MMP13-conjugated immunoglobulin

Abstract

The present invention relates to immunoglobulins, more particularly polypeptides, that specifically bind to MMP13, nucleic acids encoding such polypeptides; methods for preparing such polypeptides; and compositions comprising such polypeptides, particularly pharmaceutical compositions, for preventive, therapeutic, or diagnostic purposes. [Solution] In particular, the immunoglobulin of the present invention inhibits the activity of MMP13 and is preferably stable.

Description

[Technical Field]

[0001] 1. Field of Invention The present invention relates to immunoglobulins that bind to MMP13, and more particularly to polypeptides comprising or essentially comprising one or more such immunoglobulins (referred herein to as "the immunoglobulin of the present invention" and "the polypeptide of the present invention," respectively). The present invention also relates to constructs comprising such immunoglobulins or polypeptides, and nucleic acids encoding such immunoglobulins or polypeptides (referred herein to as "the nucleic acid of the present invention"); to methods for preparing such immunoglobulins, polypeptides and constructs; to host cells that express or are capable of expressing such immunoglobulins or polypeptides; to compositions comprising such immunoglobulins, polypeptides, constructs, nucleic acids and / or host cells, and especially to pharmaceutical compositions; and in particular to the use of immunoglobulins, polypeptides, constructs, nucleic acids, host cells and / or compositions for preventive and / or therapeutic purposes, such as the preventive and / or therapeutic purposes described herein. Other aspects, embodiments, advantages and uses of the present invention will become apparent from further description herein. [Background technology]

[0002] 2. Background of the Invention Osteoarthritis (OA) is one of the most common causes of disability worldwide. 30 million Americans suffer from it, making it the most common joint disorder. More than 20% of the US population is projected to have it by 2025. The disease is not systemic and is usually limited to a few joints. However, it can occur in all joints, most often in the knees, hips, hands, and spine. OA is characterized by the progressive erosion of articular cartilage (the cartilage covering the bone), resulting in chronic pain and impairment. Ultimately, the disease leads to the complete destruction of the articular cartilage, hardening of the underlying bone, osteophyte formation, and so on, all of which result in loss of movement and pain. Pain is the most prominent symptom of OA and is often the reason patients seek medical help. There is no cure for OA; disease management is at best limited to palliative care and does little to address the underlying causes of disease progression. Disease-modifying anti-osteoarthritis drugs (DMOADs), which can be defined as medications that inhibit the progression of structural disease and, ideally, also improve symptoms and / or function, are in high demand. Because DMOADs are likely to be prescribed over long periods in the elderly population with this chronic disease, excellent safety data are required in target populations with multiple comorbidities and potential drug interactions. Osteoarthritis can be defined as a diverse group of conditions characterized by a combination of joint symptoms, signs resulting from defects in articular cartilage, and changes in adjacent tissues, including bone, tendons, and muscles. The most abundant components of articular cartilage are proteoglycans and collagen (primarily collagen II). The main proteoglycan in cartilage is aggrecan. While disease onset can be multifactorial, cartilage destruction is thought to be a result of uncontrolled proteolytic extracellular matrix disruption (ECM).

[0003] As described above, the major component of the extracellular matrix of chondrocytes is aggrecan (Kiani et al. 2002 Cell Research 12:19-32). This molecule is important for the proper function of articular cartilage by providing a hydrated gel structure that gives cartilage load-bearing capacity. Aggrecan is a large multimodular molecule (2317 amino acids) expressed by chondrocytes. Its core protein consists of three globular domains (G1, G2, and G3) and a large extended region between G2 and G3 for glycosaminoglycan chain binding. This extended region consists of two domains: a keratan sulfate-substituted domain (KS domain) and a chondroitin sulfate-substituted domain (CS domain). The CS domain is bound to 100-150 glycosaminoglycan (GAG) chains. Aggrecan forms a large complex with hyaluronan, where 50–100 aggrecan molecules interact with one hyaluronan molecule via the G1 domain and link proteins. Upon absorbing water (depending on the GAG ​​content), these complexes form a reversibly deformable gel that resists compression. The structure, fluid retention, and function of articular cartilage are related to the matrix content of aggrecan and the amount of chondroitin sulfate bound to the intact core protein.

[0004] Type II collagen (Collagen II, Col II) makes up 50% of articular cartilage. Collagen fibrils form a network that allows cartilage to take up proteoglycans, providing strength to the tissue. Collagen is a structural protein composed of right-handed bundles of triple parallel left-handed polyproline type II (PPII) helices. Due to the dense packing of the PPII helices within the triple helix, the residue at every third position, which is an amino acid, is glycine (Gly). Glycine plays a unique role in fibrous structural proteins because it is the smallest amino acid without a side chain. In collagen, glycine is required at every third position because the assembly of the triple helix places this residue inside the helix (axis), leaving no space for a side group larger than the single hydrogen atom of glycine.

[0005] The characteristics of OA are 1) the breakdown of aggrecan, the gradual release of domains G3 and G2 (resulting in cartilage "contraction"), and finally the release of the G1 domain, and 2) the breakdown of collagen, which irreversibly destroys the cartilage structure. Compelling evidence exists demonstrating that matrix metalloproteinases (MMPs) play a significant role in tissue breakdown associated with osteoarthritis (OA). MMPs are a family of zinc-dependent endopeptidases involved in the degradation of the extracellular matrix and tissue remodeling. There are approximately 28 MMP family members, which can be classified into various subgroups, including collagenases, gelatinases, stromelicins, membrane-bound MMPs, matrilysins, and enamelicins. Collagenases, including MMP1, MMP8, MMP13, and MMP18, can degrade triple-helix fibrillary collagen into characteristic 3 / 4 and 1 / 4 fragments. Furthermore, MMP14 has also been shown to cleave fibrillary collagen, and there is evidence that MMP2 is also capable of collagen degradation. MMPs have long been considered attractive therapeutic targets for the treatment of OA. However, broad-spectrum MMP inhibitors developed for the treatment of arthritis have failed in clinical trials due to the side effect of painful arthritis, known as musculoskeletal syndrome (MSS). MSS is thought to be caused by the non-selective inhibition of multiple MMPs.

