, or cells, and can in addition, transfect using signal and stabilizing sequences for subcellularly localized expression.

In accordance with additional aspects of the invention, the polypeptide according to the invention can be used for additional applications in vivo and in vitro. For example, the polypeptides of the invention can be used for diagnostic purposes, for example, in assays designed to detect and/or quantify the presence of ADAMTS5 and/or to purify ADAMTS5. The polypeptides can also be tested in animal models of specific diseases and for toxicology, safety and dosing studies.

Finally, the invention relates to a kit containing at least one polypeptide of the invention, at least one nucleic acid sequence encoding said components, a vector or vector system of the invention, and/or a host cell of the invention. It is envisaged that the kit can be offered in various forms, for example in the form of a diagnostic kit.

The invention is now further described by means of the following non-limiting preferred aspects, examples and figures.

The entire contents of all references (including references, issued patents, published patent applications, and concurrent patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the explanations cited herein above.

The sequences are described in the main body of the specification and in a separate sequence listing, in accordance with WIPO ST.25. The SEQ ID designated by a particular number must be the same in the main body of the description and in the separate sequence listing. For example, SEQ ID no.: 1 must specify the same sequence in both the main body of the description and the separate sequence listing. In the event of a mismatch between the definition of a sequence in the main body of the description and in the separate sequence listing (if, for example, SEQ ID no.: 1 in the main body of the description erroneously matches SEQ ID no.: 2 in the separate sequence listing), then the reference to the specific sequence in application, in particular, specific embodiments, should be understood as a reference to the sequence in the main content of the application, and not in a separate sequence listing. In other words, the inconsistency between the sequence definition/designation in the main body of the description and in the separate sequence listing should be resolved by correcting the separate list of sequences in relation to the sequences and their designation described in the main body of the application, which includes the description, examples, figures and claims.

EXAMPLES

Example 1 Production of Recombinant ADAMTS5 Protein from Various Species

Various formats of recombinant ADAMTS5 protein from bovine, rat, guinea pig, mouse and cynomolgus monkey were generated in-house using the HEK293 Flp-In™ expression system using the FuGENE® HD transfection reagent (Promega). Two days after transfection, a selection medium containing 100 μg/ml hygromycin B was added to the cells to select stably expressing cells.

Stably expressing cells were expanded. Conditioned media containing secreted ADAMTS5 was harvested every day from the cells, purified by HisTrap chromatography, and followed by buffer exchange with 50 mM Hepes buffer, pH 7.5. Protein purity was confirmed by SDS-PAGE.

Example 2: Immunization of llamas with ADAMTS5 protein, cloning of heavy chain-only antibody fragment repertoires and phage production

2.1 Immunizations

After approval by the ethics committee of the Department of Veterinary Medicine (University Ghent, Belgium), 3 llamas were immunized with recombinant human ADAMTS5 protein (R&D Systems, Minneapolis, US; cat. # 2198-AD).

2.2 Cloning heavy chain-only antibody fragment repertoires and generating phage

After the last immunogen injection, blood samples were collected. From these blood samples, peripheral blood mononuclear cells (PBMC) were obtained using ficoll-hypac, according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, US). Total RNA was isolated from PBMC and used as starting material for RT-PCR to amplify VHH/nano body encoding DNA fragments, essentially as described in WO 05/044858. Phage were then obtained according to standard methods (see, for example, prior art literature and applications filed by Ablynx N.V. cited herein) and stored after sterilization by filtration at 4°C for later use.

Example 3 Selection of VHHs Specific for Human ADAMTS5 by Phage Display

VHH repertoires derived from all llamas and cloned into phage libraries were used to select human ADAMTS5-binding nanobodies. Recombinant human ADAMTS5 protein (R&D Systems, Minneapolis, US; Cat # 2198-AD) was immobilized at 5 µg/ml on a Maxisorp plate (Nunc, Wiesbaden, Germany), next to a negative control with 0 µg/ml antigen. After incubation with phage libraries and extensive washing, bound phages were eluted with trypsin (1 mg/ml) and used to infect E. coli cells. Infected E. coli cells were either used to obtain phage for the next round of selection or plated onto agar plates for analysis. In addition, synthetic libraries were used in three consecutive selections.

The yields of all selection cycles were analyzed by the enrichment factor, which is the number of phages present in the eluate compared to the control. The best selection conditions are selected for further analysis.

To screen the selection yield for specific binding members, individual colonies were selected from agar plates and grown in 1 ml 96-well deep well plates. LacZ controlled VHH expression was induced by adding IPTG. Periplasmic extracts were obtained in accordance with standard methods (see, for example, the literature of the prior art and applications filed by Ablynx N.V., cited in the present description.)

Example 4 Screening of Periplasmic Extracts for Functional Blocking Nanobodies

In the first step, the periplasmic extracts were tested for binding to recombinant human ADAMTS5 by a binding ELISA. Briefly, recombinant human ADAMTS5 (R&D Systems, Minneapolis, US; Cat # 2198-AD) at 1 μg/ml was coated in 384-well MaxiSorp plates (Nunc, Wiesbaden, Germany). The wells were blocked with casein solution (1%). After adding a 10-fold dilution of the periplasmic extracts, nanobody binding was detected using a mouse anti-Flag HRP conjugate (Sigma, St. Louis, US) and a subsequent enzyme reaction in the presence of the esTMB substrate (3,3',5,5'-tetramethylbenzidine ) (SDT, Brussels, Belgium). Clones showing ELISA signals greater than the sum of the mean signals for the control irrelevant nanobodies and 3-fold standard deviation for the control irrelevant nanobodies were considered to encode positive binding human ADAMTS5 nanobodies.

To identify nanobodies capable of preventing ADAMTS5-mediated aggrecan cleavage, clones were tested in a FRET-based human ADAMTS5 enzyme assay using 50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM CaCl ₂ *2H ₂ O, 0.1 % CHAPS, 5% glycerol as assay buffer. Briefly, periplasmic extracts containing anti-ADAMTS5 binding nanobodies were incubated in 384-well OptiPlates (PerkinElmer, Waltham, MA, US) with 10 µl of 25 µM quencher-bound fluorogenic human peptide substrate (Abz-TEGEARGSVI-Dap(Dnp)-KK -NH2, Anaspec, Serain Belgium, cat # 60431-1) and 10 µl 25 nM human ADAMTS5 (R&D Systems, Minneapolis, US; cat # 2198-AD). The ability of the nanobodies to prevent ADAMTS5-mediated cleavage was monitored every minute for 2 hours on a Tecan Infinite M1000 plate reader. Data was analyzed using Graphpad Prism software. From the enzyme reaction progression curve, the initial conversion rate for the negative control (v ₀ ) was determined, as was the conversion rate for each nanobody clone in the plate (v _i ) and the background signal (S _b ). Using the formula 100*(1 - (v _i - S _b ) /(v ₀ - S _b )), the percentage of inhibition was calculated.

A periplasmic extract containing an irrelevant nanobody was used as a negative control. Periplasmic extracts containing anti-ADAMTS5 nanobodies that were able to reduce the fluorescence signal by more than 50% compared to the negative control signal were considered inhibitory nanobodies. The DNA sequence of the positive clones was then determined.

A summary of screening data for periplasmic extracts is shown in Table 4.1. The amino acid sequences of anti-ADAMTS5 nanobodies are shown in Table A-1.

Table 4.1 Screening results for periplasmic extracts containing nanobodies against ADAMTS5

sequence ID binding ELISA FRET analysis (OD 450 nm) vi (unit/min) % ing. 2A02 1.646 2 91 2A12 1.745 -one 104 2C10 1.627 2 90 2D07 1.551 -one 104 2D12 1.354 2 90 2F03 1.842 -one 103 2G01 1.710 ten 55 3B02 2,570 -one 105 3B03 2.295 2 93 3D01 2.573 -2 111 3D02 2.581 -2 111 7B11 0.368 2 90 9A05 ND -3 113 9D03 ND -2 110 9D09 ND 0 102 9D10 ND -2 111 11B06 ND -one 106 13E02 ND 0 99 3F04 2.555 19 eight

After cloning and sequencing, different families were identified, consisting of clones with differences in CDRs but similar binding and inhibition characteristics.

Following cloning and sequencing, the 02A12 nanobody was identified as a member of the 09D03 clone family (cf. Tables A-1 and A-2). The variability in the sequence of CDR regions is shown in Tables 4.1A, 4.1B and 4.1C below. The amino acid sequences of the CDRs of clone 09D03 were used as a reference against which the CDRs of family members were compared (CDR1 starts at Kabat 26, CDR2 starts at Kabat 50, and CDR3 starts at Kabat 95).

Table 4.1A (CDR1 09D03)

09D03 CDR1* numbering according to Kabat 26 27 28 29 thirty 31 32 33 34 35 absolute numbering one 2 3 four 5 6 7 eight 9 ten mutations in the wild-type sequence G S A V S V N A M A R T F S Y G

* Up to 6 CDR1 mutations in one clone (SEQ ID NO: 22).

Table 4.1B (CDR21 09D03)

09D03 CDR2* numbering according to Kabat fifty 51 52 53 54 55 56 57 58 59 absolute numbering one 2 3 four 5 6 7 eight 9 ten mutations in the wild-type sequence G I S R S A G R T Y

* Up to 0 CDR2 mutations in one clone (SEQ ID NO: 36).

Table 4.1C (CDR3 09D03)

09D03 CDR3* numbering according to Kabat 95 96 97 98 99 100 100a absolute numbering one 2 3 four 5 6 7 eight 9 ten eleven 12 13 fourteen fifteen mutations in the wild-type sequence D L D P N R I F S R D E A A Y . . . .

* Up to 0 CDR mutations in one clone (SEQ ID NO: 54).

After cloning and sequencing, clone 09A05 was identified as a member of the clone 03B02 family (cf. tables A-1 and A-2). The variability in the sequence of CDR regions is shown in Tables 4.1D, 4.1E and 4.1F below. The amino acid sequences of CDR clone 03B02 were used as a reference against which the CDRs of family members were compared (CDR1 starts at Kabat 26, CDR1 starts at Kabat 50, and CDR3 starts at Kabat 95).

Table 4.1D (CDR1 03B02)

03B02 CDR1* numbering according to Kabat 26 27 28 29 thirty 31 32 33 34 35 absolute numbering one 2 3 four 5 6 7 eight 9 ten mutations in the wild-type sequence R R T I S S G T M G

* Up to 0 CDR1 mutations in one clone (SEQ ID NO: 33).

Table 4.1E (CDR2 03B02)

03B02 CDR2* numbering according to Kabat fifty 51 52 53 54 55 56 57 58 59 absolute numbering one 2 3 four 5 6 7 eight 9 ten mutations in the wild-type sequence A I R W S S G M P Y I T F

* Up to 3 CDR2 mutations in one clone (SEQ ID NO: 50).

Table 4.1F (CDR3 03B02)

03B02 CDR3* numbering according to Kabat 95 96 97 98 99 100 100a absolute numbering one 2 3 four 5 6 7 eight 9 ten eleven 12 13 fourteen fifteen 16 wild-type sequence D R S A F R D P S F D V N Y E Y mutations . . . L E

* Up to 2 CDR mutations in one clone (SEQ ID NO: 68).

After cloning and sequencing, clones 03D02 and 02C10 were identified as members of the 03D01 clone family (cf. Tables A-1 and A-2). The variability in the sequence of CDR regions is shown in Tables 4.1G, 4.1H and 4.1I below. The amino acid sequences of CDR clone 03D01 were used as a reference against which the CDRs of family members were compared (CDR1 starts at Kabat 26, CDR2 starts at Kabat 50, and CDR3 starts at Kabat 95).

Table 4.1G (CDR1 03D01)

03D01 CDR1* numbering according to Kabat 26 27 28 29 thirty 31 32 33 34 35 absolute numbering one 2 3 four 5 6 7 eight 9 ten mutations in the wild-type sequence G F T F S P Y Y M G

* Up to 0 CDR1 mutations in one clone (SEQ ID NO: 28).

Table 4.1H (CDR2 03D01)

03D01 CDR2* numbering according to Kabat fifty 51 52 53 54 55 56 57 58 59 absolute numbering one 2 3 four 5 6 7 eight 9 ten mutations in the wild-type sequence A I S R S R G T T Y T W I L

* Up to 3 CDR2 mutations in one clone (SEQ ID NO: 44).

Table 4.1I (CDR3 03D01)

03D01 CDR3* numbering according to Kabat 95 96 97 98 99 100 100a absolute numbering one 2 3 four 5 6 7 eight 9 ten eleven 12 13 fourteen fifteen mutations in the wild-type sequence G R S P G D P S R T Y L Y D Y S . . . E

* Up to 1 CDR mutation in one clone (SEQ ID NO: 62).

The above results show that some amino acid variability is tolerable while maintaining the binding and inhibition characteristics. Notably, ADAMTS5 binding is not predictive of ADAMTS5 activity inhibition. For example, clone 3F04 is a strong binding member but a weak inhibitor.

Example 5 Characterization of Purified Anti-ADAMTS5 Monovalent Nanobodies

Clones selected in the screening described in Example 4 were further characterized. After transformation of E. coli (TG-1) with selected nanobodies, expression was induced by adding 1 mM IPTG and allowed to continue for 4 hours at 37°C. After cell culture centrifugation, periplasmic extracts were obtained by freeze-thaw pellets followed by centrifugation. These extracts were then used as starting material for IMAC purification on HisTrap FF crude 1 ml columns (GE healthcare, Buckinghamshire, United Kingdom) followed by desalting with Zeba spin columns (Pierce, Rockford, IL, USA) to give at least 95 % purity as assessed by SDS-PAGE.

5.1 Evaluation of ADAMTS5 Blocking Nanobodies in ADAMTS5 Enzyme Assays

The ability of nanobodies to prevent ADAMTS5-mediated aggrecan cleavage was confirmed in a FRET-based enzyme assay, essentially as described in Example 4. Data were analyzed using Graphpad Prism software. From the enzyme reaction progression curve, the initial conversion rate (v _i ) was determined and these values were plotted as a function of inhibitor concentrations and IC ₅₀ values were calculated. In essence, human FRET analysis using purified nanobodies confirmed the results described in Example 4 using periplasmic extracts. IC ₅₀ values ranged between 1.8E-09 M and 3.7E-08 M. Notably, although the conventional 12F4 mAb had a slightly better IC ₅₀ of 1.0E-09 M than the monovalent nanobodies, all nanobodies showed better efficacy. (data not shown).

After the FRET-based assay, an AlphaLISA assay (Perkin Elmer, Waltham, MA, US) was performed based on human ADAMTS5 with a biotinylated 43-mer aggrecan oligopeptide as a substrate. Upon ADAMTS5 cleavage of this substrate, a biotinylated ARGSV neoepitope product is released, which can be detected by streptavidin-AlphaScreen donor beads and anti-neoepitope antibody ("ARGSV") bound to anti-mouse IgG coated AlphaLISA acceptor beads, resulting in a luminescence signal. AlphaScreen under laser excitation.

To determine the inhibitory potential of the nanobodies, serial dilutions of the purified nanobodies (together with a positive control and a negative control, an irrelevant nanobody) were incubated with human ADAMTS5 (R&D Systems, Minneapolis, US; Cat # 2198-AD) for 15 minutes at room temperature in 384-well Optiplates (Perkin Elmer, Waltham, MA, US). After addition of a biotinylated 43-mer aggrecan oligopeptide substrate (Biosyntan, Berlin, Germany) and incubation for 3 hours at 37° C., the reaction was stopped. The first stage of detection was performed by adding 5 μl of detection solution 1 containing the anti-aggrecan neoepitope antibody "ARGSV", mouse BC3 mAb (MDBioproducts, Egg, Switzerland) and AlphaScreen donor beads with streptavidin (Perkin Elmer). After 1 hour incubation at room temperature, 5 μl of a second detection solution containing anti-mouse AlphaLISA acceptor beads (Perkin Elmer) was added. The plates were incubated for 2 hours at room temperature in the dark. The measurement was performed by reading plates on an Envision Multi label Plate reader (Perkin Elmer, Waltham, MA, US).

To determine the ability of nanobodies to prevent ADAMTS5-mediated substrate degradation, signal reduction was analyzed as a function of nanobody concentration and IC ₅₀ values were calculated.

The results are summarized in Table 5.1.

Table 5.1 Activity (IC ₅₀ ) and % Inhibition of ADAMTS5 Nanobodies and Reference Compounds in Human AlphaLISA Enzyme Assay.

Connection ID AlphaLISA for humans IC50, [M] % inhibition mAb 12F4 H4L0 6.5E-11 100 2F03 7.5E-11 100 11B06 1.0E-10 100 9D03 1.1E-10 100 2A12 1.5E-10 100 2D07 1.9E-10 100 3D02 2.2E-10 100 3B02 3.0E-10 100 9A05 3.2E-10 100 3D01 3.9E-10 96 2C10 4.5E-10 100 rhTIMP3 6.1E-10 100 2D12 6.9E-10 100 9D10 9.7E-10 100 13E02 2.2E-09 100 9D09 2.6E-09 100 7B11 3.6E-09 100 2A02 4.3E-09 99 3B03 1.2E-08 100 MSC2310852A 1.4E-07 99

In the AlphaLISA assay, at least three monovalent nanobodies (2F03, 11B06 and 9D03) showed IC ₅₀ values similar to the conventional 12F4 mAb, while the rest had higher values. However, about 100% inhibition was shown for all nanobodies. Notably, the 3B03 nanobody having an IC ₅₀ value of at least 100 higher than the 12F04 mAb still has 100% inhibition.