[0006] Nam et al. (2017 Proc Natl Acad Sci USA 113:14970-14975) describe nanobodies that are clearly directed specifically to the active site of MMP14. Therapeutic interventions in osteoarthritis (OA) are also hindered by the difficulty of targeting drugs to articular cartilage. Because articular cartilage is avascular and alymphatic tissue, conventional drug delivery routes (oral, intravenous, intramuscular) ultimately rely on the transsynovial transfer of drugs from synovial capillaries to cartilage via passive diffusion. Therefore, in the absence of mechanisms for selectively targeting drugs to cartilage, it is necessary to achieve sustained intra-articular therapeutic doses by systemically exposing the body to high drug concentrations. As a result of high systemic exposure, most conventional treatments for OA have suffered from serious toxicity.

[0007] Furthermore, most newly developed DMOADs have short intra-articular residence times, even when administered intra-articular (Edwards 2011 Vet. J. 190:15-21; Larsen et al. 2008 J Pham Sci 97:4622-4654). Intra-articular (IA) delivery of therapeutic proteins is limited by rapid clearance from the joint space and insufficient retention within the cartilage. Synovial residence time of drugs in the joint is often less than 24 hours. Due to the rapid clearance of most IA-injected drugs, frequent injections are required to maintain effective concentrations (Owen et al., 1994 Br J Clin Pharmacol 38:349-355). However, frequent IA injections are undesirable because they can lead to patient compliance problems due to pain and discomfort, and also carry a risk of causing joint infections. The need for an effective DMOAD remains. [Overview of the project]

[0008] 3. Outline of the Invention The present invention aims to provide polypeptides for OA that, in addition to other advantageous properties, have improved prophylactic, therapeutic, and / or pharmacological properties (e.g., improved ease of preparation, good stability, and / or cost reduction of the product) compared to the amino acid sequences and antibodies of the prior art. In particular, the present invention aims to provide immunoglobulin single variable domains (ISVDs) and polypeptides containing the same for inhibiting MMPs, and in particular MMP13.

[0009] The inventors hypothesized that the best region for inhibiting the enzymatic activity of MMP13 would be to increase ISVD (Integrated Surgical Domain Degradation) against the catalytic pocket. However, this proved to be a major challenge. In particular, MMP13 is secreted as an inactive pro-form (proMMP13), where the pro-domain masks the catalytic pocket, preventing access to the pocket to enhance the immune response. On the other hand, activated MMP13 has a short half-life, which is mainly due to autoprotein degradation. Furthermore, even after the inventors overcame the two previous problems, it was found that high sequence conservation of the catalytic domain across various species precedes a robust immune response.

[0010] Ultimately, the inventors were able to address these challenges through the original development of new tools and unconventional screening methods. ISVD was isolated from various screening campaigns and further manipulated to possess diverse and desirable characteristics, including stability, affinity, and inhibitory activity. The monovalent ISVD of the present invention, which binds to MMP13, was superior to the comparator drug. A biparatopic polypeptide containing ISVD that was less suitable for inhibiting MMP13 activity was even more potent.

[0011] Accordingly, the present invention relates to a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that binds to a matrix metalloproteinase (MMP), preferably to the matrix metalloproteinase MMP13. The present invention also comprises a polypeptide comprising two or more ISVDs, each individually specifically binding to MMP13, wherein a) At least the "first" ISVD specifically binds to the first antigenic determinant, epitope, portion, domain, subunit, or conformation of MMP13; and here, b) At least the “second” ISVD specifically binds to a second antigenic determinant, epitope, partial, domain, subunit, or conformation of MMP13, which is distinct from the first antigenic determinant epitope, partial, domain, subunit, or conformation.

[0012] Further provided are polypeptides of the present invention comprising a single variable domain (ISVD) that binds to a matrix metalloproteinase (MMP), and a further single variable domain (ISVD) that binds to a cartilage proteoglycan, preferably aggrecan. Further aspects relate to polypeptides according to the present invention for use as pharmaceuticals. Yet another aspect relates to a method for treating and preventing a disease or disorder in an individual, for example, involving MMP13 activity, the method comprising administering the polypeptide of the present invention to the individual in an amount effective to treat or prevent the symptoms of the disease or disorder.

[0013] Other aspects, advantages, uses, and applications of polypeptides and compositions will become apparent from further disclosures herein. Several documents are referenced throughout this specification. Each of these documents (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an acknowledgment that the present invention is not entitled to any prior rights to such disclosure by prior art. [Brief explanation of the drawing]

[0014] 4. Legend for the Figure [Figure 1] Figure 1 shows the dose-response curves for profile 1 nanobodies (left graph) and profile 2 nanobodies (right graph) in a fluorescence-generating collagen assay. [Figure 2] Figure 2 shows the selectivity of MMP13 lead nanobodies. [Figure 3] Figure 3 shows a competitive ELISA of 0.6 nM biotinylated 40E09 against a panel of profile 1 and profile 2 nanobodies on full-length human MMP13 coated with a mouse anti-human MMP13 mAb (R&D Systems #MAB511). MMP13 was activated by incubation with APMA at 37°C for 90 minutes. [Figure 4] Figure 4 shows the inhibition of cartilage degradation by nanobodies in a rat MMT model.

[0015] 5. Detailed explanation The need for safe and effective OA drugs remains. These drugs need to meet a variety of frequently conflicting requirements, especially when a widely applicable format is intended. Therefore, the format should preferably be useful for a wide range of patients. The format should preferably be safe and not induce infections from frequent IA administration. Furthermore, the format should preferably be patient-friendly. For example, the format should have an extended half-life in the joint so that it is not immediately removed upon administration. However, extending the half-life should preferably not result in off-target activity or side effects, or limit efficacy. The present invention fulfills at least one of these requirements. Based on unconventional screening, characterization, and combination strategies, the inventors surprisingly observed that immunoglobulin single variable domains (ISVDs) functioned very well in in vitro and in vivo experiments.

[0016] Furthermore, the inventors were able to re-manipulate ISVD to achieve superior performance compared to the comparator drug. In the dual paratopic method, this performance was not only maintained but even improved. On the other hand, it was also demonstrated that the ISVD of the present invention is significantly more effective than the comparison molecule. The present invention provides polypeptides that antagonize MMPs, particularly MMP13, and which have improved prophylactic, therapeutic, and / or pharmacological properties, including a safer profile, compared to a comparative molecule.