5.2 Evaluation of ADAMTS5 blocking nanobodies in the bovine explant assay

To evaluate the ability of nanobodies to block cartilage degradation in an ex vivo assay in which the substrate is present in a state closer to the physiological state than biochemical assays, a bovine explant assay was performed. Briefly, freshly prepared sections of bovine cartilage explant (4 mm diameter) from bovine knee joints were obtained and incubated in 96-well plates in the presence of IL-1α to induce cartilage degradation. As an indicator of cartilage/aggrecan degradation, GAG release was detected in the supernatant after 5 days of incubation (37°C, 7.5% CO ₂ ) with the metachromatic dye 1,9-dimethylmethylene blue (radiation at 633 nm). Chondroitin sulfate was included as a standard of analysis. Efficacy was determined by IL-1α induced controls without compound (0%) and in the presence of MSC2310852A (100% effect).

The results are summarized in Table 5.2.

Table 5.2 IC ₅₀ values for monovalent nanobodies in bovine explant assay.

ID IC ₅₀ [M] Experiment ID* mAb 12F4 H4L0 3.2E-06 exp. A 2F03 1.4E-08 exp. A 11B06 4.80E-08 exp. C 9D03 5.9E-08/2.7E-07 exp. G/ exp. F 2A12 2.40E-08 exp. C 02D07 3.30E-08 exp. C 3D02 8.80E-07 exp. F 3B02 1.40E-07 exp. D 9A05 5.10E-07 exp. F 3D01 2.2E-08/1.3E-07 exp. D/ exp. G 2D12 8.00E-07 exp. D 2A02 2.40E-06 exp. D *results could only be compared within 1 analysis using the same explant, with identical experiment ID.

5.3 Grouping of epitopes

SPR-based "sandwich analysis" on a Biacore T100 device was performed to group anti-ADAMTS5 nanobodies into different epitope groups. To this end, each anti-ADAMTS5 nanobody was immobilized on a CM5 sensor chip via amino group attachment using EDC and NHS chemistry. After the 100 nM ADAMTS5 binding step, the purified nanobodies were injected at a concentration of 1 μM, each in one cycle and without regeneration, with a surface contact time of 120 seconds and a flow rate of 45 μl/minute. Curves were processed using Biacore T100 Evaluation software and evaluated for additional binding to fixed ADAMTS5 (other group) or no binding (same group).

The nanobodies could be divided into two groups: one large group ("Group 1"), which contains all inhibitory nanobodies having similar or overlapping epitopes, and a second group, which contains a binding, non-functional 3F04 nanobody recognizing a different epitope on ADAMTS5 ("Group 2").

Table 5.3 summarizes the binding epitopes of the nanobodies tested.

Table 5.3 Epitope groups of nanobodies against ADAMTS5

group 1 2A02 2A12 2C10 2D07 2D12 2F03 2G01 3B02 3B03 3D01 3D02 7B11 9A05 9D03 9D09 9D10 11B06 13E02 group 2 3F04

5.4 Cross-species reactivity

Species cross-reactivity was first assessed by SPR-based dissociation rate analysis on a Biacore T100 device. The nanobodies were tested for binding to human ADAMTS5, cynomolgus monkey ("cyno"), guinea pig, mouse and bovine. To this end, recombinant ADAMTS5 was immobilized on the CM5 chip via amino attachment using EDC and NHS. Purified nanobodies were injected over 2 minutes at a concentration of 100 nM and allowed to dissociate for 15 minutes at a flow rate of 45 μl/min. Dissociation rates for each individual nanobody were determined by matching the 1:1 interaction model (Langmuir model) to individual dissociation curves using BIA Evaluation software. As a reference, dissociation rates for human ADAMTS5 were determined in each experiment.

The results are summarized in Table 5.4A.

Table 5.4A Summary of cross-sectional data for monovalent nanobodies reactivities against ADAMTS5

Nanobody ID Dissociation rate based on SPR, kd (1/s) on ADAMTS5 Experiment 1 Experiment 2 Experiment 3 Human Javanese macaque Guinea pig Human Mouse Human Bull 2F03 3.3E-05 2.2E-05 3.4E-05 6.5E-05 2.9E-04 9.7E-05 4.3E-05 11B06 1.7E-04 1.4E-04 1.7E-04 2.7E-04 5.3E-04 3.8E-04 2.4E-04 9D03 9.8E-04 9.2E-04 1.0E-03 1.2E-03 3.7E-03 1.4E-03 9.9E-04 2A12 1.4E-04 1.0E-04 1.2E-04 2.3E-04 4.4E-04 3.1E-04 1.7E-04 2D07 1.1E-03 9.8E-04 1.1E-03 9.4E-04 1.8E-03 1.0E-03 6.2E-04 3B02 7.3E-04 7.4E-04 1.0E-03 8.5E-04 2.0E-03 1.2E-03 1.1E-03 3D01 2.3E-04 2.0E-04 2.7E-04 3.2E-04 6.4E-04 4.6E-04 3.5E-04

For each nanobody tested, similar dissociation rates were observed for all species tested.

In addition, cross-reactivity for ADAMTS5 cynomolgus monkey and guinea pig was also investigated by activity determination using AlphaLISA, essentially as described in Example 5.1, but using biotinylated cynomolgus monkey 43-aggrecan oligopeptide substrate and biotinylated marine 43-aggrecan oligopeptide substrate. pigs. To determine the ability of nanobodies to prevent ADAMTS5-mediated substrate degradation, signal reduction as a function of nanobody concentration was analyzed and IC ₅₀ values were calculated.

The activity obtained is summarized in Table 5.4B.

Table 5.4B Summary of cross-sectional data for reactivity species of monovalent nanobodies against ADAMTS5

All nanobodies and control compounds blocked human and cynomolgus ADAMTS5 with similar activity. In guinea pig AlphaLISA, the most active nanobodies show slightly lower activity than human AlphaLISA, but this is most likely the result of lower assay sensitivity due to increased enzyme concentration.

Rat cross-reactivity of nanobodies 2A12, 2F03, 093 and 049 was assessed by SPR based affinity determination using Biacore T100. As a control, the affinity for human ADAMTS5 was also determined. For this, rat and human ADAMTS5 were immobilized on the CM5 chip via amino group attachment using EDC and NHS chemistries. Purified nanobodies were injected over 2 minutes at various concentrations (between 1 and 1000 nM) and allowed to dissociate for 25 minutes at a flow rate of 45 μl/min. Kinetic constants were calculated from sensograms using BIAEvaluation software (for 1:1 interaction).

As shown in Table 5.4C, similar affinities for human ADAMTS5 and rat ADAMTS5 were obtained for all nanobodies tested. The affinity of the extended half-life nanobody 069 (cf. below) was similar to that of its monovalent equivalent (which is nanobody 049), indicating that the addition of ALB11 at the C-terminus had no effect on affinity. The affinity of the extended half-life biparatope nanobody 130 was higher for both species (15-fold for human ADAMTS5 and 10-fold for rat ADAMTS5), compared to the affinity of extended half-life nanobody 069 (Table 5.4C).

Table 5.4C Affinity Summary for Human ADAMTS5 and Rat ADAMTS5

ID Description KD (nM) for human ADAMTS5 KD (nM) for rat ADAMTS5 2A12 1.9 one 2F03 1.6 0.7 093 _3F04 (N100fQ) 4.9 5.3 049 11B06 (N52S) 3.5 1.7 069 049-35GS-ALB11 2.7 1.3 130 049-35GS-093-35GS-ALB11 0.18 0.13

5.5 Inhibition of MMP-1 and MMP-14 activity

To confirm the selectivity of nanobodies for ADAMTS5, inhibition of MMP-1 and MMP-14 enzyme activity was assessed by FRET-based assays using the respective enzymes. Briefly, activated human MMP-1 or MMP-14 was incubated for 30 minutes at room temperature with 10 μl of nanobody serial dilutions. After incubation, 20 μl of respectively 5 μM or 2.5 μM fluorogenic peptide substrate (Mca-PLGL-Dpa-AR-NH2 fluorogenic substrate for MMP (R&D Systems, cat. #ES001)) was added. The ability of the nanobodies to prevent MMP-1 and MMP-14 mediated cleavage was monitored every minute for 2 hours at 37° C. on a Tecan Infinite M1000 plate reader.

The resulting titration curves are shown in figure 1.

While natural inhibitors of TIMP2 and TIMP3 inhibited the activity of MMP-1 and MMP-14, none of the nanobodies tested showed inhibition.

5.6 Inhibition of human ADAMTS4 activity

To assess inhibition of human ADAMTS4, an assay similar to AlphaLISA for human ADAMTS5 was performed essentially as described in Example 5.1, but using human ADAMTS4 (R&D Systems, Minneapolis, US; Cat # 4307-AD). To determine the ability of nanobodies to prevent human ADAMTS4-mediated substrate degradation, signal reduction as a function of nanobody concentration was analyzed and IC ₅₀ values were calculated.

The results are shown in figure 2.

While the small molecule MSC2310852A inhibited human ADAMTS4 activity, none of the 12F4 nanobodies or mAbs tested showed inhibitory activity (see Figure 2). The monoclonal antibody 12F4 (H4L0) is described as selective over ADAMTS4 in WO 2011/002968.

Example 6 Nanobody Formatting

6.1 Knockout of N-glycosylation motifs

N-glycosylation motifs present in the 11B06 nanobody (N52 position) and 3F04 nanobody (N110f position) were knocked out before formatting. Plots were randomized with the NNK codon library. Because these positions are localized in the CDR, mutation of the motif can potentially affect binding characteristics. Accordingly, libraries were screened for possible substitutions that did not affect the binding property by dissociation rate analysis based on SPR for human ADAMTS5 using the Biacore T100 device, essentially as described in Example 5.4.

Surprisingly, changing amino acid N52 to serine in the 11B06 nanobody and amino acid _N100F to glutamine in the 3F04 nanobody had no effect on binding (data not shown).

Thus, as building blocks for further formatting, nanobodies 049 (=11B06 (N52S)) and 093 (3F04 (N100 _F Q)) were used to represent nanobodies 11B06 and 3F04, respectively (see Table A-1).

6.2 Preparation of formatted nanobodies

Nanobodies 2A12, 2D07, 2F03, 049, 9D03, and 3B02 were fused with anti-human serum albumin (HSA) ALB11 nanobodies to obtain nanobody constructs with extended half-life that block the activity of human ADAMTS5 aggrecanase activity (see Table 6.3). In addition, extended half-life ALB11 constructs were also made from a combination of two nanobodies for different binding epitopes (a combination of a group 1 member and a group 2 member). The formatted nanobody constructs were expressed in Pichia pastoris, secreted into culture medium, and affinity purified on Poros MabCaptureA Protein A beads (Applied Biosystems, Bleiswijk, Netherlands Cat # 4374729). The integrity of the nanobody structures was confirmed.

6.3 Activity in AlphaLISA in the absence and presence of HSA

The formatted nanobody constructs were tested in AlphaLISA as described in Example 5.1. In addition, the effect of HSA binding on the activity of the nanobody was investigated by conducting an experiment in the presence of an excess of HSA (4.2 μM final concentration).

IC ₅₀ values for all extended half-life nanobody designs are listed in Table 6.3.

Table 6.3. Formatted nanobody construct activity (+/- HSA) as defined by AlphaLISA

Nanobody ID Description IC50 (M) Nanobody ID IC50 (M) - HSA + HSA - HSA 004 2A12-35GS-ALB11 1.3E-10 1.1E-10 2A12 1.5E-10 005 2D07-35GS-ALB11 2.9E-10 3.4E-10 2D07 1.9E-10 006 2F03-35GS-ALB11 7.0E-11 9.2E-11 2F03 7.5E-11 069 049-35GS-ALB11 6.9E-11 9.8E-11 049 1.0E-10 070 9D03-35GS-ALB11 4.1E-10 6.0E-10 9D03 1.1E-10 071 3B02-35GS-ALB11 2.8E-10 4.1E-10 3B02 3.0E-10 129 2F03-35GS-093*-35GS-ALB11 1.1E-10 ND 130 049 ² *-35GS-093-35GS-ALB11 9.7E-11 ND 131 9D03-35GS-093-35GS-ALB11 8.1E-11 ND

ND=not determined; 093*=3F04 (N100 _f Q); 049 ² *=11B06 (N52S).

The results show that neither ALB11 formatting nor HSA binding affects the activity of the nanobody constructs.

6.4 HSA binding

The affinity of the ALB11 nanobody for HSA was determined by SPR on a Biacore T100. To this end, HSA was immobilized on the CM5 chip via amino attachment and processed essentially as described in Example 5.3. As a reference, the affinity of monovalent ALB11 was also determined and was 3.2 nM.

The results are summarized in Table 6.4.

Table 6.4 Affinity of HLE nanobody constructs for HSA

ID from FNb* KD (nM) FNb-ALB11 FNb-093 ³ *-ALB11 FNb-ALB11-093 2F03 46 66 52 049 ² * 40 55 40 2A12 27 ND ND

ND=not determined; *FNb=functional nanobody; 049 ² *=11B06 (N52S); 093 ³ *=3F04 (N100 _f Q).

All ADAMTS5 nanobody constructs with extended half-life had a similar affinity (see Table 6.4), which was lower than that of monovalent ALB11 for HSA.

6.5 Affinity determination for human ADAMTS5 using KinExA

The affinity of formatted nanobody construct 069 (049-35GS-ALB11) and nanobody construct 130 (049-35GS-093-ALB11) (see also Table 6.5) was determined in solution using a KinExA3000 kinetic exclusion assay (Sapidyne, Boise, USA) . To this end, human ADAMTS5 was coupled to PMMA beads according to the manufacturer's instructions detailed in Darling and Brault (2004 Assay Drug Dev Technol. 2:647-57). A fixed concentration of the nanobody construct (50 nM nanobody construct 069 or 5 nM nanobody construct 130) was added to serial dilutions of human ADAMTS5 ranging from 2 nM to 0.2 nM and incubated at room temperature for 16 hours until equilibrium was reached. The mixtures were then injected with a KinExA autosampler onto a column packed with human ADAMTS5-conjugated PMMA beads for binding of free nanobody constructs on the beads and detection using labeled Alexa647 HSA. The percent free nanobody construct was plotted as a function of titrated human ADAMTS5 and fitted to an appropriate curve using KinExA Pro v3.2.6 software.

The results are shown in Table 6.5.

Table 6.5 Affinity of 2 nanobody constructs for binding to human ADAMTS5 as determined by KinExA

nanobody Description KD (nM) 069 049*-35GS-ALB11 20.4 130 049-35GS-093*-ALB11 1.2

049*=11B06 (N52S); 093 ² *=3F04 (N100 _f Q).

Approximately 20-fold higher affinity was observed for nanobody construct 130 compared to nanobody construct 069. These data support avid binding to human ADAMTS5 of nanobody construct 130.

Example 7 Sequence Optimization (SO) of Nanobodies

Exemplary nanobody 2F03, nanobody 049 (11B06 (N52S)) and nanobody 093 (3F04 (N100 _f Q)) were subjected to a sequence optimization process. Sequence optimization is a method in which the sequence of the original nanobody is subjected to mutagenesis, and this method includes humanizing (i) the nanobody and knocking out post-translational modifications (ii) as well as epitopes for potential pre-existing antibodies (iii). The ALB11 nanobody was also mutated to knock out epitopes for potential preexisting antibodies.

(i) for humanization purposes, the sequence of the original nanobody is subjected to mutagenesis to obtain a nanobody sequence that is more identical to the human germline IGHV3-IGHJ consensus sequence. Specific amino acids in the framework regions (with the exception of the so-called characteristic residues) that differ between the nanobody and the human IGHV3-IGHJ germline consensus are altered to human equivalents, so as to keep intact the structure, activity and stability of the protein. It is known that some characteristic residues are critical for the stability, activity and affinity of the nanobody, and thus, they are not subjected to mutagenesis.

(ii) 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 as to inactivate the PTM site, while maintaining intact the structure, activity and stability of the protein.

(iii) the sequence of the nanobody is optimized, without affecting the structure, activity and stability of the protein, to minimize the binding of any naturally occurring pre-existing antibodies and reduce the potential to stimulate a treatment-induced immunogenic response.

To obtain sequence-optimized formatted 581 nanobody constructs and 579 nanobody constructs, the respective building blocks were connected via 35GS linkers. This resulted in nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) and nanobody construct 579 (2F3 ^SO -35GS linker-093 ^SO -35GS linker-Alb11), respectively (cf. Table A-1). The constructs were produced in Pichia pastoris as delabeled proteins and purified by protein A affinity chromatography followed by desalting. The integrity of the nanobody structures was confirmed.