[0017] Accordingly, the present invention relates to ISVDs and polypeptides that can be directed to and / or specifically bound to MMPs (as defined herein), preferably the MMP being selected from the group consisting of MMP13 (collagenase), MMP8 (collagenase), MMP1 (collagenase), MMP19 (matrix metalloproteinase RASI), and MMP20 (enamelicin), preferably the MMP being MMP13, and a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that modulates its activity, and in particular specifically binds to MMP13, wherein binding to MMP13 modulates the activity of MMP13.

[0018] definition Unless otherwise indicated or defined, all terms used have their usual meanings in this art, which would be obvious to those skilled in the art. See, for example, the following standard handbook: Sambrook et al. (Molecular Cloning: A Laboratory Manual (2 ndEd.) 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 (6 th Ed.) Mosby / Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's Essential Immunology (10 th Ed.) Blackwell Publishing, UK, 2001), and Janeway et al. (Immunobiology (6 th (ed.) Garland Science Publishing / Churchill Livingstone, New York, 2005), and the general background technology cited herein.

[0019] Unless otherwise indicated, all methods, steps, techniques, and operations not described in particular detail can and have been performed in a manner known by itself, and this would be obvious to those skilled in the art. For example, the standard handbooks and general background techniques mentioned herein, as well as the further references cited therein, are again referred to; also, for example, the following reviews are also referred to: 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); these describe protein engineering techniques such as affinity maturation, and other techniques for improving the specificity and other desirable properties of proteins such as immunoglobulins.

[0020] It should be noted that, as used herein, the singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. For example, a reference to one “reagent” includes one or more such different reagents, and a reference to a “method” includes references to equivalent steps and methods known to those skilled in the art, which may be modified or replaced by the methods described herein. Unless otherwise specified, the term “at least” preceding a set of elements should be understood to refer to all of those elements. Those skilled in the art will be able to recognize or confirm, by routine experimentation alone, many equivalents to specific embodiments of the invention described herein. Such equivalents are intended to be included in the invention.

[0021] The terms "and / or" as used herein always include the meanings of "and," "or," and "all or any other combination of the elements connected by the aforementioned terms." As used herein, the terms “about” or “approximately” mean within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range. Throughout this specification and the subsequent claims, unless the context specifically requires otherwise, the word “comprising,” and variations such as “comprising” and “including,” mean to include the integer or step or group of integers or steps mentioned, but not to exclude any other integer or step or group of integers or steps. As used herein, the term “comprising” may be replaced by the terms “containing” or “including,” or, as may be the case herein, the term “having.”

[0022] As used herein, the term "sequence" (e.g., "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V") HH The term "sequence" or "protein sequence" should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence that encodes it, unless the context requires a more specific interpretation. An amino acid sequence is interpreted, depending on the context, as meaning a single amino acid or an unbranched sequence of two or more amino acids. A nucleotide sequence is interpreted as meaning an unbranched sequence of three or more nucleotides.

[0023] The amino acids are L-amino acids commonly found in natural proteins. Amino acid residues are indicated according to standard three-letter or one-letter amino acid codes. See, for example, Table A-2 on page 48 of WO 08 / 020079. These amino acid sequences containing D-amino acids are not intended to be included in this definition. Amino acid sequences containing post-translational modified amino acids may be described as the first translated amino acid sequence using the symbols shown in this Table A-2 and modification sites such as hydroxylation or glycosylation, however these modifications must not be explicitly indicated in the amino acid sequence. All peptides or proteins that can be expressed as sequence-modified links, crosslinks and end caps, non-peptidyl links, etc., are included in this definition.

[0024] The terms “protein,” “peptide,” “protein / peptide,” and “polypeptide” are used interchangeably throughout this disclosure and each has the same meaning for the purposes of this disclosure. Each term refers to an organic compound consisting of a linear chain of two or more amino acids. Compounds may have 10 or more amino acids, 25 or more amino acids; 50 or more amino acids; 100 or more amino acids; 200 or more amino acids; and even 300 or more amino acids. Those skilled in the art will understand that: polypeptides generally contain fewer amino acids than proteins, however there is no art-recognized cutoff point for the number of amino acids that distinguishes polypeptides from proteins; polypeptides can be prepared by chemical synthesis or recombinant methods; and proteins are generally prepared in vitro or in vivo by recombinant methods known in the art. By convention, the amide bonds in the primary structure of a polypeptide are in the order in which the amino acids are written, where the amine terminus (N-terminus) of the polypeptide is always on the left and the acid terminus (C-terminus) is on the right.

[0025] A nucleic acid or amino acid sequence is considered to be "(in essence) isolated" if it has been separated from at least one other component that is normally associated with the source or medium, for example, another nucleic acid, another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or trace component, compared to the reaction medium or culture medium from which it was obtained. In particular, a nucleic acid or amino acid sequence is considered "(in essence) isolated" if it has been purified to at least 2-fold, especially at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. A nucleic acid or amino acid "(in essence) isolated" is preferably in essence homogeneous, as determined using appropriate techniques, for example, by appropriate chromatographic techniques such as polyacrylamide gel electrophoresis.

[0026] Where a nucleotide sequence or amino acid sequence is said to "contain" or "become essentially" of another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence is incorporated into each of the first-referenced nucleotide sequences or amino acid sequences. More generally, however, this generally means that the first-referenced nucleotide sequence or amino acid sequence contains within its sequence each of stretches of nucleotide or amino acid residues having the same nucleotide sequence or amino acid sequence as the latter sequence, regardless of how the first-referenced sequence was actually produced or obtained (e.g., by any suitable method described herein). By non-limiting example, where a polypeptide of the present invention is said to contain an immunoglobulin monovariable domain ("ISVD"), this may mean that the immunoglobulin monovariable domain sequence is incorporated into the sequence of the polypeptide of the present invention. More generally, however, this generally means that the polypeptide of the present invention contains within its sequence the sequence of an immunoglobulin monovariable domain, regardless of how the polypeptide of the present invention was produced or obtained. Furthermore, when a nucleic acid or nucleotide sequence is said to contain another nucleotide sequence, the first nucleic acid or nucleotide sequence, when expressed in an expression product (e.g., a polypeptide), has amino acid sequences encoded by the latter nucleotide sequence that form part of the expression product (in other words, the latter nucleotide sequence is in the same reading frame as the larger nucleic acid or nucleotide sequence initially mentioned).Furthermore, when a construct of the present invention is said to contain a polypeptide or ISVD, this may mean that the construct includes at least the polypeptide or ISVD, respectively, but more generally, it means that the construct includes, in addition to the polypeptide or ISVD, groups, residues (e.g., amino acid residues), parts and / or binding units, regardless of how the polypeptide or ISVD is attached to the groups, residues (e.g., amino acid residues), parts and / or binding units, and regardless of how the construct is produced or obtained.

[0027] "Essentially consisting of" means that the immunoglobulin monovariable domain used in the method of the present invention is either identical to the immunoglobulin monovariable domain of the present invention or corresponds to the following immunoglobulin monovariable domain of the present invention: an immunoglobulin monovariable domain having a limited number of amino acid residues, e.g., 1 to 20 amino acid residues, e.g., 1 to 10 amino acid residues, preferably 1 to 6 amino acid residues, e.g., 1, 2, 3, 4, 5, or 6 amino acid residues, attached to the amino terminus, carboxy terminus, or both the amino and carboxy terminus of the immunoglobulin monovariable domain. To compare two or more nucleotide sequences, the percentage of "sequence identity" between a first and second nucleotide sequence can be calculated as follows: [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotide at the corresponding position in the second nucleotide sequence] is divided by [the total number of nucleotides in the first nucleotide sequence] and multiplied by [100%]; where each deletion, insertion, substitution, or addition of nucleotides in the second nucleotide sequence compared to the first nucleotide sequence is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings with known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Several other techniques, computer algorithms, and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768. Typically, for the purpose of determining the percentage of “sequence identity” between two nucleotide sequences according to the calculation method outlined above, the nucleotide sequence with the largest number of nucleotides is designated as the “first” nucleotide sequence, and the other nucleotide sequence is designated as the “second” nucleotide sequence.

[0028] To compare two or more amino acid sequences, the percentage of “sequence identity” (also referred to herein as “amino acid identity”) between a first amino acid sequence and a second amino acid sequence can be calculated as follows: [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] is divided by [the total number of amino acid residues in the first amino acid sequence] and multiplied by [100%]; here, each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., an “amino acid difference” as defined herein. Alternatively, the degree of sequence identity between two or more amino acid sequences can be calculated again using standard settings with known computer algorithms for determining the degree of sequence identity of nucleotide sequences, such as those described above. Typically, for the purpose of determining the percentage of “sequence identity” between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the “first” amino acid sequence, and the other amino acid sequences are designated as the “second” amino acid sequences.

[0029] Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue of a similar chemical structure, but 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 may be selected based on relevant teachings from, for example, WO 04 / 037999 and WO 98 / 49185 and further references cited therein.

[0030] Such conservative substitutions are preferably those in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, 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 nonpolar residues: Met, Leu, Ile, Val, and Cys; (e) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Gln to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0031] Any amino acid substitution applied to the polypeptides described herein may also be based on the analysis of the frequency of amino acid mutations between homologous proteins of different species developed by Schulz et al. ("Principles of Protein Structure", Springer-Verlag, 1978), the analysis of conformational probability developed by Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol., 47: 45-149, 1978), and the analysis of the hydrophobic pattern of 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 hereby incorporated by reference in their entirety. Information regarding the primary, secondary and tertiary structures of nanobodies is described in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of the V HH domain from llama is given, for example, by 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 regarding some of the amino acid residues that form the V H / V H interface in the conventional V L domain, and potential camelization substitutions at these positions, can be found in the prior art cited above.

[0032] Amino acid sequences and nucleic acid sequences are said to be "completely identical" if they have 100% sequence identity (as defined herein) over their entire length. When comparing two amino acid sequences, the term “amino acid difference” refers to the insertion, deletion, or substitution of a single amino acid residue at the position of 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 ISVD and / or polypeptides of the present invention, the term “amino acid difference” refers to the insertion, deletion, or substitution of a single amino acid residue at the position of the CDR sequence identified in b), d), or f), compared to the CDR sequence in a), c), or e), respectively. It is understood that the CDR sequences in b), d), and f), compared to the CDR sequence in a), c), or e), respectively, may contain one, two, three, four, or up to five such amino acid differences.

[0033] The "amino acid difference" can be any one, two, three, four, or up to five substitutions, deletions, or insertions, or any combination thereof, which improves the properties of the MMP13 binder of the present invention, such as the polypeptide of the present invention, or does not reduce, at least excessively, the desired properties of the MMP13 binder of the present invention, such as the polypeptide of the present invention, or the balance or combination of desired properties. In this regard, the MMP13 binders obtained in the present invention, such as the polypeptide of the present invention, should bind to MMP13 with at least the same, nearly the same, or higher affinity than polypeptides containing one or more CDR sequences without one, two, three, four, or up to 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.

[0034] In this regard, the amino acid sequences of the CDR according to b), d), and / or f) may be amino acid sequences derived from the amino acid sequences of a), c), and / or e), respectively, using affinity maturation that is known in itself or using one or more affinity maturation techniques as described in the Examples. For example, depending on the host organism used to express the polypeptide of the present invention, such deletions and / or substitutions may be designed to remove one or more sites for post-translational modification (such as one or more glycosylation sites), which would be within the capabilities of those skilled in the art (see Examples). As used herein, “represented by” in the context of any sequence number is equivalent to “contains or consists of” the sequence number “contains,” and preferably is equivalent to “consists of.” The terms "nanobody family" and "V" used herein refer to the following: HH "Family" or "Family" refers to nanobodies and / or V-type bodies of the same length. HH This refers to a group of sequences (i.e., they have the same number of amino acids within their sequence), of which the amino acid sequence between position 8 and position 106 (according to Kabat numbering) has more than 89% amino acid sequence identity.