7.1 Affinity for human ADAMTS5

The affinity of the 581 nanobody construct (2F3 ^SO -35GS linker-Alb11) was determined using KinExA as described in Example 6.5 with a few minor adaptations to the method described. A fixed concentration of 20 nM nanobody construct 581 was incubated with serial dilutions of human ADAMTS5 (2.2-fold serial dilutions ranging between 20 nM and 0.32 nM and a blank control without ADAMTS5). To test the effect of albumin, HSA was added to this pre-incubation mixture in select experiments with human ADAMTS5, at a concentration 100 times its KD for nanobody construct 581. The mixtures were incubated and allowed to equilibrate for 24 hours prior to injection with a KinExA a column packed with human ADAMTS5-conjugated PMMA beads. The associated 581 nanobody construct was detected using an AF647 labeled anti-nanobody recognizing the 581 nanobody construct (obtained from Ablynx).

The results are presented in table 7.1.

Table 7.1 Affinity of Nanobody Construct 581 for Human ADAMTS5 in the Presence and Absence of HSA

+ or - HSA K _D (nM) 95%CI for K _D (nM) - HSA (n=3) 3.65 (CV 20%) 2.27-5.86 + HSA (n=3) 4.84 (CV 22%) 3.45-6.80

The results show that the affinity of the 581 nanobody construct for human ADAMTS5 is 3.65 nM without HSA and 4.84 nM with HSA.

7.2 Affinity for serum albumin

The affinity of the ALB11 building block in nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) for binding to human, cynomolgus monkey, guinea pig, mouse and rat serum albumin (SA) was determined using SPR as described in Example 5.3.

The results are shown in table 7.2.

Table 7.2 Affinity (K _D , nM) of nanobody construct 581 for binding to human, cynomolgus, guinea pig, mouse, and rat serum albumin

Nanobody ID human SA SA cynomolgus monkey SA guinea pig SA mice SA rats 581 48 51 420 790 > 7500*

* dissociation rate is out of detection limit: > 5.0E-01 (1/s).

7.3 Binding of pre-Ab

Nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) and nanobody construct 579 (2F3 ^SO -35GS linker-093 ^SO -35GS linker-Alb11) were screened for binding of a pre-existing antibody (pre-Ab) from 3 corresponding groups of donor serum samples ( samples from healthy subjects, OA samples, and a biased group of samples with known residual pre-Ab binding to other nanobodies), via SPR technology on the ProteOn XPR36 device. Blood samples were analyzed for binding to anti-ADAMTS5 nanobody constructs that were associated with HSA. The levels of binding of preexisting antibodies were determined after the correction of the sensogram according to two standards, by setting the registration points at 125 seconds (5 seconds after the end of binding). Analysis was performed using ProteOn manager 3.1 (Bio-Rad Laboratories, Inc.).

The results are shown in figure 3.

Both nanobody constructs had low residual levels of pre-Ab binding, with nanobody construct 581 clearly showing the least residual pre-Ab binding (cf. figure 3.).

7.4 Functionality in Human AlphaLISA

AlphaLISA was performed using the nanobody construct 581 (2F3SO- ^35GS linker-Alb11) using human ADAMTS5, essentially as described in Example 5.1.

The results are shown in Table 7.4.

Table 7.4 Activity (nM) of nanobody construct 581 as determined by AlphaLISA.

Sample IC50 (nM) 95% CI (nM) mAb 12F4H4L0 120 102-140 581 188 157-226

The results show that the activity of the 581 nanobody construct is 188 nM.

7.5 Obtaining Immunogenicity Profiles in the Dendritic Cell - T Cell Assay

Relative immunogenicity was determined in a dendritic cell proliferation assay - T cells. As such, the nanobodies were tested on a group of 50 healthy donor cell samples containing the most common HLA class II alleles, thus representing the majority of the global population. The immune response was assessed using T cell proliferation as a surrogate marker for antibody production against the antibody drug. Limpet hemocyanin (KLH) was used as a positive control. A positive KLH control resulted in a positive response in all 50 donors. None of the donors tested positive for the 581 nanobody construct (2F3 ^SO -35GS linker-Alb11). Nanobody construct 579 (2F3 ^SO -35GS linker-093 ^SO -35GS linker-Alb11) resulted in a positive response in three donors, or 6%. Under null conditions (medium only), 2 out of 50 donors, or 4%, have a positive response. These results were used to establish an overall response threshold of more than 3 out of 50 donors corresponding to an overall response threshold of >6%. The overall immunogenic potential of the tested nanobodies was considered low because the percentage of significant, positive responses was ≤6% (see Table 7.5).

Table 7.5 Immunogenicity profiling result as determined by DC-T cell assay

Tested product # positive responses Immunogenic potential Zero 2 (4%) / KLH 50 (100%) / 581 0 (0%) Short 579 3 (6%) Short Control Nb 12%) Short

7.6 Binding to human ADAMTS1

Binding to human ADAMTS1 was tested in a binding ELISA (see Figure 4AA). Briefly, 18.5 nM human ADAMTS1 (R&D Systems, Minneapolis, US; cat. #2197-AD) and human ADAMTS5 (R&D Systems, Minneapolis, US; cat. #2198-AD) were coated on 384-well MaxiSorp plates ( Nunc, Wiesbaden, Germany). The wells were blocked with casein solution (1%) and serial dilutions of nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) and anti-human ADAMTS1 mAb (R&D Systems, Minneapolis, US; Cat # MAB2197) were tested for binding to both coatings. proteins. After detection using an HRP-conjugated anti-nanobody recognizing nanobody construct 581 (produced at Ablynx) and an HRP-conjugated anti-mouse antibody (Abcam, Cambridge, UK), respectively, and subsequent enzymatic reaction in the presence of esTMB substrate (3, 3',5,5'-tetramethylbenzidine) (SDT, Brussels, Belgium), measured OD450 nm.

A dose-response curve was observed for the 581 nanobody construct using human ADAMTS5 and for the anti-human ADAMTS1 mAb using human ADAMTS1, showing that the 581 nanobody construct did not bind to human ADAMTS1.

7.7 Binding to human ADAMTS4 and ADAMTS15

Binding to human ADAMTS4 and ADAMTS15 was tested in a binding ELISA (see Figure 4B). Briefly, using 1 μg/mL human ADAMTS4 (R&D Systems, Minneapolis, US; cat. # 4307-AD) or human ADAMTS15 (R&D Systems, Minneapolis, US; cat. # 5149-AD) was coated overnight at 1 μg /ml 96-well Maxisorp ELISA plates (Nunc, Wiesbaden, Germany). After blocking the plates using SuperBlock T20 (PBS) (Thermo Scientific Pierce; cat. # 37516), trituration for the dose-response curve of nanobody construct 581 ("C011400581") or anti-ADAMTS4 positive control mAb (R&D Systems, Minneapolis, US; cat. # MAB4307), or a positive control anti-ADAMTS15 mAb (R&D Systems, Minneapolis, US; Cat # MAB5149) was applied to the coated plate starting at 1 μM and incubated for 1 hour at room temperature. Bound nanobody C011400581 was detected using a biotinylated anti-nanobody agent recognizing nanobody design 581 (produced at Ablynx), while positive control mAbs were detected using biotinylated goat anti-mouse pAb (Jackson Immuno Research; Cat # 115-065-062 ). For both, streptavidin-HRP was used as a secondary detection agent (Thermo Scientific Pierce; cat. # 21126). The plates were then visualized by adding sTMB) (SDT, Brussels, Belgium). All dilutions and detection tools were prepared in assay diluent (=PBS+10% Superblock+0.05% Tween20). The staining reaction was stopped after 2.5 minutes by adding 1M HCl. The absorbance was measured at 450 nm using 620 nm as the reference wavelength.

7.8 Species cross-reactivity using KinExA affinity determination

The affinity of the 581 nanobody construct (2F3 ^SO -35GS linker-Alb11) for several species of ADAMTS5 was determined using KinExA as described in Example 7.1. For all species, human ADAMTS5-conjugated PMMA beads were prepared and used to bind the free 581 nanobody construct as described above. . The associated 581 nanobody construct was detected using an AF647 labeled anti-nanobody recognizing the 581 nanobody construct (obtained from Ablynx). The results are shown in table 7.7

Table 7.7 Affinity of 581 nanobody construct for ADAMTS5 cynomolgus monkey, rat, mouse, and bovine

Types of ADAMTS5 K _D (nM) 95%CI for K _D (nM) Javanese macaque (n=1) 2.22 0.88-4.76 Rat (n=1) 2.77 1.48-5.07 Mouse (n=1) 2.81 0.30-11.42 Bull (n=1) 7.66 3.33-19.88

Example 8 Activity in Human Explant Assay

Nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) was evaluated for its ability to block cartilage degradation in an ex vivo assay, essentially as described in Example 5.2, but now in a human explant assay. Briefly, freshly prepared human cartilage explant sections (4 mm diameter) from human knee joints were obtained and incubated in 96-well plates in the presence of 10 ng/ml IL-1α to induce cartilage degradation). As an indicator of cartilage/aggrecan degradation, GAG release was detected in the supernatant after 7 days of incubation (37°C, 7.5% CO ₂ ) with the metachromatic dye 1,9-dimethylmethylene blue (radiation at 633 nm). chondroitin sulfate was included as a standard of analysis. Efficacy was determined by induced IL-1β and OSM control without compound and in the presence of MSC2310852A. CRB0017 is an anti-ADAMTS5 mAb under investigation in phase I clinical trials (Rottapharm).

The results are summarized in Figure 5 and Table 8.

Table 8

Compound Analysis IC50 GAG [µM] 581 Human 0.0025-0.0079 581 Bullish 0.035 CRB0017 Bullish 0.33

Nanobodies showed high activity and 100% inhibition of cartilage degradation (GAG loss) in human explant assay. As shown in Table 8, the nanobodies are at least 10 times better than CRB0017 in the bovine explant assay.

Example 9 Modulation of C2M, C3M and exAGNX1 in a bovine co-culture model

The effect of nanobody construct 581 ("581" in the figures) on the release and content of GAG, C2M (collagen II degradation marker), C3M (collagen III degradation marker) and exAGNx1 (aggrecan degradation marker characterized by TEGE neoepitope) in a bovine co-culture system was investigated. In these co-culture systems, cartilage explants from a bovine knee were incubated with synovial membrane from the same animal for 28 days. The highest release of GAG was detected after 7 days in culture (Figure 6A). The 581 nanobody construct was tested at three concentrations: 1 nM, 10 nM, and 100 nM. Incubation of cartilage explants with synovial membrane resulted in increased GAG release. The 581 nanobody design inhibited GAG release (Figure 6B).

Consistent with GAG release, analysis of the remaining GAG content of the cartilage explant (after papain digestion) revealed a decrease in GAG content after co-incubation of the explants with the synovial membrane, compared to the explants alone (FIG. 6C).

In parallel with the GAG release assay, exAGNx1 release was measured as a specific marker of aggrecan degradation. Area under the curve (AUC) analysis showed a strong induction of exAGNx1 release when cartilage explants and synovial membrane were incubated together. In addition, the 581 nanobody construct inhibited the release of exAGNx1 in a concentration-dependent manner with complete inhibition at 100 nM 581 (Figure 7).

To determine the effect of the 581 nanobody construct on the collagen network of cartilage in a co-culture system, C2M was determined as a marker of collagen II degradation. In contrast to markers of aggrecan network degradation (GAG and exAGNx1), C2M levels began to increase between days 14 and 21. Incubation of the 581 nanobody construct in a co-culture system inhibited C2M release (Figure 8).

An additional marker of synovial inflammation, C3M (ie, a marker of type III collagen degradation) was determined. C3M release time-dependence analysis showed that C3M levels began to increase after 14 days, peaking at approximately day 21. Treatment with the 581 nanobody construct inhibited C3M at all three concentrations (Figure 9).

Example 10 Inhibition of Aggrecanase Activity by NHP

The ability of nanobody construct 581 (2F3 ^SO -35GS linker-Alb11; "C011400581") to inhibit aggrecanase activity in vivo was evaluated in a cynomolgus monkey, non-human primate (NHP) model. Briefly, nanobody construct 581 was administered to groups of 3 male and 3 female cynomolgus monkeys once a week via subcutaneous (sc) administration at dosage levels of 6, 30, or 150 mg/kg for 4 weeks. A related control group was treated with vehicle, ie. 20 mM histidine, 8% sucrose, 0.01% Tween 20 pH 6.0.

Inhibition of aggrecanase activity was measured in serum samples collected at several time points by determining the level of aggrecan degradation fragments characterized by the presence of an ARGS neoepitope in serum. All animals treated with the 581 nanobody construct showed a similar profile, i. ARGS levels decreased after the first dosing and reached very low levels near or below the lower measurement range limit (LLMR) of the assay (0.08 nM) between 48 hours and 120 hours. Then, ARGS levels showed a sustained maximum decrease at or below the LLMR and did not return to baseline until the end of the study at 4 weeks.

A summary of the results is shown in figure 10.

In conclusion, ADAMTS5-specific nanobodies target after systemic administration and actively modulate ARGS neoepitope levels in vivo at all dosage levels tested. In addition, unlike prior art mAbs, no test drug-associated abnormal arrhythmias were observed. In addition, there was no evidence of any test-related ST elevation in male and female monkeys treated with nanobodies at any of the dosage levels tested.

Example 11 Inhibition of Cartilage Degeneration

To further evaluate the ability of nanobodies and nanobody constructs to inhibit cartilage degeneration in vivo, a DMM (medial meniscus destabilization) model in mice was used. Briefly, the medial meniscus was subjected to surgical destabilization. An exemplary 581 nanobody construct (2F3 ^SO -35GS linker-Alb11) was either administered subcutaneously 3 days prior to DMM induction (prophylactically) or administered 3 days after DMM induction (therapeutically), at various concentrations. After 8 weeks of treatment, the animals were sacrificed and the knee joints were removed. Knee joints were embedded in paraffin blocks, sectioned and stained using toluidine blue. Sections were then scored for several parameters, including total medial cartilage degeneration.

The results are shown in figure 11.

The results show that there is a structural advantage of up to 50% for both prophylactic and therapeutic treatment (s.c.) using nanobodies in the DMM mouse model.

Example 12 Symptomatic Benefit in a Rat Surgical Model of OA

To assess the ability of nanobodies to provide symptomatic benefit, a rat surgical model of OA was used. Briefly, rats were surgically debridemented with ACLt and tMx to induce OA at day 0. In the surgical model of ACLt and tMx (anterior cruciate ligament dissection expanded using medial meniscectomy), the 581 nanobody construct was injected with s.c. every second day from day 3 onwards. On a weekly basis, symptomatic benefit was determined by gait analysis (on the catwalk) as well as reduction in joint diameter.

The results are shown in figure 12.

Osteoarthritis was induced (on day 0) in adult male rats (Lister Hooded; mean body weight 346±20 g) by anterior cruciate ligament transection (ACLT)+medial meniscus resection (tMx) on the right knee. Treatment with vehicle or test drug (sc) was started on day 3 post-surgery and continued every other day until day 42. The healthy control group did not undergo surgery but was administered sc vehicle at the same time points as the dosing groups. (A) Gait disturbance over time was calculated as "% advantage over wearer". Mean healthy + placebo = 100% advantage. Mean for the ACLT tMx+vehicle group = 0% advantage. (B) Mean "advance over vehicle" at all time points examined after the start of treatment. Mean±SEM for 14-15 rats/group is shown. *= p<0.05 calculated using one-way ANOVA and Dunnett's test.

Early treatment with nanobodies produced a dose dependent, significant and significant symptomatic benefit in ACLT+tMx induced OA.

Example 13 Toxicity studies in cynomolgus monkeys

A 4-week subchronic toxicity study in cynomolgus monkeys was performed to obtain information on systemic toxicity, local tolerability, and pharmacological safety of nanobodies. An exemplary nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) was administered sc in groups of 3 male and 3 female cynomolgus monkeys (Macaca fascicularis) (once a week via dorsal subcutaneous injection at doses of 6, 30, or 150 mg/kg within 4 weeks A companion control group was treated with vehicle, 20 mM histidine, 8% sucrose, 0.01% Tween 20 pH 6.0.

No test drug-related signs of local intolerance were noted at any of the dose levels tested. There were no test drug-related effects on behavior, body weight, food intake, hematological and biochemical parameters, lymphocyte typing, CRP levels, urinalysis parameters, visual and auditory function, and organ weight in any of the animals at any dosage level (data not presented). Neither macroscopic examination at necropsy nor histopathological examination revealed any local or systemic organ changes associated with treatment with the test drug in any of the animals studied at any dosage level tested.

Since mAb 12F4 has been reported to show focal endocardial hemorrhage, a dose-dependent increase in blood pressure without evidence of reversibility, as well as cardiac conduction abnormalities (ST elevation and ventricular arrhythmias) (Larkin, et al., The highs and lows of translational drug development : Antibody-mediated inhibition of ADAMTS-5 for osteoarthritis disease modification, OARSI conference 2014: Paris; Renninger et al., Identification of Altered Cardiovascular Function Produced by a Novel Biologic Compound in a Stand Alone Safety Pharmacology Primate Study, in SPS meeting, 2013 ), extensive non-invasive telemetry using the EMKA system was performed on animals.

There was no test drug related effect on any of the telemetry parameters (using the non-invasive EMKA system) in any of the animals at any dosage level. In particular, no pathological arrhythmias were observed and there was no evidence of ST elevation associated with any test drug in male and female monkeys treated with nanobodies at any dose levels tested (data not shown).

These studies confirm that ADAMTS5-specific nanobodies can be considered safe.

Example 14 In Vivo Studies in the MMT DMOAD Rat Model

To demonstrate the in vivo efficacy of ADAMTS5 inhibitors fused to the CAP binding molecule of the invention, a surgically induced medial meniscus tear (MMT) rat model was used. Briefly, the anti-ADAMTS5 nanobody was coupled to a CAP binding molecule (designated nanobody construct C010100954 or nanobody construct 954). Rats underwent surgery on one knee to induce OA-like symptoms. Treatment was started 3 days after surgery, by IA injection. Histopathology was performed on day 42 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 was the percent reduction in cartilage degeneration. Used 20 animals per group.

A subcartilaginous defect in the medial tibia is shown in Figure 13.

The results show that cartilage width was significantly reduced by the ADAMTS5-CAP construct after 42 days compared to vehicle. These results further suggest that

(a) the CAP group does not adversely affect the activity of the nanobody against ADAMTS5;

(b) the CAP group provides retention of the nanobody against ADAMTS5; and

(c) The anti-ADAMTS5 nanobody has a positive effect on cartilage width, even after the attachment of the CAP group.

Example 15

Methods: Bovine cartilage explants from four animals (BEX), as well as human cartilage explants from eight surgically replaced knee joints (HEX) and from one healthy human knee joint (hHEX), were cultured for up to 21 days in medium (w /o), in the presence of pro-inflammatory cytokines (oncostatin M [10 ng/mL]+TNFα [20 ng/mL] (O+T)) or O+T with an exemplary design of nanobody 581 (2F3 ^SO -35GS linker-Alb11) [ 1 μM - 1 nM]. Cartilage and synovial membrane from bovine (bCC) and from 4 replacement osteoarthritis-affected human knees (hCC) were co-cultured ex vivo for up to 28 days in medium alone (w/o), with O+T or O+T plus illustrative nanobody design 581 (2F3 ^SO -35GS linker-Alb11) [1 μM - 0.6 nM]. Cartilage was cut into equal size explants using a biopsy punch. Synovial membranes were cut into equal size explants (30 mg [±3 mg]) with a scalpel. The metabolic activity of the explants was assessed by Alamar blue. Cartilage tissue renewal was assessed using enzyme-linked immunosorbent assay (ELISA) to measure well-characterized biomarkers of degradation (huARGS, exAGNxI, C2M) and formation (ProC2) in conditioned media. ProC2 and C2M are metabolites of type II collagen formation and degradation, respectively, while exAGNxI and huARGS are metabolites of ADAMTS-5 degraded aggrecan. Means and standard error of the mean (SEM) are published. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Dunn's multiple comparison test or two-way ANOVA with Dunnett's multiple comparison test, assuming a normal distribution.

Results: The metabolic activity of BEX, HEX and bCC was stable throughout the culture period, while the metabolic activity of hCC and hHEX dropped significantly from day 14 under O+T treatment conditions compared to w/o. In cultures stimulated with O+T, ADAMTS-5 degraded aggrecan metabolites peaked within the first week of culture, except for hHEX, in which huARGS and exAGNxI levels increased slightly later. Degradation of type II collagen, C2M, by O+T, peaked after day 19. Formation of type II collagen, Pro-C2, remained relatively stable throughout culture compared to w/o controls.

Treatment with exemplary nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) in combination with O+T dose-dependently reduced huARGS levels at day 5 in BEX (highest dose: 8% of O+T), HEX (highest dose: 40 % of O+T), bCC (highest dose: 10% of O+T), hCC (highest dose: 40% of O+T), and hHEX (highest dose: 24% of O+T) (Figure 14) . The IC50 of an exemplary 581 nanobody construct (2F3 ^SO -35GS linker-Alb11), based on the reduction in huARGS, ranged from 300 nM in BEX to <15 nM in HEX, hHEX, bCC and hCC (Figure 14). The effect of the exemplary 581 nanobody construct (2F3 ^SO -35GS linker-Alb11) on exAGNxI was similar to that of huARGS in cultures tested. The exemplary 581 nanobody construct ( ^2F3SO -35GS linker-Alb11) also reduced C2M (a marker of type II collagen degradation) in a significant and dose-dependent manner, although the effect was smaller than for markers of aggrecan degradation. The exemplary nanobody construct 581 (2F3 ^SO -35GS linker-Alb11) showed no effect on the type II collagen formation metabolite Pro-C2 under any of the conditions tested.

Conclusions: In this study, the present inventors showed that the exemplary nanobody construct 581 (2F3SO- ^35GS linker-Alb11) exerted protective effects on cartilage through its dose-dependent inhibition of ADAMTS-5 mediated aggrecan degradation and MMP mediated type II collagen degradation. under pro-inflammatory conditions in cultures of bovine and human cartilage ex vivo and in co-cultures of cartilage and synovial membrane. 2F3 ^SO -35GS linker-Alb (SEQ ID NO: 129) is one of the preferred embodiments of the invention.

Example 16 Nanobody Designs of the Present Invention Are Superior to Prior Art Nanobody Designs

WO 2008/074840 describes various nanobodies against ADAMTS5. In the present experiment, a comparison was made between the 26 nanobodies described in WO2008/074840 and the nanobodies of the present invention represented by the exemplary 2F3 ^so (2F3*) nanobody.

All constructs were tested in the AlphaLISA assay as described in Example 5.1. The 2F3 ^so nanobody (2F3*) is an ADAMTS5-binding monovalent building block from C011400581 (SEQ ID NO: 129). An unrelated nanobody IRR00028 was used as a negative control.

First, the activity of prior art nanobodies was determined in AlphaLISA. The results are shown in Table 16.1.

Table 16.1 Activity of prior art nanobodies in AlphaLISA

no Name Length CDR3 length Results in AlphaLISA one 36A01 149 17 blocking 2 36A06 147 fifteen Gain 3 36C06 150 17 Gain four 36D06 146 fourteen blocking 5 36E01 147 fifteen Gain 6 36F01 149 17 blocking 7 37B01 146 fifteen Gain eight 37B06 145 13 blocking 9 37B12 151 16 Gain ten 37C06 150 eighteen Gain eleven 37C12 150 eighteen Gain 12 37D06 146 fifteen Gain 13 37E06 151 16 Lack of activity fourteen 37F01 146 fifteen Gain fifteen 37F12 150 eighteen Gain 16 37G01 150 eighteen Blocking 17 37G06 146 fifteen Gain eighteen 40A07 151 16 Lack of activity 19 40B08 146 fifteen Lack of activity twenty 40D07 146 fourteen Lack of activity 21 40E08 147 fifteen Lack of activity 22 40F07 146 fourteen Blocking 23 40F08 151 19 Lack of activity 24 40G07 146 fourteen blocking 25 40G08 151 16 Lack of activity 26 40H07 146 fourteen Lack of activity

The results show that only 7 prior art nanobodies have blocking activity. The rest of the prior art nanobodies had no activity or enhanced activity.

In the second phase of this comparison experiment, the blocking nanobodies were compared individually with an exemplary monovalent nanobody 2F3 ^so (2F3*).

IC ₅₀ values for nanobody designs are listed in Table 16.2. The fold difference with the exemplary monovalent nanobody 2F3 ^so (2F3*) is also indicated for ease of comparison.

Table 16.2 Activity of prior art nanobody constructs versus 2F3 ^so (2F3*) nanobody as determined by AlphaLISA

ID exp. ID Nb IC50 [M] The fold difference compared to 2F3 ^so (2F3*) one 2F3 ^so (2F3*) 2.0E-10 [1.2E-10; 3.1E-10] - 36D06 1.4E-09 [8.5E-10; 3.6E-09] 7x 36F01 7.0E-09 [2.3E-09] 35x 36B06 2.2E-09 [1.4E-09; 3.7E-09] 11x 2 2F3 ^so (2F3*) 2.7E-10 [2.2E-10; 3.4E-10] - 37G01 7.3E-09 [5.2E-09; 1.2E-08] 27x 3 2F3 ^so (2F3*) 1.9E-10 [1.5E-10; 2.3E-10] - 40F07 1.9E-09 [1.4E-09; 2.8E-09] 10x 40G07 1.7E-09 [1.2E-09; 2.4E-09] 9x four 2F3 ^so (2F3*) 4.4E-10 [3.6E-10; 5.3E-10] C011400581 3.5E-10 [2.8E-10; 4.4E-10] 36A01 Partial inhibition

It can be concluded that the nanobody constructs of the present invention are superior to prior art nanobodies against ADAMTS5.

Table A-1. Name and short description ("ID"), SEQ ID NO: ("SEQ") and amino acid sequences of monovalent and multivalent nanobodies against ADAMTS5

ID SEQ Subsequence 2A02 5 EVQLVESGGGLVQPGGSLRLSCAASRRTFSSYVMAWFRQAPGKEREFVAAISRSGDSTYYYDSLEGRFTISRDNAKNTVHLQMNSLKPEDTAVYICAASRAPSFRTIDAINYYDYWGQGTLVTVSS 2A12 one EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERDFVAGISRSAGRTYYVDSVKGRFTISRDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSS 2C10 9 EVQLVESGGGLVQAGGSLRLSCATSGGFTFSPYYMGWFRQAPGKERDFVAAITRSSRGTTYYLDSTEGRFTISRDNAKNTMYLQMNSLNPEDTAVYYCAAGRSPGDPSRTYLYEYWGQGTLVTVSS 2D07 6 EVQLVESGGGLVQAGGSLRLSCSFSGPGRTFARYAMGWFRQAPGKNRDFITGISGSGDSTYYVYPMKDRFTISRDNAKNMVYLQMNALKPEDTAVYYCAADREINRIANDKELDFWGQGTLVTVSS 2D12 four EVQLVESGGGLVQAGDSLRLSCAASGRTFSTYFVGWFRQAPGKERDFVAAISRNGARTYYYDSVAGLFTISRDNAKNTVYLQMSSLKPEDTAVYYCAAARISPSDPSNEDGYDYWGQGTLVTVSS 2F03 2 EVQLVESGGGLVQAGGSLRLSCAASGRTVSSYAMGWFRLAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDSAKNTVYLHMNRLKPEDTAVYYCAADLDPNRIFSREEYAYWGQGTLVTVSS 2G01 7 EVQLVESGGGLVQAGGSLRLSCAASGRTTFSSYAMGWFRQAPGKERAFVATIWSGGLTVYADSAKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEAVGTYYTPDGWTYWGQGTLVTVSS 3B02 16 EVQLVESGGGFVQAGGSLRLSCVASRRTISSGTMGWFRQAPGKEREFVAAIRWSSGMPYYLDSVMDRFTISRDNAKNTVSLQMNSLQPEDTAVYYCAADRSAFRDPSFDVNYEYWGRGTLVTVSS 3B03 13 EVQLVESGGDLVQPGGSLRLSCAASGSDVVVNDMGWYRQAPGKQRELVADITTGGRTNYADSVKGRFTISRDNVKNTVYLQMNSLKPEDTAVYYCNAQVGDSDDDVWYAYWGQGTLVTVSS 3D01 ten EVQLVESGGGLVQAGGSLRLSCAPSGFTFSPYYMGWFRQAPGKERDFVAAAISRSRGTTYYLDSTEGRFTISRDNANDTVYLQMNSLNPEDTAVYYCAAGRSPGDPSRTYLYDYWGQGTLVTVSS 3D02 eleven EVQLVESGGGLVQAGASLRLSCATSGFTFSPYYMGWFRQAPGKERDFVAAISWSRGILYYTDSTEGRFTISRDNAKNTMYLQMDNLNPEDTAVYYCAASRSPGDPSRTYLYDYWGQGTLVTVSS 7B11 fourteen EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAIISGGRTNYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNAEVDAGIYAYGYWGQGTLVTVSS 9A05 17 EVQLVESGGGFVQAGGSLRLSCAASRRTISSGTMGWFRQAPGKEREFVAAIRWSSGITFYPDSVEGRFTISGDNAKNTVSLQMNSLKPEDTAVYYCAADRSALRDPSFEVNYEYWGRGTLVTVSS 9D03 3 EVQLVESGGGLVQSGGSLRLSCAASGSAVSVNAMAWYRQAPGKQRDFVAGISRSAGRTYYTDSVKDRFTIARDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSS 9D09 fifteen EVQLVESGGGLVQAGGSLRLSCAASGLTFSSYTMGWFRQAPGQEREFVSAISWNTFTTYYVDSVKDRFTVSRDNAKNTLYLRMNSLKPEDTAVYYCAAAGGSPRQHEPYEYEYRVWGQGTLVTVSS 9D10 12 EVQLVESGGGLVQAGGSLRLSCAASGRALSSSIMGWFRQAPGKEREFVAAITWSGGRAYYADVSDFEKGRFTISRDNGKNTVNLQMKGLKPEDTAVYYCAAALAIPVTMSPHEYPYWGQGTLVTVSS 11B06 eight EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGINWSGGTTYYVDSVKGRFTISRDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSS 13E02 eighteen EVQLVESGGGLVQAGGSLRLSCVASGSIFSIDAMGWYRQAPGKERELVASVTTGASPNYGDSVTGRFTASRDRAKNALYLQMNSLKPEDTAVYYCNLIMTIPGGSQIMYWGQGTLVTVSS 3F04 19 EVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDNYSDWGQGTLVTVSS construction 049 11B06:N52 117 EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGISWSGGTTYYVDSVKGRFTISRDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSS design 093 3F04:N100fQ 116 EVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSS design 004 2A12-Alb 120 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERDFVAGISRSAGRTYYVDSVKGRFTISRDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS design 005 2D7-Alb 121 EVQLVESGGGLVQAGGSLRLSCSFSGPGRTFARYAMGWFRQAPGKNRDFITGISGSGDSTYYVYPMKDRFTISRDNAKNMVYLQMNALKPEDTAVYYCAADREINRIANDKELDFWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS design 006 2F3-Alb 122 EVQLVESGGGLVQAGGSLRLSCAASGRTVSSYAMGWFRLAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDSAKNTVYLHMNRLKPEDTAVYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construction 069 049-Alb 123 EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGISWSGGTTYYVDSVKGRFTISRDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS design 070 9D3-Alb 124 EVQLVESGGGLVQSGGSLRLSCAASGSAVSVNAMAWYRQAPGKQRDFVAGISRSAGRTYYTDSVKDRFTIARDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS design 071 3B2-Alb 125 EVQLVESGGGFVQAGGSLRLSCVASRRTISSGTMGWFRQAPGKEREFVAAIRWSSGMPYYLDSVMDRFTISRDNAKNTVSLQMNSLQPEDTAVYYCAADRSAFRDPSFDVNYEYWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS design 129 2F3-093-Alb 126 EVQLVESGGGLVQAGGSLRLSCAASGRTVSSYAMGWFRLAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDSAKNTVYLHMNRLKPEDTAVYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construction 130 049-093-Alb 127 EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGISWSGGTTYYVDSVKGRFTISRDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS design 131 9D3-093-Alb 128 EVQLVESGGGLVQSGGSLRLSCAASGSAVSVNAMAWYRQAPGKQRDFVAGISRSAGRTYYTDSVKDRFTIARDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construction 577=581 2F3*-Alb 129 DVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA design 579 2F3*-093*-Alb 130 DVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVKGRFTISRDYSKNTLYLQMNSLRPEDTALYYCAGDPFGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA

* SO (Sequence Optimized) variant.