[0035] The interchangeable terms “epitope” and “antigenic determinant” refer to a part of a macromolecule, such as an antigen-binding molecule, a polypeptide or protein, that is recognized by an antigen-binding molecule, more specifically by the antigen-binding site of the molecule, such as immunoglobulins, conventional antibodies, immunoglobulin monovariate domains, and / or polypeptides of the present invention. An epitope defines the minimal binding site for immunoglobulins and therefore represents the target of immunoglobulin specificity. Some antigen-binding molecules that recognize epitopes (such as immunoglobulins, conventional antibodies, immunoglobulin monovariate domains, and / or polypeptides of the present invention) are called "paratopes." An amino acid sequence (an immunoglobulin monovariate domain, antibody, polypeptide, or generally its antigen-binding protein or polypeptide or fragment) that can “bind” or “specifically bind” to a particular epitope, antigen, or protein (or at least a part thereof, fragment or epitope), has “affinity,” and / or “specificity” to such epitope, antigen, or protein is said to be “directed” to or “directed” to such epitope, antigen, or protein, or is said to be a “binding” molecule to such epitope, antigen, or protein, or is said to be an “anti”epitope, “anti”antigen, or “anti”protein (e.g., “anti”MMP13).

[0036] Affinity indicates the strength or stability of molecular interactions. Affinity is usually expressed as K D The affinity is given as the association constant K, or as the dissociation constant, which has units of moles / liter (or M). A It can also be expressed as, which is 1 / K D Equivalent to (moles / liter) -1 (or M -1 ) has units of ). In this specification, the stability of the interaction between two molecules is mainly determined by the K of their interaction. D It is expressed in terms of a value. K A = 1 / K D Considering the relationship, the strength of the molecular interaction is its K D By specifying by value, the corresponding K A It is obvious to those skilled in the art that the value can be calculated. D The value also characterizes the strength of molecular interactions in a thermodynamic sense, but this is K D The value is related to the change in the bond's free energy (ΔG) by a well-known relationship: ΔG = RTln(K D )(Equivalently, ΔG = -RTln(K A This is because they are related by the equation (where R represents the gas constant, T represents the absolute temperature, and ln represents the natural logarithm). K for biological interactions considered important (e.g., specific) D Usually 10 -12 M(0.001nM)~10 -5The range is M (10000 nM). The stronger the interaction, the greater the K D It will decrease.

[0037] K D is, k off The dissociation rate constant of the complex represented by and k on It can also be expressed as a ratio to the association rate represented by (therefore K D =k off / k on and K A =k on / k off Off-rate k off The unit is s -1 (s is the SI unit for seconds). On rate k on The unit is M -1 s -1 The on-rate is 10 2 M -1 s -1 ~about 10 7 M -1 s -1 It changes between and approaches the diffusion-limited association rate constant of bimolecular interactions. The off-rate is related to t 1 / 2 =ln(2) / k off This is related to the half-life of specific molecular interactions. The off-rate is 10 -6 s -1 (t for multiple days) 1 / 2 (Similar to an irreversible complex) ~1s -1 (t 1 / 2 It can vary between 0.69s. The specific binding of antigen-binding proteins such as ISVDs to antigens or antigenic determinants can be determined by any suitable method known on its own, including, for example, saturated binding assays and / or competitive binding assays, such as radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and sandwich competitive assays, as well as various variations thereof known on its own in the art, and other techniques referred to herein.

[0038] The affinity of molecular interactions between two molecules can be measured by different known techniques, such as the well-known surface plasmon resonance (SPR) biosensor technique (e.g., Ober et al. 2001, Intern. Immunology 13: 1551-1559), where one molecule is immobilized on a biosensor chip and the other molecule is k on , k off Measurement, therefore K D (or K A Under flow conditions that yield a value, the molecule passes over an immobilized molecule. This can be performed, for example, using the well-known BIACORE® instrument (Pharmacia Biosensor AB, Uppsala, Sweden). The Kinetic Exclusion Assay (KINEXA® instrument) (Drake et al. 2004, Analytical Biochemistry 328: 35-43) is based on measuring binding events in solution without labeling of the binding partner and kinetically excluding complex dissociation. In-solution affinity analysis can also be performed using the GYROLAB® immunoassay system (Fraley et al. 2013, Bioanalysis 5: 1765-74), which provides a platform for automated bioanalysis and rapid sample turnaround.

[0039] Furthermore, if the measurement process, for example, involves artifacts related to the coating of a biosensor of a certain molecule, which in some way affects the intrinsic binding affinity of the molecule being measured, then the measured K D The apparent K D It will also be apparent to those skilled in the art that this can be addressed. Furthermore, when one molecule contains multiple recognition sites for other molecules, the apparent K D The affinity may be measured. In such a situation, the measured affinity may be affected by the binding force of the interaction between the two molecules. In particular, K D The accurate measurement of K can be very labor-intensive, and as a result, the apparent K DThe value is often determined to assess the binding strength of two molecules. As long as all measurements are performed in a consistent manner (e.g., without changing assay conditions), the apparent K D Measurement true K D Since it can be used as an approximation, this book uses K D and apparent K D They should be treated with equal importance or relevance.

[0040] 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 the polypeptide or ISVD of the present invention) can bind, as described, for example, in paragraph n) on pages 53–56 of WO 08 / 020079. The specificity of an antigen-binding protein can be determined based on affinity and / or binding strength, as described on pages 53–56 of WO 08 / 020079 (incorporated herein by reference), which also describes several preferred techniques for measuring binding between an antigen-binding molecule (such as the polypeptide or ISVD of the present invention) and the associated antigen. Typically, an antigen-binding protein (such as the ISVD and / or polypeptide of the present invention) has a dissociation constant (K D ) as 10 -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, and more preferably 10 -8 ~10 -12 In moles / liter, the binding to those antigens (i.e., the association constant (K) A ) as 10 5 ~10 12 Liters / mol or more, preferably 10 7 ~10 12 Liters / moles or more, and more comfortably 10 8 ~10 12 (liters / moles). 10 -4 Any K greater than moles / liter D Value (or 10) 4 Any K less than liters / mol AThe value) is generally considered to indicate non-specific binding. Preferably, the monovalent ISVD of the present invention will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM, such as 10 to 5 pM or less.

[0041] An immunoglobulin single variable domain and / or polypeptide is said to be "specific" for a (first) target or antigen compared to another (second) target or antigen when the immunoglobulin single variable domain and / or polypeptide binds to the first antigen with an affinity of at least 10-fold, such as at least 100-fold, preferably at least 1000-fold or more, compared to the affinity when the immunoglobulin single variable domain and / or polypeptide binds to the second target or antigen. (As described above, and appropriately expressed as the K D value, K A value, K off rate and / or K on rate). For example, an immunoglobulin single variable domain and / or polypeptide binds to the first target or antigen with a K D value that is at least one-tenth, such as at least one-hundredth, and preferably at least one-thousandth, or even smaller than the K D value when the immunoglobulin single variable domain and / or polypeptide binds to the second target or antigen. Preferably, when an immunoglobulin single variable domain and / or polypeptide is "specific" for a first target or antigen compared to a second target or antigen, (as defined herein) this is directed to the first target or antigen but not to the second target or antigen.

[0042] The specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined by any suitable method known in itself, including, for example, saturated binding assays, scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays, and different variations thereof known in the art; as well as by other techniques referred herein. The dissociation constant may be an actual or apparent dissociation constant, as will be obvious to those skilled in the art and as described on pages 53-56 of WO 08 / 020079. Methods for determining the dissociation constant are obvious to those skilled in the art and include, for example, the techniques described on pages 53-56 of WO 08 / 020079.

[0043] Another preferred approach that can be used to assess affinity is the two-step ELISA (enzyme-linked immunosorbent assay) procedure described by Friguet et al. 1985 (J. Immunol. Methods 77: 305-19). This method establishes a liquid-phase bond equilibrium measurement and avoids possible artifacts associated with the adsorption of a single molecule to a support such as plastic. As will be apparent to those skilled in the art, and as described on pages 53-56 of WO 08 / 020079, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining the dissociation constant will be apparent to those skilled in the art and include, for example, the techniques described on pages 53-56 of WO 08 / 020079. In one aspect, the present invention relates to MMP13 binders such as ISVD and polypeptides, wherein the MMP13 binders do not bind to MMP1 or MMP14 (membrane type).

[0044] Finally, it should be noted that in many situations, an experienced scientist may determine that it is convenient to determine the binding affinity for a certain reference molecule. For example, to evaluate the binding strength between molecules A and B, a reference molecule C can be used, which is known to bind to, for example, B and is preferably labeled with a fluorophore or chromophore or other chemical component, such as biotin for ELISA or FACS (fluorescence-activated cell sorting) or other formats (fluorophore for fluorescence detection, chromophore for light absorption detection, biotin for streptavidin-mediated ELISA detection). Usually, the reference molecule C is maintained at a fixed concentration, and the concentration of A is varied relative to a specific concentration or amount of B. As a result, an IC 50 value is obtained corresponding to the concentration of A at which the signal measured for C in the absence of A is halved. If the K D which is K Dref , and the total concentration c ref of the reference molecule are known, the apparent K D of the interaction A - B is obtained from the following equation: K D = IC 50 / (1 + c ref / K Dref ). Note that when c ref << K Dref , K D ≈ IC 50 . If the measurement of IC 50 is performed in a consistent manner for the binding agents being compared (e.g., fixing c ref ), the differences in the strength or stability of the molecular interactions can be evaluated by comparing the IC 50 , and this measurement is judged to be equivalent to K D or the apparent K D throughout this text.

[0045] Half maximal inhibitory concentration (IC 50) is also a measure of the effectiveness of a compound in inhibiting biological or biochemical functions, such as pharmacological effects. This quantitative measure indicates the amount of polypeptide or ISVD (such as nanobodies) required to inhibit half of a certain biological process (or component of the process, i.e., enzymes, cells, cell receptors, chemotaxis, anaplasia, translocation, invasiveness, etc.). In other words, this is the maximum half (50%) inhibitory concentration (IC) of a substance (50% IC, or IC). 50 ) is IC 50 The value can be calculated by determining the concentration required to inhibit half of the maximum biological response of a given antagonist, such as the polypeptide or ISVD (e.g., nanobody) of the present invention. D This can be determined by creating a dose-response curve and investigating the effect of different concentrations of the polypeptide or ISVD (e.g., nanobody) of the present invention on reversing agonist activity.

[0046] Half-effective concentration (EC) 50 The term EC4 refers to the concentration of a compound that elicits baseline and maximum intermediate reactions after a specified exposure time. In this context, it is used as a measure of the potency of polypeptides or ISVDs (e.g., nanobodies). EC4 of a stepwise dose-response curve 50 This represents the concentration of the compound at which 50% of the maximum effect is observed. The concentration is preferably expressed in moles. In biological systems, small changes in ligand concentration typically result in a rapid change in response following an S-shape. The inflection point where the increase in response with increasing ligand concentration begins to slow down is the EC (Electrical Cycle). 50 This can be mathematically determined by deriving the fitting line. Relying on graphs for estimation is convenient in most cases. EC 50 If provided in the Examples section, the experiment is K D It is designed to reflect EC as accurately as possible. 50 The value is K D It is considered a value. The term "average K" D" is obtained from at least one, but preferably more than one, for example, at least two experiments, with an average K D Regarding values: The term "average" refers to the mathematical term "average" (the sum of data divided by the number of data items).

[0047] This is also IC, a measure of the inhibition (50% inhibition) of the compound. 50 It is also related to competitive binding assays and functional antagonist assays, IC 50 is the most common summary measure for dose-response curves. For agonist / stimulant assays, the most common summary measure is EC 50 That is the case. The inhibition constant Ki is an indicator of how potent an inhibitor is; it is the concentration required to produce half-number inhibition. IC50 may vary depending on experimental conditions. 50 Unlike (but see above), Ki is an absolute value and is often called the inhibitory constant of a drug. The inhibitory constant Ki can be calculated using the following Cheng-Prusoff equation:

number

[0048] As used herein, the term “potency” of the polypeptide of the present invention is a function of the amount of the polypeptide of the present invention required to produce its particular effect. This is simply the IC of that polypeptide 50It is measured as the reciprocal of . This refers to the ability of the polypeptide of the present invention to modulate and / or partially or completely inhibit the activity of MMP13. More specifically, it may refer to the ability of the polypeptide to reduce or completely inhibit the MMP13 activity as defined herein. Thus, this may refer to the ability of the polypeptide to inhibit proteolytic activity such as protease activity and endopeptidase activity, and / or to inhibit binding to substrates such as aggrecan, collagen II, collagen I, collagen III, collagen IV, collagen IX, collagen X, collagen XIV, and gelatin. The potency may be measured by any suitable assay known in the art or described herein.