Table A-2. CDR and framework sequences, plus preferred combinations as shown in formula I, namely FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (following terms: "ID" refers to this SEQ ID NO)

Table B. Other amino acid sequences: Name and short description ("ID"), SEQ ID NO: ("SEQ") and amino acid sequences ("sequences")

view SEQ ID Amino acid sequence man, ADAM-TS5, Q9UNA0-1 149 MLLGWASLLLCAFRLPLAAVGPAATPAQDKAGQPPTAAAAAQPRRRQGEEVQERAEPPGHPHPLAQRRRSKGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDGSVGIAGFVPAGGGTSAPWRHRSHCFYRGTVDGSPRSLAVFDLCGGLDGFFAVKHARYTLKPLLRGPWAEEEKGRVYGDGSARILHVYTREGFSFEALPPRASCETPASTPEAHEHAPAHSNPSGRAALASQLLDQSALSPAGGSGPQTWWRRRRRSISRARQVELLLVADASMARLYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEETFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPRNNGRYCTGKRAIYRSCSLMPCPPNGKSFRHEQCEAKNGYQSDAKGVKTFVEWVPKYAGVLPADVCKLTCRAKGTGYYVVFSPKVTDGTECRLYSNSVCVRGKCVRTGCDGIIGSKLQYDKCGVCGGDNSSCTKIVGTFNKKSKGYTDVVRIPEGATHIKVRQFKAKDQTRFTAYLALKKKNGEYLINGKYMISTSETIIDINGTVMNYSGWSHRDDFLHGMGYSATKEILIVQILATDPTKPLDVRYSFFVPKKSTPKVNSVTSHGSNKVGSHTSQPQWVTGPWLACSRTCDTGWHTRTVQCQDGNRKLAKGCPLSQRPSAFKQCLLKKC bull (AK remnants 1-626) 150 MLLGWAALMLCALRLPPVAAGPTAAPAQDKAGQPRAAAVAAAAQPRGRRGEEAQEPAEPPGHPHPLAPQRGSRGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDDSVGAAGLVPAGGGPNATRRHRGHCFYRGTVDGSPRSLAVFDLCGGLDGFFAVKRARYTLQPLLRGPWAEAEGDARVYGDESARILHVYTREGFSFEALPPRTSCETHASPPGARERPPAPSRPDGRWALAPQQLPGQSAPSSDGSQGPRTWWRRRRRSISRARQVELLLVADASMARMYGRGLQHYLLTLASIANKLYSHASIENHIRLVVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQIPGPEELPGQTYDASQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPRNNGRYCTGKRAIYRSCSVTPCPHHHHHHHHHH rat, (AK residues 1-619) 151 MRLEWASLLLLLLLLCASCLALAADNPAAAPAQDKTRQPRAAAAAAQPDQRQWEETQERGHPQPLARQRRSSGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDDTVGAAGGIVTAGGLSASSGHRGHCFYRGTVDGSPRSLAVFDLCGGLDGFFAVKHARYTLKPLLRGSWAESERVYGDGSSRILHVYTREGFSFEALPPRTSCETPASPSGAQESPSVHSSSRRRTELAPQLLDHSAFSPAGNAGPQTWWRRRRRSISRARQVELLLVADSSMAKMYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLTDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDVPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGPWGQCSRSCGGGVQFAYRHCNNPAPRNSGRYCTGKRAIYRSCSVIPCPHHHHHHHHHH guinea pig, (AK remnants 1-622) 152 MLLGWASLLLCAFRLPQAAASAAAAPAQDKAGQPRAAAAAPQPRRRQGEHAPLRVEPPGHPHALAPQRRGRGLLQSIDRLYSGGGKVGYLVYAGGRRFLLDLERDGSVGAAGLFPAGGGLSAPRRHRSHCFYRGTVDGSPRSLAVFDLCGGLRGFFAVKHARYTVKPLLRGPWAEADTPRVYGDESARIPHVYTREGFSFEALPPRASCETPASQPGPHERPPAHNSPGRHSTVDPQLPELSALSPAGDPGQQIWWRRRRRSISRARQVELLLVADGSMAKMYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGLTEDKRLMSSILTSIDASKPWSKCTSATMTEFLDDGHGNCLLDVPRKQIPSPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGPWGQCSRSCGGGVQFAYRHCNNPAPRNSGRYCTGKRAIYRSCSVTPCPHHHHHHHHHH mouse (AK remnants 1-622) 153 MRLEWAPLLLLLLLLSASCLSLAADSPAAAPAQDKTRQPQAAAAAAEPDQPQGEETRERGHLQPLAGQRRSGGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDDTVGAAGSIVTAGGGLSASSGHRGHCFYRGTVDGSPRSLAVFDLCGGLDGFFAVKHARYTLKPLLRGSWAEYERIYGDGSSRILHVYNREGFSFEALPPRASCETPASPSGPQESPSVHSRSRRRSALAPQLLDHSAFSPSGNAGPQTWWRRRRRSISRARQVELLLVADSSMARMYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLTDKDTSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGTTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRVCLQGKCVDKTKKKYYSTSSHGNWGSWGPWGQCSRSCGGGVQFAYRHCNNPAPRNSGRYCTGKRAIYRSCSVTPCPHHHHHHHHHH cynomolgus macaque (AK remains 1-622) 154 MLLGWASLLLCAFRLPLAAAGPAAAPAQDKAGQPATAAAAAQPRRRQGEEVQERTEPPGHPHPLAQRRSSKGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDGSVGTAGFVPTEGGTSAPWRHRSHCFYRGTVDGSPRSLAVFDLCGGLDGFFAVKHARYTLKPLLRGPWAEEETRRVYGDGSARILHVYTREGFSFEALQPRASCETPASTPEPHERPPAHSNPGGRAALASQLLDQSAVSPAGGPGPQTWWRRRRRSISRARQVELLLVADASMARLYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEETFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDQPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPRNNGRYCTGKRAIYRSCGLMPCPHHHHHHHHHH human aggrecan 155 MTTLLWVFVTLRVITAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEVQSLRSNDSGVYRCEVMHGIEDSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGHVYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYVHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAPEIGATAFAEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGPTRYSLTFEEAQQACPGTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSTETYDVYCFVDRLEGEVFFATRLEQFTFQEALEFCESHNATATTGQLYAAWSRGLDKCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGISAVPSPGEEEGGTPTSPSGVEEWIVTQVVPGVAAVPVEEETTAVPSGETTAILEFTTEPENQTEWEPAYTPVGTSPLPGILPTWPPTGAETEESTEGPSATEVPSASEEPSPSEVPFPSEEPSPSEEPFPSVRPFPSVELFPSEEPFPSKEPSPSEEPSASEEPYTPSPPEPSWTELPSSGEESGAPDVSGDFTGSGDVSGHLDFSGQLSGDRASGLPSGDLDSSGLTSTVGSGLTVESGLPSGDEERIEWPSTPTVGELPSGAEILEGSASGVGDLSGLPSGEVLETSASGVGDLSGLPSGEVLETTAPGVEDISGLPSGEVL ETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETTAPGVEEISGLPSGEVLETTAPGVDEISGLPSGEVLETTAPGVEEISGLPSGEVLETSTSAVGDLSGLPSGGEVLEISVSGVEDISGLPSGEVVETSASGIEDVSELPSGEGLETSASGVEDLSRLPSGEEVLEISASGFGDLSGVPSGGEGLETSASEVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDLVGSASGDLDLGKLPSGTLGSGQAPETSGLPSGFSGEYSGVDLGSGPPSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTIGISGAGEISGLPSSELDISGRASGLPSGTELSGQASGSPDVSGEIPGLFGVSGQPSGFPDTSGETSGVTELSGLSSGQPGVSGEASGVLYGTSQPFGITDLSGETSGVPDLSGQPSGLPGFSGATSGVPDLVSGTTSGSGESSGITFVDTSLVEVAPTTFKEEEGLGSVELSGLPSGEADLSGKSGMVDVSGQFSGTVDSSGFTSQTPEFSGLPSGIAEVSGESSRAEIGSSLPSGAYYGSGTPSSFPTVSLVDRTLVESVTQAPTAQEAGEGPSGILELSGAHSGAPDMSGEHSGFLDLSGLQSGLIEPSGEPPGTPYFSGDFASTTNVSGESSVAMGTSGEASGLPEVTLITSEFVEGVTEPTISQELGQRPPVTHTPQLFESSGKVSTAGDISGATPVLPGSGVEVSSVPESSSETSAYPEAGFGASA APEASREDSGSPDLSETTSAFHEANLERSSGLGVSGSTLTFQEGEASAAPEVSGESTTTSDVGTEAPGLPSATPTASGDRTEISGDLSGHTSQLGVVISTSIPESEWTQQTQRPAETHLEIESSSLLYSGEETHTVETATSPTDASIPASPEWKRESESTAAAPARSCAEEPCGAGTCKETEGHVICLCPPGYTGEHCNIDQEVCEEGWNKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGEPPVVEHARTFGQKKDRYEINSLVRYQCTEGFVQRHMPTIRCQPSGHWEEPRITCTDATTYKRRLQKRSSRHPRRSRPSTAH 00745 PEA114F08 156 EVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTVYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 00747 PEA604F02 157 EVQLVESGGGVVQPGGSLRLSCAASGRTFSSYTMGWFRQAPGKEREFVAAISWSGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAYRRRRASSNRGLWDYWGQGTLVTVSSA

Table C. Various linker sequences ("ID" refers to SEQ ID NO, within the scope of the invention)

Name ID Amino acid sequence A3 158 AAA Linker 5GS 159 GGGGS Linker 7GS 160 sggsggs Linker 8GS 161 Gggggggs Linker 9GS 162 GGGGSGGGS Linker 10GS 163 GGGGSGGGGS Linker 15GS 164 GGGGSGGGGSGGGGS Linker 18GS 165 GGGGSGGGGSGGGGGGGS Linker 20GS 166 GGGGSGGGGSGGGGSGGGGS Linker 25GS 167 GGGGSGGGGSGGGGSGGGGSGGGGS Linker 30GS 168 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Linker 35GS 169 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Linker 40GS 170 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Hinge G1 171 EPKSCDKTHTCPPCP Hinge 9GS-G1 172 GGGGSGGGSEPKSCDKTHTCPPCP Lama's upper long hinged area 173 epktpkpqpaaa Hinge G3 174 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

Table D. ISVD Serum Albumin Binding Sequences ("ID" refers to SEQ ID NO, within the scope of the invention), including CDR sequences

Name ID Amino acid sequence Alb8 131 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 132 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 133 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 134 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 135 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11(S112K)-A 136 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 137 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 138 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 139 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 140 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 141 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSSQGTLVTVSSG Alb82-GG 142 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 143 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG Alb92 144 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 145 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA ALB CDR1 146 SFGMS ALB CDR2 147 SISGSGSDTLYADSVKG ALB CDR3 148 GGSLSSR

--->

SEQUENCE LIST

<110> Merck Patent GmbH

Ablynx N.V.

<120> ADAMTS-binding immunoglobulins

<130> MEPAT.0103

<150>EP17174403.0

<151> 2017-06-02

<160> 174

<170> BiSSAP 1.3.6

<210> 1

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid 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 Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120

<210> 2

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 2

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 Thr Val Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120

<210> 3

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120

<210> 4

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 5

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 5

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 Arg Arg Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His

65 70 75 80

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

85 90 95

Ala Ala Ser Arg Ala Pro Ser Phe Arg Thr Ile Asp Ala Ile Asn Tyr

100 105 110

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

115 120 125

<210> 6

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 6

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 Ser Phe Ser Gly Pro Gly Arg Thr Phe Ala

20 25 30

Arg Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asn Arg Asp

35 40 45

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

50 55 60

Pro Met Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met

65 70 75 80

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

85 90 95

Tyr Cys Ala Ala Asp Arg Glu Ile Asn Arg Ile Ala Asn Asp Lys Glu

100 105 110

Leu Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 7

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 7

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 Thr Thr Phe Ser Ser

20 25 30

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

35 40 45

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

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 Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Glu Ala Val Gly Thr Tyr Tyr Thr Pro Asp Gly Trp Thr Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 8

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 Leu Thr Phe Arg Arg Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Asp Ile Gly Thr Leu Val Asn Asp Glu Asn Pro Arg

100 105 110

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

115 120

<210> 9

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 9

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 Thr Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120

<210> 10

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 10

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 Pro Ser Gly Phe Thr Phe Ser Pro Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120

<210> 11

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120

<210> 12

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 12

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 Ala Leu Ser Ser Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Tyr Cys Ala Ala Ala Leu Ala Ile Pro Val Thr Met Ser Pro His

100 105 110

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

115 120 125

<210> 13

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 13

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asp Val Val Val Asn

20 25 30

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

35 40 45

Ala Asp 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 Val 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

Ala Gln Val Gly Asp Ser Asp Asp Asp Val Trp Tyr Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 14

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 Ile Asn

20 25 30

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

35 40 45

Ala Ala Ile Ile Ser 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 Asp Ala Gly Ile Tyr Ala Tyr Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 15

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 15

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 Leu Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Ala Gly Gly Ser Pro Arg Gln His Glu Pro Tyr Glu Tyr Arg

100 105 110

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

115 120

<210> 16

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 16

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

1 5 10 15

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

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 Arg Trp Ser Ser Gly Met Pro Tyr Tyr Leu Asp Ser Val

50 55 60

Met Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser

65 70 75 80

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

85 90 95

Ala Ala Asp Arg Ser Ala Phe Arg Asp Pro Ser Phe Asp Val Asn Tyr

100 105 110

Glu Tyr Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 17

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 17

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

1 5 10 15

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

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Glu Tyr Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 18

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 18

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 Val Ala Ser Gly Ser Ile Phe Ser Ile Asp

20 25 30

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

35 40 45

Ala Ser Val Thr Thr Gly Ala Ser Pro Asn Tyr Gly Asp Ser Val Thr

50 55 60

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

Leu Ile Met Thr Ile Pro Gly Gly Ser Gln Ile Met Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 19

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Tyr Pro Met Ala Trp

20 25 30

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

35 40 45

Trp Gly Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val Gln Gly Arg Phe

50 55 60

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

65 70 75 80

Ser Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys Ala Gly Asp Pro

85 90 95

Trp Gly Arg Leu Phe Arg Val Lys Asp Asn Tyr Ser Asp Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 20

Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly

1 5 10

<210> 21

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 21

Gly Arg Thr Val Ser Ser Tyr Ala Met Gly

1 5 10

<210> 22

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 22

Gly Ser Ala Val Ser Val Asn Ala Met Ala

1 5 10

<210> 23

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 23

Gly Arg Thr Phe Ser Thr Tyr Phe Val Gly

1 5 10

<210> 24

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 24

Arg Arg Thr Phe Ser Ser Tyr Val Met Ala

1 5 10

<210> 25

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 25

Gly Pro Gly Arg Thr Phe Ala Arg Tyr Ala Met Gly

1 5 10

<210> 26

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 26

Gly Arg Thr Thr Phe Ser Ser Tyr Ala Met Gly

1 5 10

<210> 27

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 27

Gly Leu Thr Phe Arg Arg Asn Ala Met Gly

1 5 10

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 28

Gly Phe Thr Phe Ser Pro Tyr Tyr Met Gly

1 5 10

<210> 29

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 29

Gly Arg Ala Leu Ser Ser Ser Ile Met Gly

1 5 10

<210> 30

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 30

Gly Ser Asp Val Val Val Asn Asp Met Gly

1 5 10

<210> 31

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 31

Gly Ser Ile Phe Ser Ile Asn Val Met Gly

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 32

Gly Leu Thr Phe Ser Ser Tyr Thr Met Gly

1 5 10

<210> 33

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 33

Arg Arg Thr Ile Ser Ser Gly Thr Met Gly

1 5 10

<210> 34

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 34

Gly Ser Ile Phe Ser Ile Asp Ala Met Gly

1 5 10

<210> 35

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 35

Gly Arg Tyr Pro Met Ala

fifteen

<210> 36

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 36

Gly Ile Ser Arg Ser Ala Gly Arg Thr Tyr

1 5 10

<210> 37

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 37

Gly Ile Ser Arg Ser Ala Glu Arg Thr Tyr

1 5 10

<210> 38

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 38

Ala Ile Ser Arg Asn Gly Ala Arg Thr Tyr Tyr

1 5 10

<210> 39

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 39

Ala Ile Ser Arg Ser Gly Asp Ser Thr Tyr

1 5 10

<210> 40

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 40

Gly Ile Ser Gly Ser Gly Asp Ser Thr Tyr

1 5 10

<210> 41

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 41

Thr Ile Trp Ser Gly Gly Leu Thr Val

fifteen

<210> 42

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 42

Gly Ile Asn Trp Ser Gly Gly Thr Thr Tyr

1 5 10

<210> 43

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 43

Ala Ile Thr Arg Ser Arg Gly Thr Thr Tyr

1 5 10

<210> 44

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 44

Ala Ile Ser Arg Ser Arg Gly Thr Thr Tyr

1 5 10

<210> 45

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 45

Ala Ile Ser Trp Ser Arg Gly Ile Leu Tyr

1 5 10

<210> 46

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 46

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

1 5 10

<210> 47

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 47

Asp Ile Thr Thr Gly Gly Arg Thr Asn

fifteen

<210> 48

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 48

Ala Ile Ile Ser Gly Gly Arg Thr Asn

fifteen

<210> 49

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 49

Ala Ile Ser Trp Asn Thr Phe Thr Thr Tyr

1 5 10

<210> 50

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 50

Ala Ile Arg Trp Ser Ser Gly Met Pro Tyr

1 5 10

<210> 51

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 51

Ala Ile Arg Trp Ser Ser Gly Ile Thr Phe

1 5 10

<210> 52

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 52

Ser Val Thr Thr Gly Ala Ser Pro Asn

fifteen

<210> 53

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 53

Gly Val Ser Trp Gly Gly Asp Arg Thr Tyr

1 5 10

<210> 54

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 54

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

1 5 10 15

<210> 55

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 55

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

1 5 10 15

<210> 56

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 56

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

1 5 10 15

<210> 57

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 57

Ser Arg Ala Pro Ser Phe Arg Thr Ile Asp Ala Ile Asn Tyr Tyr Asp

1 5 10 15

Tyr

<210> 58

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 58

Asp Arg Glu Ile Asn Arg Ile Ala Asn Asp Lys Glu Leu Asp Phe

1 5 10 15

<210> 59

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 59

Glu Ala Val Gly Thr Tyr Tyr Thr Pro Asp Gly Trp Thr Tyr

1 5 10

<210> 60

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 60

Asp Gly Asp Ile Gly Thr Leu Val Asn Asp Glu Asn Pro Arg Tyr

1 5 10 15

<210> 61

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 61

Gly Arg Ser Pro Gly Asp Pro Ser Arg Thr Tyr Leu Tyr Glu Tyr

1 5 10 15

<210> 62

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 62

Gly Arg Ser Pro Gly Asp Pro Ser Arg Thr Tyr Leu Tyr Asp Tyr

1 5 10 15

<210> 63

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 63

Ser Arg Ser Pro Gly Asp Pro Ser Arg Thr Tyr Leu Tyr Asp Tyr

1 5 10 15

<210> 64

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 64

Ala Leu Ala Ile Pro Val Thr Met Ser Pro His Glu Tyr Pro Tyr

1 5 10 15

<210> 65

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 65

Gln Val Gly Asp Ser Asp Asp Asp Val Trp Tyr Ala Tyr

1 5 10

<210> 66

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 66

Glu Val Asp Ala Gly Ile Tyr Ala Tyr Gly Tyr

1 5 10

<210> 67

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 67

Ala Gly Gly Ser Pro Arg Gln His Glu Pro Tyr Glu Tyr Arg Val

1 5 10 15

<210> 68

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 68

Asp Arg Ser Ala Phe Arg Asp Pro Ser Phe Asp Val Asn Tyr Glu Tyr

1 5 10 15

<210> 69

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 69

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

1 5 10 15

<210> 70

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 70

Ile Met Thr Ile Pro Gly Gly Ser Gln Ile Met Tyr

1 5 10

<210> 71

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 71

Asp Pro Trp Gly Arg Leu Phe Arg Val Lys Asp Asn Tyr Ser Asp

1 5 10 15

<210> 72

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 72

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> 73

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 73

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 74

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 74

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 75

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 75

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> 76

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 76

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 Ser Phe Ser

20 25

<210> 77

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 77

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 Thr Ser

20 25

<210> 78

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 78

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 Pro Ser

20 25

<210> 79

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 79

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser

20 25

<210> 80

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 80

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro 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> Amino acid sequence