[0049] The "efficacy" of the polypeptide of the present invention is measured at the maximum intensity of the effect itself at the saturated polypeptide concentration. Efficacy represents the maximum response achievable from the polypeptide of the present invention. This refers to the polypeptide's ability to produce the desired (therapeutic) effect. In one aspect, the present invention relates to the polypeptide described herein, wherein the polypeptide is determined to be MMP13, for example by KinExA, K D As 1E -07 M~1E -13 During M, for example, 1E -08 M~1E -12 Between M, preferably up to 1E -07 M, preferably 1E -08 M or 1E -09 Less than M, or 1E -10 Less than M, for example, 5E -11 M, 4E -11 M, 3E -11 M, 2E -11 M, 1.7E -11 M, 1E -11 M, or 5E -12 M, 4E -12 M, 3E -12 M, 1E -12 Combine with M, etc.

[0050] In one aspect, the present invention relates to polypeptides described herein, wherein the polypeptide is determined, for example, by competitive ELISA, competitive TIMP-2 ELISA, fluorescence peptide assay, fluorescence collagen assay, or collagen degradation assay, as detailed in the Examples section. 50 ga 1E -07 M~1E -12 During M, for example, 1E -08 M~1E -;11 During M, it inhibits the activity of MMP13. In one aspect, the present invention relates to a polypeptide described herein, wherein the polypeptide is IC 50 up to 1E -07 M, preferably 1E -08 M, 5E -09 M or 4E -9 M, 3E -9 M, 2E -9 M, for example, 1E -9 M inhibits the activity of MMP13. In one aspect, the present invention relates to polypeptides described herein, wherein the polypeptide is determined, for example, by competitive ELISA, competitive TIMP-2 ELISA, fluorescence-generating peptide assay, fluorescence-generating collagen assay or collagen degradation assay, EC 50 ga 1E -07 M~1E -12 During M, for example, 1E -08 M~1E -11 It connects to MMP13 between M.

[0051] In one aspect, the present invention relates to a polypeptide described herein, wherein the polypeptide is determined, for example, by SPR, and the offrate is 1E -04 (s -1 ) is bound to MMP13 if it is less than ). An amino acid sequence such as an ISVD or polypeptide is said to be “cross-reactive” to two different antigens or antigenic determinants (e.g., MMP13 from different mammalian species, e.g., human MMP13, canine MMP13, bovine MMP13, rat MMP13, porcine MMP13, mouse MMP13, rabbit MMP13, cynomolgus monkey MMP13, and / or rhesus monkey MMP13) if it is specific to these different antigens or antigenic determinants (as defined herein). It will be understood that an ISVD or polypeptide can be considered cross-reactive if it is specific to these different antigens or antigenic determinants (as defined herein), even if its binding affinity to the two different antigens may differ by a factor of 2, 5, 10, 50, 100 or more.

[0052] MMP13 is also known as CLG3 or collagenase 3, MANDP1, MMP13, matrix metallopeptidase 13, or MDST. Relevant structural information about MMP13 can be found, for example, in the UniProt accession number, as shown in Table 1 below (see Table B). Table 1 [Table 1]

[0053] "Human MMP13" refers to MMP13 containing the amino acid sequence of SEQ ID NO: 115. In one aspect, the polypeptide of the present invention specifically binds to MMP13 from Human sapiens, Mus musculus, Canis lupus, Bos taurus, Macaca mulatta, Rattus norvegicus, Gallus gallus, and / or P. troglodytes, preferably specifically to human MMP13, preferably to SEQ ID NO: 115. The terms “(cross)blocking,” “(cross)blocked,” “(cross)blocking,” “competitive binding,” “(cross)competing,” “(cross)competing,” and “(cross)competition” are used interchangeably herein and mean the ability of an immunoglobulin, antibody, ISVD, polypeptide, or other binder to interfere with the binding of other immunoglobulins, antibodies, ISVD, polypeptides, or binders to a given target. The extent to which an immunoglobulin, antibody, ISVD, polypeptide, or other binder can interfere with the binding of another to a target, and therefore whether it can be said to cross-block according to the present invention, can be determined using competitive binding assays common in the art, for example, by screening purified ISVD against ISVD presented on a phage in a competitive ELISA, as described in the Examples. Methods for determining whether an immunoglobulin, antibody, immunoglobulin monovariate domain, polypeptide, or other binder directed to a target (cross-block) is capable of (cross-blocking), competitively binding, or (cross-competing) as defined herein are described, for example, in Xiao-Chi Jia et al. (Journal of Immunological Methods 288: 91-98, 2004), Miller et al. (Journal of Immunological Methods 365: 118-125, 2011) and / or the methods described herein (see, for example, Example 7). The present invention relates to polypeptides represented herein, for example, by SEQ ID NOs: 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8, wherein the polypeptide competes with other polypeptides, for example, as determined by competitive ELISA.

[0054] The present invention relates to a method for determining competing substances, such as polypeptides, that compete with the polypeptides described herein, wherein the polypeptides described herein are represented by any one of SEQ ID NOs: 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8, wherein the polypeptides described herein compete with or cross-block competing substances, such as polypeptides, for binding to MMP13, for example, human MMP13 (SEQ ID NO: 115), wherein the binding of the competing substance to MMP13 is reduced by at least 5%, for example, 10%, 20%, 30%, 40%, 50%, or more, for example, 80%, 90%, or 100%, in the presence of the polypeptides of the present invention compared to binding to MMP13 in the absence of the polypeptides of the present invention (i.e., substantially undetectable in a particular assay). Competition and cross-blocking can be determined by any means known in the art, such as competitive ELISA. In one aspect, the present invention relates to a polypeptide, wherein the polypeptide cross-blocks the binding of at least one polypeptide represented by SEQ ID NOs: 111, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8 to MMP13, and / or the binding of the polypeptide to MMP13 is cross-blocked by at least one polypeptide represented by SEQ ID NOs: 111, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8.