<400> 81

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser

20 25

<210> 82

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 82

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Val Gln Ala 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> Amino acid sequence

<400> 83

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 Val Ala Ser

20 25

<210> 84

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 84

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala 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> Amino acid sequence

<400> 85

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

1 5 10

<210> 86

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 86

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

1 5 10

<210> 87

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 87

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

1 5 10

<210> 88

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 88

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

1 5 10

<210> 89

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 89

Trp Phe Arg Gln Ala Pro Gly Lys Asn Arg Asp Phe Ile Thr

1 5 10

<210> 90

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 90

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

1 5 10

<210> 91

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 91

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

1 5 10

<210> 92

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 92

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

1 5 10

<210> 93

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 93

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

1 5 10

<210> 94

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 94

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

1 5 10

<210> 95

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 95

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 96

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 96

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

1 5 10 15

Lys Asn Thr Val Tyr Leu His Met Asn Arg Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 97

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 97

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 98

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 98

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

1 5 10 15

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

20 25 30

Val Tyr Tyr Cys Ala Ala

35

<210> 99

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 99

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

1 5 10 15

Lys Asn Thr Val His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Ile Cys Ala Ala

35

<210> 100

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 100

Tyr Val Tyr Pro Met Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 101

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 101

Tyr Ala Asp Ser Ala 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 Arg Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 102

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 102

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

1 5 10 15

Lys Asn Thr Val Asp Leu Gln Met Ile Ser Pro Lys 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> Amino acid sequence

<400> 103

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 104

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 104

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 105

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 105

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

1 5 10 15

Lys Asn Thr Met Tyr Leu Gln Met Asp Asn Leu Asn Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 106

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 106

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 107

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 107

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

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 Ala

35

<210> 108

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 108

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> 109

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 109

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 110

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 110

Tyr Leu Asp Ser Val Met Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 111

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 111

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 112

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 112

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Asn Leu

35

<210> 113

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 113

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

1 5 10 15

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

20 25 30

Ala Val Tyr Tyr Cys Ala Gly

35

<210> 114

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 114

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 115

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 115

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 116

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 116

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Tyr Pro Met Ala Trp

20 25 30

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

35 40 45

Trp Gly Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val Gln Gly Arg Phe

50 55 60

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

65 70 75 80

Ser Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys Ala Gly Asp Pro

85 90 95

Trp Gly Arg Leu Phe Arg Val Lys Asp Gln Tyr Ser Asp Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 117

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 117

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 Leu Thr Phe Arg Arg Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Asp Ile Gly Thr Leu Val Asn Asp Glu Asn Pro Arg

100 105 110

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

115 120

<210> 118

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 118

Asp Pro Trp Gly Arg Leu Phe Arg Val Lys Asp Gln Tyr Ser Asp

1 5 10 15

<210> 119

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 119

Gly Ile Ser Trp Ser Gly Gly Thr Thr Tyr

1 5 10

<210> 120

<211> 274

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 120

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 Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Ser

<210> 121

<211> 276

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 121

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 Ser Phe Ser Gly Pro Gly Arg Thr Phe Ala

20 25 30

Arg Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asn Arg Asp

35 40 45

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

50 55 60

Pro Met Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met

65 70 75 80

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

85 90 95

Tyr Cys Ala Ala Asp Arg Glu Ile Asn Arg Ile Ala Asn Asp Lys Glu

100 105 110

Leu Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Thr Val Ser Ser

275

<210> 122

<211> 274

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 122

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 Thr Val Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Ser

<210> 123

<211> 274

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 123

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 Leu Thr Phe Arg Arg Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Asp Ile Gly Thr Leu Val Asn Asp Glu Asn Pro Arg

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Ser

<210> 124

<211> 274

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 124

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Ser

<210> 125

<211> 275

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 125

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

1 5 10 15

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

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 Arg Trp Ser Ser Gly Met Pro Tyr Tyr Leu Asp Ser Val

50 55 60

Met Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser

65 70 75 80

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

85 90 95

Ala Ala Asp Arg Ser Ala Phe Arg Asp Pro Ser Phe Asp Val Asn Tyr

100 105 110

Glu Tyr Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Val Ser Ser

275

<210> 126

<211> 429

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 126

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 Thr Val Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Tyr Pro Met Ala Trp Phe

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys Ala Gly Asp Pro Trp

245 250 255

Gly Arg Leu Phe Arg Val Lys Asp Gln Tyr Ser Asp Trp Gly Gln Gly

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425

<210> 127

<211> 429

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 127

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 Leu Thr Phe Arg Arg Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Asp Ile Gly Thr Leu Val Asn Asp Glu Asn Pro Arg

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Tyr Pro Met Ala Trp Phe

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys Ala Gly Asp Pro Trp

245 250 255

Gly Arg Leu Phe Arg Val Lys Asp Gln Tyr Ser Asp Trp Gly Gln Gly

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425

<210> 128

<211> 429

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 128

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Tyr Pro Met Ala Trp Phe

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys Ala Gly Asp Pro Trp

245 250 255

Gly Arg Leu Phe Arg Val Lys Asp Gln Tyr Ser Asp Trp Gly Gln Gly

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425

<210> 129

<211> 275

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 129

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Ser Ala

275

<210> 130

<211> 430

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 130

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Tyr Pro Met Ala Trp Phe

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gly Arg Leu Phe Arg Val Lys Asp Gln Tyr Ser Asp Trp Gly Gln Gly

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

<210> 131

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 131

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> 132

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 132

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> 133

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 133

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> 134

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 134

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> 135

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 135

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> 136

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid 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 Lys

100 105 110

Val Ser Ser Ala

115

<210> 137

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 137

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> 138

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid 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 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> 139

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 139

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> 140

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 140

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> 141

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 141

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> 142

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid 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 Gly Gly

115

<210> 143

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid 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 Gly Gly Gly

115

<210> 144

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 144

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> 145

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 145

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> 146

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 146

Ser Phe Gly Met Ser

fifteen

<210> 147

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 147

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

1 5 10 15

gly

<210> 148

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 148

Gly Gly Ser Leu Ser Arg

fifteen

<210> 149

<211> 930

<212> PRT

<213> Homo sapiens

<220>

<223> Amino acid sequence

<400> 149

Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys Ala Phe Arg Leu Pro

1 5 10 15

Leu Ala Ala Val Gly Pro Ala Ala Thr Pro Ala Gln Asp Lys Ala Gly

20 25 30

Gln Pro Pro Thr Ala Ala Ala Ala Ala Gln Pro Arg Arg Arg Gln Gly

35 40 45

Glu Glu Val Gln Glu Arg Ala Glu Pro Pro Gly His Pro His Pro Leu

50 55 60

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

65 70 75 80

Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly Arg

85 90 95

Arg Phe Leu Leu Asp Leu Glu Arg Asp Gly Ser Val Gly Ile Ala Gly

100 105 110

Phe Val Pro Ala Gly Gly Gly Thr Ser Ala Pro Trp Arg His Arg Ser

115 120 125

His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser Leu Ala

130 135 140

Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val Lys His

145 150 155 160

Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Pro Trp Ala Glu Glu

165 170 175

Glu Lys Gly Arg Val Tyr Gly Asp Gly Ser Ala Arg Ile Leu His Val

180 185 190

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

195 200 205

Cys Glu Thr Pro Ala Ser Thr Pro Glu Ala His Glu His Ala Pro Ala

210 215 220

His Ser Asn Pro Ser Gly Arg Ala Ala Leu Ala Ser Gln Leu Leu Asp

225 230 235 240

Gln Ser Ala Leu Ser Pro Ala Gly Gly Ser Gly Pro Gln Thr Trp Trp

245 250 255

Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu

260 265 270

Leu Val Ala Asp Ala Ser Met Ala Arg Leu Tyr Gly Arg Gly Leu Gln

275 280 285

His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His

290 295 300

Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val

305 310 315 320

Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr

325 330 335

Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly

340 345 350

Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu

355 360 365

Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val

370 375 380

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

385 390 395 400

Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu

405 410 415

Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu Thr Phe Gly Ser

420 425 430

Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala

435 440 445

Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Ile Thr Glu Phe Leu

450 455 460

Asp Asp Gly His Gly Asn Cys Leu Leu Asp Leu Pro Arg Lys Gln Ile

465 470 475 480

Leu Gly Pro Glu Glu Leu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln

485 490 495

Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp

500 505 510

Val Cys Ala Arg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val

515 520 525

Cys Leu Thr Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys

530 535 540

Gly Arg Ile Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys

545 550 555 560

Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Ser Trp

565 570 575

Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg

580 585 590

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

595 600 605

Lys Arg Ala Ile Tyr Arg Ser Cys Ser Leu Met Pro Cys Pro Pro Asn

610 615 620

Gly Lys Ser Phe Arg His Glu Gln Cys Glu Ala Lys Asn Gly Tyr Gln

625 630 635 640

Ser Asp Ala Lys Gly Val Lys Thr Phe Val Glu Trp Val Pro Lys Tyr

645 650 655

Ala Gly Val Leu Pro Ala Asp Val Cys Lys Leu Thr Cys Arg Ala Lys

660 665 670

Gly Thr Gly Tyr Tyr Val Val Phe Ser Pro Lys Val Thr Asp Gly Thr

675 680 685

Glu Cys Arg Leu Tyr Ser Asn Ser Val Cys Val Arg Gly Lys Cys Val

690 695 700

Arg Thr Gly Cys Asp Gly Ile Ile Gly Ser Lys Leu Gln Tyr Asp Lys

705 710 715 720

Cys Gly Val Cys Gly Gly Asp Asn Ser Ser Cys Thr Lys Ile Val Gly

725 730 735

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

740 745 750

Glu Gly Ala Thr His Ile Lys Val Arg Gln Phe Lys Ala Lys Asp Gln

755 760 765

Thr Arg Phe Thr Ala Tyr Leu Ala Leu Lys Lys Lys Asn Gly Glu Tyr

770 775 780

Leu Ile Asn Gly Lys Tyr Met Ile Ser Thr Ser Glu Thr Ile Ile Asp

785 790 795 800

Ile Asn Gly Thr Val Met Asn Tyr Ser Gly Trp Ser His Arg Asp Asp

805 810 815

Phe Leu His Gly Met Gly Tyr Ser Ala Thr Lys Glu Ile Leu Ile Val

820 825 830

Gln Ile Leu Ala Thr Asp Pro Thr Lys Pro Leu Asp Val Arg Tyr Ser

835 840 845

Phe Phe Val Pro Lys Lys Ser Thr Pro Lys Val Asn Ser Val Thr Ser

850 855 860

His Gly Ser Asn Lys Val Gly Ser His Thr Ser Gln Pro Gln Trp Val

865 870 875 880

Thr Gly Pro Trp Leu Ala Cys Ser Arg Thr Cys Asp Thr Gly Trp His

885 890 895

Thr Arg Thr Val Gln Cys Gln Asp Gly Asn Arg Lys Leu Ala Lys Gly

900 905 910

Cys Pro Leu Ser Gln Arg Pro Ser Ala Phe Lys Gln Cys Leu Leu Lys

915 920 925

Lys Cys

930

<210> 150

<211> 636

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 150

Met Leu Leu Gly Trp Ala Ala Leu Met Leu Cys Ala Leu Arg Leu Pro

1 5 10 15

Pro Val Ala Ala Gly Pro Thr Ala Ala Pro Ala Gln Asp Lys Ala Gly

20 25 30

Gln Pro Arg Ala Ala Ala Val Ala Ala Ala Ala Gln Pro Arg Gly Arg

35 40 45

Arg Gly Glu Glu Ala Gln Glu Pro Ala Glu Pro Pro Gly His Pro His

50 55 60

Pro Leu Ala Pro Gln Arg Gly Ser Arg Gly Leu Val Gln Asn Ile Asp

65 70 75 80

Gln Leu Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly

85 90 95

Gly Arg Arg Phe Leu Leu Asp Leu Glu Arg Asp Asp Ser Val Gly Ala

100 105 110

Ala Gly Leu Val Pro Ala Gly Gly Gly Pro Asn Ala Thr Arg Arg His

115 120 125

Arg Gly His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser

130 135 140

Leu Ala Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val

145 150 155 160

Lys Arg Ala Arg Tyr Thr Leu Gln Pro Leu Leu Arg Gly Pro Trp Ala

165 170 175

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

180 185 190

Leu His Val Tyr Thr Arg Glu Gly Phe Ser Phe Glu Ala Leu Pro Pro

195 200 205

Arg Thr Ser Cys Glu Thr His Ala Ser Pro Pro Gly Ala Arg Glu Arg

210 215 220

Pro Pro Ala Pro Ser Arg Pro Asp Gly Arg Trp Ala Leu Ala Pro Gln

225 230 235 240

Gln Leu Pro Gly Gln Ser Ala Pro Ser Ser Asp Gly Ser Gln Gly Pro

245 250 255

Arg Thr Trp Trp Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln

260 265 270

Val Glu Leu Leu Leu Val Ala Asp Ala Ser Met Ala Arg Met Tyr Gly

275 280 285

Arg Gly Leu Gln His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Lys

290 295 300

Leu Tyr Ser His Ala Ser Ile Glu Asn His Ile Arg Leu Val Val Val

305 310 315 320

Lys Val Val Val Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys

325 330 335

Asn Ala Ala Thr Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His

340 345 350

Asn Gln Leu Gly Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu

355 360 365

Phe Thr Arg Glu Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly

370 375 380

Met Ala Asp Val Gly Thr Ile Cys Ser Pro Glu Arg Ser Cys Ala Val

385 390 395 400

Ile Glu Asp Asp Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile

405 410 415

Gly His Leu Leu Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu

420 425 430

Asn Phe Gly Ser Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr

435 440 445

Ser Ile Asp Ala Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Ile

450 455 460

Thr Glu Phe Leu Asp Asp Gly His Gly Asn Cys Leu Leu Asp Leu Pro

465 470 475 480

Arg Lys Gln Ile Pro Gly Pro Glu Glu Leu Pro Gly Gln Thr Tyr Asp

485 490 495

Ala Ser Gln Gln Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser Val Cys

500 505 510

Pro Gly Met Asp Val Cys Ala Arg Leu Trp Cys Ala Val Val Arg Gln

515 520 525

Gly Gln Met Val Cys Leu Thr Lys Lys Leu Pro Ala Val Glu Gly Thr

530 535 540

Pro Cys Gly Lys Gly Arg Ile Cys Leu Gln Gly Lys Cys Val Asp Lys

545 550 555 560

Thr Lys Lys Lys Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser

565 570 575

Trp Gly Ser Trp Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln

580 585 590

Phe Ala Tyr Arg His Cys Asn Asn Pro Ala Pro Arg Asn Asn Gly Arg

595 600 605

Tyr Cys Thr Gly Lys Arg Ala Ile Tyr Arg Ser Cys Ser Val Thr Pro

610 615 620

Cys Pro His His His His His His His His His His His

625 630 635

<210> 151

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 151

Met Arg Leu Glu Trp Ala Ser Leu Leu Leu Leu Leu Leu Leu Leu Cys

1 5 10 15

Ala Ser Cys Leu Ala Leu Ala Ala Asp Asn Pro Ala Ala Ala Pro Ala

20 25 30

Gln Asp Lys Thr Arg Gln Pro Arg Ala Ala Ala Ala Ala Ala Gln Pro

35 40 45

Asp Gln Arg Gln Trp Glu Glu Thr Gln Glu Arg Gly His Pro Gln Pro

50 55 60

Leu Ala Arg Gln Arg Arg Ser Ser Gly Leu Val Gln Asn Ile Asp Gln

65 70 75 80

Leu Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly

85 90 95

Arg Arg Phe Leu Leu Asp Leu Glu Arg Asp Asp Thr Val Gly Ala Ala

100 105 110

Gly Gly Ile Val Thr Ala Gly Gly Leu Ser Ala Ser Ser Gly His Arg

115 120 125

Gly His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser Leu

130 135 140

Ala Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val Lys

145 150 155 160

His Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Ser Trp Ala Glu

165 170 175

Ser Glu Arg Val Tyr Gly Asp Gly Ser Ser Arg Ile Leu His Val Tyr

180 185 190

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

195 200 205

Glu Thr Pro Ala Ser Pro Ser Gly Ala Gln Glu Ser Pro Ser Val His

210 215 220

Ser Ser Ser Arg Arg Arg Thr Glu Leu Ala Pro Gln Leu Leu Asp His

225 230 235 240

Ser Ala Phe Ser Pro Ala Gly Asn Ala Gly Pro Gln Thr Trp Trp Arg

245 250 255

Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu Leu

260 265 270

Val Ala Asp Ser Ser Met Ala Lys Met Tyr Gly Arg Gly Leu Gln His

275 280 285

Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His Ala

290 295 300

Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val Leu

305 310 315 320

Thr Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr Thr Leu Lys

325 330 335

Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly Asp Asp His

340 345 350

Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu Asp Leu Cys

355 360 365

Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val Gly Thr Ile

370 375 380

Cys Ser Pro Glu Arg Ser Cys Ala Val Ile Glu Asp Asp Gly Leu His

385 390 395 400

Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu Gly Leu Ser

405 410 415

His Asp Asp Ser Lys Phe Cys Glu Glu Asn Phe Gly Ser Thr Glu Asp

420 425 430

Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala Ser Lys Pro

435 440 445

Trp Ser Lys Cys Thr Ser Ala Thr Ile Thr Glu Phe Leu Asp Asp Gly

450 455 460

His Gly Asn Cys Leu Leu Asp Val Pro Arg Lys Gln Ile Leu Gly Pro

465 470 475 480

Glu Glu Leu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln Cys Asn Leu

485 490 495

Thr Phe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp Val Cys Ala

500 505 510

Arg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val Cys Leu Thr

515 520 525

Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys Gly Arg Ile

530 535 540

Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys Tyr Tyr Ser

545 550 555 560

Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Pro Trp Gly Gln Cys

565 570 575

Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg His Cys Asn

580 585 590

Asn Pro Ala Pro Arg Asn Ser Gly Arg Tyr Cys Thr Gly Lys Arg Ala

595 600 605

Ile Tyr Arg Ser Cys Ser Val Ile Pro Cys Pro His His His His His

610 615 620

His His His His His His

625

<210> 152

<211> 632

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 152

Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys Ala Phe Arg Leu Pro

1 5 10 15

Gln Ala Ala Ala Ser Ala Ala Ala Ala Pro Ala Gln Asp Lys Ala Gly

20 25 30

Gln Pro Arg Ala Ala Ala Ala Ala Pro Gln Pro Arg Arg Arg Gln Gly

35 40 45

Glu His Ala Pro Leu Arg Val Glu Pro Pro Gly His Pro His Ala Leu

50 55 60

Ala Pro Gln Arg Arg Gly Arg Gly Leu Leu Gln Ser Ile Asp Arg Leu

65 70 75 80

Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly Arg

85 90 95

Arg Phe Leu Leu Asp Leu Glu Arg Asp Gly Ser Val Gly Ala Ala Gly

100 105 110

Leu Phe Pro Ala Gly Gly Gly Leu Ser Ala Pro Arg Arg His Arg Ser

115 120 125

His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser Leu Ala

130 135 140

Val Phe Asp Leu Cys Gly Gly Leu Arg Gly Phe Phe Ala Val Lys His

145 150 155 160

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

165 170 175

Asp Thr Pro Arg Val Tyr Gly Asp Glu Ser Ala Arg Ile Pro His Val

180 185 190

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

195 200 205

Cys Glu Thr Pro Ala Ser Gln Pro Gly Pro His Glu Arg Pro Pro Ala

210 215 220

His Asn Ser Pro Gly Arg His Ser Thr Val Asp Pro Gln Leu Pro Glu

225 230 235 240

Leu Ser Ala Leu Ser Pro Ala Gly Asp Pro Gly Gln Gln Ile Trp Trp

245 250 255

Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu

260 265 270

Leu Val Ala Asp Gly Ser Met Ala Lys Met Tyr Gly Arg Gly Leu Gln

275 280 285

His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His

290 295 300

Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val

305 310 315 320

Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr

325 330 335

Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly

340 345 350

Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu

355 360 365

Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val

370 375 380

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

385 390 395 400

Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu

405 410 415

Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu Asn Phe Gly Leu

420 425 430

Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala

435 440 445

Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Met Thr Glu Phe Leu

450 455 460

Asp Asp Gly His Gly Asn Cys Leu Leu Asp Val Pro Arg Lys Gln Ile

465 470 475 480

Pro Ser Pro Glu Glu Leu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln

485 490 495

Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp

500 505 510

Val Cys Ala Arg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val

515 520 525

Cys Leu Thr Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys

530 535 540

Gly Arg Ile Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys

545 550 555 560

Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Pro Trp

565 570 575

Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg

580 585 590

His Cys Asn Asn Pro Ala Pro Arg Asn Ser Gly Arg Tyr Cys Thr Gly

595 600 605

Lys Arg Ala Ile Tyr Arg Ser Cys Ser Val Thr Pro Cys Pro His His

610 615 620

His His His His His His His His His

625 630

<210> 153

<211> 632

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 153

Met Arg Leu Glu Trp Ala Pro Leu Leu Leu Leu Leu Leu Leu Leu Ser

1 5 10 15

Ala Ser Cys Leu Ser Leu Ala Ala Asp Ser Pro Ala Ala Ala Pro Ala

20 25 30

Gln Asp Lys Thr Arg Gln Pro Gln Ala Ala Ala Ala Ala Ala Glu Pro

35 40 45

Asp Gln Pro Gln Gly Glu Glu Thr Arg Glu Arg Gly His Leu Gln Pro

50 55 60

Leu Ala Gly Gln Arg Arg Ser Gly Gly Leu Val Gln Asn Ile Asp Gln

65 70 75 80

Leu Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly

85 90 95

Arg Arg Phe Leu Leu Asp Leu Glu Arg Asp Asp Thr Val Gly Ala Ala

100 105 110

Gly Ser Ile Val Thr Ala Gly Gly Gly Leu Ser Ala Ser Ser Gly His

115 120 125

Arg Gly His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser

130 135 140

Leu Ala Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val

145 150 155 160

Lys His Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Ser Trp Ala

165 170 175

Glu Tyr Glu Arg Ile Tyr Gly Asp Gly Ser Ser Arg Ile Leu His Val

180 185 190

Tyr Asn Arg Glu Gly Phe Ser Phe Glu Ala Leu Pro Pro Arg Ala Ser

195 200 205

Cys Glu Thr Pro Ala Ser Pro Ser Gly Pro Gln Glu Ser Pro Ser Val

210 215 220

His Ser Arg Ser Arg Arg Arg Ser Ala Leu Ala Pro Gln Leu Leu Asp

225 230 235 240

His Ser Ala Phe Ser Pro Ser Gly Asn Ala Gly Pro Gln Thr Trp Trp

245 250 255

Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu

260 265 270

Leu Val Ala Asp Ser Ser Met Ala Arg Met Tyr Gly Arg Gly Leu Gln

275 280 285

His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His

290 295 300

Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val

305 310 315 320

Leu Thr Asp Lys Asp Thr Ser Leu Glu Val Ser Lys Asn Ala Ala Thr

325 330 335

Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly

340 345 350

Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu

355 360 365

Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val

370 375 380

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

385 390 395 400

Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu

405 410 415

Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu Asn Phe Gly Thr

420 425 430

Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala

435 440 445

Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Ile Thr Glu Phe Leu

450 455 460

Asp Asp Gly His Gly Asn Cys Leu Leu Asp Leu Pro Arg Lys Gln Ile

465 470 475 480

Leu Gly Pro Glu Glu Leu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln

485 490 495

Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp

500 505 510

Val Cys Ala Arg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val

515 520 525

Cys Leu Thr Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys

530 535 540

Gly Arg Val Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys

545 550 555 560

Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Pro Trp

565 570 575

Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg

580 585 590

His Cys Asn Asn Pro Ala Pro Arg Asn Ser Gly Arg Tyr Cys Thr Gly

595 600 605

Lys Arg Ala Ile Tyr Arg Ser Cys Ser Val Thr Pro Cys Pro His His

610 615 620

His His His His His His His His His

625 630

<210> 154

<211> 632

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 154

Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys Ala Phe Arg Leu Pro

1 5 10 15

Leu Ala Ala Ala Gly Pro Ala Ala Ala Pro Ala Gln Asp Lys Ala Gly

20 25 30

Gln Pro Ala Thr Ala Ala Ala Ala Ala Gln Pro Arg Arg Arg Gln Gly

35 40 45

Glu Glu Val Gln Glu Arg Thr Glu Pro Pro Gly His Pro His Pro Leu

50 55 60

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

65 70 75 80

Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly Arg

85 90 95

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

100 105 110

Phe Val Pro Thr Glu Gly Gly Thr Ser Ala Pro Trp Arg His Arg Ser

115 120 125

His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser Leu Ala

130 135 140

Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val Lys His

145 150 155 160

Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Pro Trp Ala Glu Glu

165 170 175

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

180 185 190

Tyr Thr Arg Glu Gly Phe Ser Phe Glu Ala Leu Gln Pro Arg Ala Ser

195 200 205

Cys Glu Thr Pro Ala Ser Thr Pro Glu Pro His Glu Arg Pro Pro Ala

210 215 220

His Ser Asn Pro Gly Gly Arg Ala Ala Leu Ala Ser Gln Leu Leu Asp

225 230 235 240

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

245 250 255

Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu

260 265 270

Leu Val Ala Asp Ala Ser Met Ala Arg Leu Tyr Gly Arg Gly Leu Gln

275 280 285

His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His

290 295 300

Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val

305 310 315 320

Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr

325 330 335

Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly

340 345 350

Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu

355 360 365

Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val

370 375 380

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

385 390 395 400

Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu

405 410 415

Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu Thr Phe Gly Ser

420 425 430

Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala

435 440 445

Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Ile Thr Glu Phe Leu

450 455 460

Asp Asp Gly His Gly Asn Cys Leu Leu Asp Gln Pro Arg Lys Gln Ile

465 470 475 480

Leu Gly Pro Glu Glu Leu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln

485 490 495

Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp

500 505 510

Val Cys Ala Arg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val

515 520 525

Cys Leu Thr Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys

530 535 540

Gly Arg Ile Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys

545 550 555 560

Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Ser Trp

565 570 575

Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg

580 585 590

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

595 600 605

Lys Arg Ala Ile Tyr Arg Ser Cys Gly Leu Met Pro Cys Pro His His

610 615 620

His His His His His His His His His

625 630

<210> 155

<211> 2415

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 155

Met Thr Thr Leu Leu Trp Val Phe Val Thr Leu Arg Val Ile Thr Ala

1 5 10 15

Ala Val Thr Val Glu Thr Ser Asp His Asp Asn Ser Leu Ser Val Ser

20 25 30

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

35 40 45

Ile Pro Cys Tyr Phe Ile Asp Pro Met His Pro Val Thr Thr Ala Pro

50 55 60

Ser Thr Ala Pro Leu Ala Pro Arg Ile Lys Trp Ser Arg Val Ser Lys

65 70 75 80

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val

85 90 95

Asn Ser Ala Tyr Gln Asp Lys Val Ser Leu Pro Asn Tyr Pro Ala Ile

100 105 110

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

115 120 125

Gly Val Tyr Arg Cys Glu Val Met His Gly Ile Glu Asp Ser Glu Ala

130 135 140

Thr Leu Glu Val Val Val Lys Gly Ile Val Phe His Tyr Arg Ala Ile

145 150 155 160

Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg Ala Gln Arg Ala Cys Leu

165 170 175

Gln Asn Ser Ala Ile Ile Ala Thr Pro Glu Gln Leu Gln Ala Ala Tyr

180 185 190

Glu Asp Gly Phe His Gln Cys Asp Ala Gly Trp Leu Ala Asp Gln Thr

195 200 205

Val Arg Tyr Pro Ile His Thr Pro Arg Glu Gly Cys Tyr Gly Asp Lys

210 215 220

Asp Glu Phe Pro Gly Val Arg Thr Tyr Gly Ile Arg Asp Thr Asn Glu

225 230 235 240

Thr Tyr Asp Val Tyr Cys Phe Ala Glu Glu Met Glu Gly Glu Val Phe

245 250 255

Tyr Ala Thr Ser Pro Glu Lys Phe Thr Phe Gln Glu Ala Ala Asn Glu

260 265 270

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

275 280 285

Ala Trp Gln Ala Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp

290 295 300

Arg Ser Val Arg Tyr Pro Ile Ser Lys Ala Arg Pro Asn Cys Gly Gly

305 310 315 320

Asn Leu Leu Gly Val Arg Thr Val Tyr Val His Ala Asn Gln Thr Gly

325 330 335

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

340 345 350

Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly Gly Glu Glu

355 360 365

Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu

370 375 380

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

385 390 395 400

Val Lys Pro Ile Phe Glu Val Ser Pro Ser Pro Leu Glu Pro Glu Glu

405 410 415

Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe Ala Glu Val

420 425 430

Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe Pro Thr Pro

435 440 445

Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu Val Val Gln

450 455 460

Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly Val Val Phe

465 470 475 480

His Tyr Arg Pro Gly Pro Thr Arg Tyr Ser Leu Thr Phe Glu Glu Ala

485 490 495

Gln Gln Ala Cys Pro Gly Thr Gly Ala Val Ile Ala Ser Pro Glu Gln

500 505 510

Leu Gln Ala Ala Tyr Glu Ala Gly Tyr Glu Gln Cys Asp Ala Gly Trp

515 520 525

Leu Arg Asp Gln Thr Val Arg Tyr Pro Ile Val Ser Pro Arg Thr Pro

530 535 540

Cys Val Gly Asp Lys Asp Ser Ser Pro Gly Val Arg Thr Tyr Gly Val

545 550 555 560

Arg Pro Ser Thr Glu Thr Tyr Asp Val Tyr Cys Phe Val Asp Arg Leu

565 570 575

Glu Gly Glu Val Phe Phe Ala Thr Arg Leu Glu Gln Phe Thr Phe Gln

580 585 590

Glu Ala Leu Glu Phe Cys Glu Ser His Asn Ala Thr Ala Thr Thr Gly

595 600 605

Gln Leu Tyr Ala Ala Trp Ser Arg Gly Leu Asp Lys Cys Tyr Ala Gly

610 615 620

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

625 630 635 640

Ala Cys Gly Gly Asp Lys Pro Gly Val Arg Thr Val Tyr Leu Tyr Pro

645 650 655

Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg His His Ala Phe Cys

660 665 670

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

675 680 685

Thr Pro Thr Ser Pro Ser Gly Val Glu Glu Trp Ile Val Thr Gln Val

690 695 700

Val Pro Gly Val Ala Ala Val Pro Val Glu Glu Glu Thr Thr Ala Val

705 710 715 720

Pro Ser Gly Glu Thr Thr Ala Ile Leu Glu Phe Thr Thr Glu Pro Glu

725 730 735

Asn Gln Thr Glu Trp Glu Pro Ala Tyr Thr Pro Val Gly Thr Ser Pro

740 745 750

Leu Pro Gly Ile Leu Pro Thr Trp Pro Pro Thr Gly Ala Glu Thr Glu

755 760 765

Glu Ser Thr Glu Gly Pro Ser Ala Thr Glu Val Pro Ser Ala Ser Glu

770 775 780

Glu Pro Ser Pro Ser Glu Val Pro Phe Pro Ser Glu Glu Pro Ser Pro

785 790 795 800

Ser Glu Glu Pro Phe Pro Ser Val Arg Pro Phe Pro Ser Val Glu Leu

805 810 815

Phe Pro Ser Glu Glu Pro Phe Pro Ser Lys Glu Pro Ser Pro Ser Glu

820 825 830

Glu Pro Ser Ala Ser Glu Glu Pro Tyr Thr Pro Ser Pro Pro Glu Pro

835 840 845

Ser Trp Thr Glu Leu Pro Ser Ser Gly Glu Glu Ser Gly Ala Pro Asp

850 855 860

Val Ser Gly Asp Phe Thr Gly Ser Gly Asp Val Ser Gly His Leu Asp

865 870 875 880

Phe Ser Gly Gln Leu Ser Gly Asp Arg Ala Ser Gly Leu Pro Ser Gly

885 890 895

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

900 905 910

Val Glu Ser Gly Leu Pro Ser Gly Asp Glu Glu Arg Ile Glu Trp Pro

915 920 925

Ser Thr Pro Thr Val Gly Glu Leu Pro Ser Gly Ala Glu Ile Leu Glu

930 935 940

Gly Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Glu

945 950 955 960

Val Leu Glu Thr Ser Ala Ser Gly Val Gly Asp Leu Ser Gly Leu Pro

965 970 975

Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser

980 985 990

Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu

995 1000 1005

Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro

1010 1015 1020

Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr

1025 1030 1035 1040

Thr Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val

1045 1050 1055

Leu Glu Thr Thr Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser

1060 1065 1070

Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly

1075 1080 1085

Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp

1090 1095 1100

Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly

1105 1110 1115 1120

Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala

1125 1130 1135

Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu

1140 1145 1150

Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly

1155 1160 1165

Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu

1170 1175 1180

Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile

1185 1190 1195 1200

Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala Pro Gly Val

1205 1210 1215

Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Ala Ala

1220 1225 1230

Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu

1235 1240 1245

Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro Ser Gly Glu

1250 1255 1260

Val Leu Glu Thr Ala Ala Pro Gly Val Glu Asp Ile Ser Gly Leu Pro

1265 1270 1275 1280

Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Glu Glu Ile Ser

1285 1290 1295

Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro Gly Val Asp

1300 1305 1310

Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr Thr Ala Pro

1315 1320 1325

Gly Val Glu Glu Ile Ser Gly Leu Pro Ser Gly Glu Val Leu Glu Thr

1330 1335 1340

Ser Thr Ser Ala Val Gly Asp Leu Ser Gly Leu Pro Ser Gly Gly Glu

1345 1350 1355 1360

Val Leu Glu Ile Ser Val Ser Gly Val Glu Asp Ile Ser Gly Leu Pro

1365 1370 1375

Ser Gly Glu Val Val Glu Thr Ser Ala Ser Gly Ile Glu Asp Val Ser

1380 1385 1390

Glu Leu Pro Ser Gly Glu Gly Leu Glu Thr Ser Ala Ser Gly Val Glu

1395 1400 1405

Asp Leu Ser Arg Leu Pro Ser Gly Glu Glu Val Leu Glu Ile Ser Ala

1410 1415 1420

Ser Gly Phe Gly Asp Leu Ser Gly Val Pro Ser Gly Gly Glu Gly Leu

1425 1430 1435 1440

Glu Thr Ser Ala Ser Glu Val Gly Thr Asp Leu Ser Gly Leu Pro Ser

1445 1450 1455

Gly Arg Glu Gly Leu Glu Thr Ser Ala Ser Gly Ala Glu Asp Leu Ser

1460 1465 1470

Gly Leu Pro Ser Gly Lys Glu Asp Leu Val Gly Ser Ala Ser Gly Asp

1475 1480 1485

Leu Asp Leu Gly Lys Leu Pro Ser Gly Thr Leu Gly Ser Gly Gln Ala

1490 1495 1500

Pro Glu Thr Ser Gly Leu Pro Ser Gly Phe Ser Gly Glu Tyr Ser Gly

1505 1510 1515 1520

Val Asp Leu Gly Ser Gly Pro Ser Gly Leu Pro Asp Phe Ser Gly

1525 1530 1535

Leu Pro Ser Gly Phe Pro Thr Val Ser Leu Val Asp Ser Thr Leu Val

1540 1545 1550

Glu Val Val Thr Ala Ser Thr Ala Ser Glu Leu Glu Gly Arg Gly Thr

1555 1560 1565

Ile Gly Ile Ser Gly Ala Gly Glu Ile Ser Gly Leu Pro Ser Ser Glu

1570 1575 1580

Leu Asp Ile Ser Gly Arg Ala Ser Gly Leu Pro Ser Gly Thr Glu Leu

1585 1590 1595 1600

Ser Gly Gln Ala Ser Gly Ser Pro Asp Val Ser Gly Glu Ile Pro Gly

1605 1610 1615

Leu Phe Gly Val Ser Gly Gln Pro Ser Gly Phe Pro Asp Thr Ser Gly

1620 1625 1630

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

1635 1640 1645

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

1650 1655 1660

Phe Gly Ile Thr Asp Leu Ser Gly Glu Thr Ser Gly Val Pro Asp Leu

1665 1670 1675 1680

Ser Gly Gln Pro Ser Gly Leu Pro Gly Phe Ser Gly Ala Thr Ser Gly

1685 1690 1695

Val Pro Asp Leu Val Ser Gly Thr Thr Ser Gly Ser Gly Glu Ser Ser

1700 1705 1710

Gly Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Ala Pro Thr Thr

1715 1720 1725

Phe Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu Pro

1730 1735 1740

Ser Gly Glu Ala Asp Leu Ser Gly Lys Ser Gly Met Val Asp Val Ser

1745 1750 1755 1760

Gly Gln Phe Ser Gly Thr Val Asp Ser Ser Gly Phe Thr Ser Gln Thr

1765 1770 1775

Pro Glu Phe Ser Gly Leu Pro Ser Gly Ile Ala Glu Val Ser Gly Glu

1780 1785 1790

Ser Ser Arg Ala Glu Ile Gly Ser Ser Leu Pro Ser Gly Ala Tyr Tyr

1795 1800 1805

Gly Ser Gly Thr Pro Ser Ser Phe Pro Thr Val Ser Leu Val Asp Arg

1810 1815 1820

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

1825 1830 1835 1840

Glu Gly Pro Ser Gly Ile Leu Glu Leu Ser Gly Ala His Ser Gly Ala

1845 1850 1855

Pro Asp Met Ser Gly Glu His Ser Gly Phe Leu Asp Leu Ser Gly Leu

1860 1865 1870

Gln Ser Gly Leu Ile Glu Pro Ser Gly Glu Pro Pro Gly Thr Pro Tyr

1875 1880 1885

Phe Ser Gly Asp Phe Ala Ser Thr Thr Asn Val Ser Gly Glu Ser Ser

1890 1895 1900

Val Ala Met Gly Thr Ser Gly Glu Ala Ser Gly Leu Pro Glu Val Thr

1905 1910 1915 1920

Leu Ile Thr Ser Glu Phe Val Glu Gly Val Thr Glu Pro Thr Ile Ser

1925 1930 1935

Gln Glu Leu Gly Gln Arg Pro Val Thr His Thr Pro Gln Leu Phe

1940 1945 1950

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

1955 1960 1965

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

1970 1975 1980

Ser Ser Glu Thr Ser Ala Tyr Pro Glu Ala Gly Phe Gly Ala Ser Ala

1985 1990 1995 2000

Ala Pro Glu Ala Ser Arg Glu Asp Ser Gly Ser Pro Asp Leu Ser Glu

2005 2010 2015

Thr Thr Ser Ala Phe His Glu Ala Asn Leu Glu Arg Ser Ser Gly Leu

2020 2025 2030

Gly Val Ser Gly Ser Thr Leu Thr Phe Gln Glu Gly Glu Ala Ser Ala

2035 2040 2045

Ala Pro Glu Val Ser Gly Glu Ser Thr Thr Thr Ser Asp Val Gly Thr

2050 2055 2060

Glu Ala Pro Gly Leu Pro Ser Ala Thr Pro Thr Ala Ser Gly Asp Arg

2065 2070 2075 2080

Thr Glu Ile Ser Gly Asp Leu Ser Gly His Thr Ser Gln Leu Gly Val

2085 2090 2095

Val Ile Ser Thr Ser Ile Pro Glu Ser Glu Trp Thr Gln Gln Thr Gln

2100 2105 2110

Arg Pro Ala Glu Thr His Leu Glu Ile Glu Ser Ser Ser Leu Leu Tyr

2115 2120 2125

Ser Gly Glu Glu Thr His Thr Val Glu Thr Ala Thr Ser Pro Thr Asp

2130 2135 2140

Ala Ser Ile Pro Ala Ser Pro Glu Trp Lys Arg Glu Ser Glu Ser Thr

2145 2150 2155 2160

Ala Ala Ala Pro Ala Arg Ser Cys Ala Glu Glu Pro Cys Gly Ala Gly

2165 2170 2175

Thr Cys Lys Glu Thr Glu Gly His Val Ile Cys Leu Cys Pro Pro Gly

2180 2185 2190

Tyr Thr Gly Glu His Cys Asn Ile Asp Gln Glu Val Cys Glu Glu Gly

2195 2200 2205

Trp Asn Lys Tyr Gln Gly His Cys Tyr Arg His Phe Pro Asp Arg Glu

2210 2215 2220

Thr Trp Val Asp Ala Glu Arg Arg Cys Arg Glu Gln Gln Ser His Leu

2225 2230 2235 2240

Ser Ser Ile Val Thr Pro Glu Glu Gln Glu Phe Val Asn Asn Asn Ala

2245 2250 2255

Gln Asp Tyr Gln Trp Ile Gly Leu Asn Asp Arg Thr Ile Glu Gly Asp

2260 2265 2270

Phe Arg Trp Ser Asp Gly His Pro Met Gln Phe Glu Asn Trp Arg Pro

2275 2280 2285

Asn Gln Pro Asp Asn Phe Phe Ala Ala Gly Glu Asp Cys Val Val Met

2290 2295 2300

Ile Trp His Glu Lys Gly Glu Trp Asn Asp Val Pro Cys Asn Tyr His

2305 2310 2315 2320

Leu Pro Phe Thr Cys Lys Lys Gly Thr Val Ala Cys Gly Glu Pro Pro

2325 2330 2335

Val Val Glu His Ala Arg Thr Phe Gly Gln Lys Lys Asp Arg Tyr Glu

2340 2345 2350

Ile Asn Ser Leu Val Arg Tyr Gln Cys Thr Glu Gly Phe Val Gln Arg

2355 2360 2365

His Met Pro Thr Ile Arg Cys Gln Pro Ser Gly His Trp Glu Glu Pro

2370 2375 2380

Arg Ile Thr Cys Thr Asp Ala Thr Thr Tyr Lys Arg Arg Leu Gln Lys

2385 2390 2395 2400

Arg Ser Ser Arg His Pro Arg Arg Ser Arg Pro Ser Thr Ala His

2405 2410 2415

<210> 156

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 156

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> 157

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 157

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> 158

<400> 158

000

<210> 159

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 159

Gly Gly Gly Gly Ser

fifteen

<210> 160

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 160

Ser Gly Gly Ser Gly Gly Ser

fifteen

<210> 161

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 161

Gly Gly Gly Gly Gly Gly Gly Ser

fifteen

<210> 162

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 162

Gly Gly Gly Gly Ser Gly Gly Gly Ser

fifteen

<210> 163

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 163

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 164

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 164

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

1 5 10 15

<210> 165

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 165

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

1 5 10 15

Gly Ser

<210> 166

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid 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

twenty

<210> 167

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 167

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> 168

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 168

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> 169

<211> 35

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 169

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> 170

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 170

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> 171

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 171

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

1 5 10 15

<210> 172

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 172

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

1 5 10 15

Thr His Thr Cys Pro Pro Cys Pro

twenty

<210> 173

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 173

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

1 5 10

<210> 174

<211> 62

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence

<400> 174

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

<---

Claims (30)

1. A disintegrin and metalloproteinase-binding polypeptide with thrombospondin motifs 5 (ADAMTS5) comprising at least 1 single immunoglobulin variable domain (ISVD) binding ADAMTS5, wherein said ADAMTS5-binding ISVD contains 3 complementarity-determining regions, where CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37 and CDR3 is SEQ ID NO: 55. 2. The polypeptide of claim 1, wherein said ISVD consists of, or substantially consists of, 4 framework regions and said 3 complementarity-determining regions. 3. A polypeptide according to claim 1, wherein said polypeptide has the sequence of SEQ ID NO: 129 or 130, or a sequence having at least 95% sequence identity to SEQ ID NO: 129 or 130. 4. The polypeptide according to any one of paragraphs. 1-3, where the specified polypeptide binds to ADAMTS5 with a K _D of less than 1E ^-08 M. 5. The polypeptide according to any one of paragraphs. 1-3, where the specified polypeptide inhibits the enzyme activity of ADAMTS5 with an IC _{50 of} not more than 1E ^-9 M. 6. The polypeptide according to any one of paragraphs. 1-3, where the indicated polypeptide modulates ADAMTS5 with an EC ₅₀ between 1E– ⁰⁷ M and 1E ^–12 M. 7. The polypeptide according to any one of paragraphs. 1-3, where the indicated polypeptide binds to ADAMTS5 with a dissociation rate of less than 1E– ⁰⁴ s– ¹ . 8. The polypeptide according to any one of paragraphs. 1-3 wherein said ADAMTS5 is human ADAMTS5 has the sequence of SEQ ID NO: 149. 9. The polypeptide according to any one of paragraphs. 1-3, where the specified polypeptide is an antagonist of ADAMTS5 aggrecanase activity. 10. The polypeptide according to any one of paragraphs. 1-3, wherein said polypeptide inhibits ADAMTS5 binding to aggrecan by at least 20%. 11. The polypeptide according to any one of paragraphs. 1-3, where the specified polypeptide inhibits the protease activity of ADAMTS5. 12. The polypeptide according to any one of paragraphs. 1-3, additionally containing a second ISVD. 13. The polypeptide of claim 12, wherein the second ISVD specifically binds to ADAMTS5. 14. The polypeptide according to any one of paragraphs. 1-3, additionally containing ISVD, which binds to serum albumin. 15. The polypeptide of claim 14, wherein said serum albumin-binding ISVD primarily consists of 4 framework regions and 3 complementarity determining regions, wherein CDR1 has the sequence of SEQ ID NO: 146, CDR2 has the sequence of SEQ ID NO: 147, and CDR3 has the sequence SEQ ID NO: 148. 16. The polypeptide of claim 14, wherein said serum albumin binding ISVD is selected from the group consisting of ALB8 having the sequence of SEQ ID NO: 131, ALB23 having the sequence of SEQ ID NO: 132, ALB129 having the sequence of SEQ ID NO: 133, ALB132 having SEQ ID NO: 134, ALB11 having SEQ ID NO: 135, ALB11 having (S112K)-A SEQ ID NO: 136, ALB82 having SEQ ID NO: 137, ALB82-A having the sequence of SEQ ID NO: 138, ALB82-AA, having the sequence of SEQ ID NO: 139, ALB82-AAA, having the sequence of SEQ ID NO: 140, ALB82-G, having the sequence of SEQ ID NO: 141, ALB82-GG, having the sequence of SEQ ID NO: 142, ALB82-GGG having the sequence of SEQ ID NO: 143, ALB92 having the sequence of SEQ ID NO: 144 and ALB223 having the sequence of SEQ ID NO: 145. 17. The polypeptide according to claim 14, having a sequence selected from the group: SEQ ID NO: 129 (clone 577 ^2F3SO -Alb), SEQ ID NO: 130 (clone 579 2F3 ^SO -093-Alb), SEQ ID NO: 122 (clone 6 2F3-Alb), and SEQ ID NO: 126 (clone 129 2F3-093-Alb). 18. The polypeptide of claim 12, wherein the ADAMTS5-binding ISVD is linked directly or via a linker to a second ISVD. 19. A polypeptide according to claim 18, wherein said linker has any of the sequences of SEQ ID NOs: 158-174. 20. The polypeptide according to any one of paragraphs. 1-3, wherein said polypeptide has at least 90% sequence identity with any of SEQ ID NOs: 2, 129, or 130. 21. A method of treating or preventing a disease or disorder in an individual in which ADAMTS5 activity is involved, comprising administering a polypeptide according to any one of paragraphs. 1-20 to the specified individual in an amount effective to treat or prevent a symptom of the specified disease or disorder. 22. The method of claim 21, wherein said disease or disorder is selected from the group consisting of arthropathy, chondrodystrophy, arthritic disease, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration disease, degenerative joint disease, relapsing polychondritis, dissecting osteochondritis and aggrecanopathy. 23. The method of claim 22, wherein the arthritic disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture, and detachment. 24. The polypeptide according to any one of paragraphs. 1-20 for use as a drug. 25. The polypeptide according to any one of paragraphs. 1-20 for use in the treatment or prevention in an individual of a disease or disorder in which ADAMTS5 activity is involved, wherein the disease or disorder is selected from chondrodystrophy arthropathy, arthritic disease, achondroplasia, costal chondritis, spondyloepimetaphyseal dysplasia, herniated disc, lumbar disc degeneration disease, degenerative joint disease, relapsing polychondritis, osteochondritis dissecans and aggrecanopathy. 26. The polypeptide of claim 25, wherein the arthritic disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic rupture, and detachment.

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