[0055] The present invention also relates to a competing substance that competes with the polypeptides described herein, such as SEQ ID NOs: 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8, wherein the competing substance competes with or cross-blocks the polypeptides described herein for binding to MMP13, wherein the binding of the polypeptide of the present invention to MMP13 is reduced in the presence of the competing substance by at least 5%, e.g., 10%, 20%, 30%, 40%, 50%, or more, e.g., 80%, or even more, e.g., at least 90% or 100%, compared to the binding of the polypeptide of the present invention to MMP13 in the absence of the competing substance (i.e., substantially undetectable in a particular assay). In one aspect, the present invention relates to a polypeptide, such as one of SEQ ID NOs: 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8, which cross-blocks and / or blocks the binding of the polypeptide to MMP13. The present invention relates to a polypeptide that is cross-blocked by at least one of the polypeptides of the present invention, such as 4, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8, wherein the polypeptide preferably comprises at least one VH, VL, dAb, or immunoglobulin single variable domain (ISVD) that specifically binds to MMP13, wherein the binding to MMP13 modulates the activity of MMP13.

[0056] "MMP13 activity" and "activity of MMP13" (these terms are used interchangeably herein) include, but are not limited to, protease activity (also called proteinase or peptidase activity) and endopeptidase activity, and binding to substrates by, for example, hemopexin-like domains and peptidoglycan-binding domains. MMP13 activity includes binding to and / or proteolysis of substrates such as aggrecan, collagen II, collagen I, collagen III, collagen IV, collagen IX, collagen X, collagen XIV, and gelatin. As used herein, proteolysis is the breakdown of a protein into smaller polypeptides or amino acids by hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain. In the context of the present invention, “modulate” or “regulate” generally means a change in the activity of MMP13 as measured using a suitable in vitro, cellular, or in vivo assay (e.g., those referenced herein). In particular, “modulate” or “regulate” may mean reducing or inhibiting the activity of MMP13 as measured using a suitable in vitro, cellular, or in vivo assay (e.g., those referenced herein) by 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 the activity of MMP13 in the same assay, under the same conditions, but without the ISVD or polypeptide of the present invention. Therefore, the present invention relates to a polypeptide described herein, wherein the polypeptide modulates, and preferably inhibits, the activity of MMP13.

[0057] Accordingly, the present invention relates to polypeptides described herein, wherein the polypeptide inhibits the protease activity of MMP13, thereby inhibiting the proteolysis of substrates such as aggrecan, collagen II, collagen I, collagen III, collagen IV, collagen IX, collagen X, collagen XIV and / or gelatin. Therefore, the present invention relates to the polypeptide described herein, wherein the polypeptide blocks the binding of MMP13 to a substrate such as aggrecan, collagen II, collagen I, collagen III, collagen IV, collagen IX, collagen X, collagen XIV and / or gelatin, wherein the collagen is preferably collagen II.

[0058] In one aspect, the present invention relates to the polypeptides described herein, wherein the polypeptides block the binding of MMP13 to collagen and / or aggrecan by at least 20%, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, as determined by, for example, an ELISA-based competitive assay (see Howes et al. 2014 J. Biol. Chem. 289:24091-24101). In one aspect, the present invention relates to a polypeptide described herein, wherein the polypeptide antagonizes or inhibits the activity of MMP13, for example, (i) protease activity, preferably cleavage of aggrecan and / or collagen (wherein the collagen is preferably collagen II); or (ii) binding of collagen to the hemopexin-like domain. Accordingly, the present invention relates to the polypeptide described herein, wherein the polypeptide inhibits the protease activity of MMP13 by any suitable method known in the art, for example, a competitive assay, or as determined in the Examples section, preferably by at least 5%, for example 10%, 20%, 30%, 40%, 50%, or more, for example at least 60%, 70%, 80%, 90%, 95%, or more.

[0059] ISVD Unless otherwise specified, the terms “immunoglobulin” and “immunoglobulin sequence” are used herein to refer to a full-size antibody, both its individual chains, and all its parts, domains, or fragments (each, an antigen-binding domain or fragment, e.g., V HH Domain or V H / V L It is used as a general term that includes, but is not limited to, domains, etc.

[0060] As used herein, the term “domain” (of a polypeptide or protein) refers to a folded protein structure that has the ability to maintain its tertiary structure independently of the rest of the protein. Generally, domains are involved in the individual functional properties of a protein and can often be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or domain. As used herein, the term “immunoglobulin domain” refers to a globular region of an antibody chain (e.g., the chains of a conventional four-chain or heavy-chain antibody), or a polypeptide essentially derived from such a globular region. The immunoglobulin domain is characterized by its ability to retain the immunoglobulin folds characteristic of the antibody molecule, which consist of a two-layer sandwich of approximately seven antiparallel β-chains arranged in two β-sheets, optionally stabilized by conserved disulfide bonds.

[0061] As used herein, the term “immunoglobulin variable domain” means an immunoglobulin domain essentially consisting of four “framework regions,” which are referred to herein and herein as “framework region 1” or “FR1”; “framework region 2” or “FR2”; “framework region 3” or “FR3”; and “framework region 4” or “FR4”; the framework regions are interrupted by three “complementarity-determining regions” or “CDR,” which are referred herein and herein herein as “complementarity-determining region 1” or “CDR1”; “complementarity-determining region 2” or “CDR2”; and “complementarity-determining region 3” or “CDR3”; respectively. Thus, the general structure or sequence of an immunoglobulin variable domain may be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain that confers specificity to an antigen to an antibody by possessing an antigen-binding site. In preferred embodiments of all aspects of the present invention, the immunoglobulin monovariate domain (ISVD) according to the present invention preferably comprises, or essentially comprises, four framework regions (FR1-FR4, respectively) and three complementarity-determining regions CDR1, CDR2, and CDR3 of the general structure outlined above. Preferred framework sequences are outlined, for example, in Table A-2 below and can be used in the ISVD of the present invention. Preferably, the CDRs shown in Table A-2 correspond to the respective framework regions of the same ISVD construct.

[0062] The term “Immunoglobulin single variable domain” (hereinafter abbreviated as “ISVD” or “ISV”) is used interchangeably with “single variable domain” and defines a molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain. Therefore, an immunoglobulin single variable domain is defined separately from “conventional” immunoglobulins or fragments thereof, where two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, conventional immunoglobulins have heavy chain variable domains (V H ) and light chain variable domain (V L ) interact to form an antigen-binding site. In the latter case, V H and V L Both complementarity-determining regions (CDRs) contribute to the antigen-binding site; that is, a total of six CDRs are involved in the formation of the antigen-binding site.

[0063] Considering the above definition, the antigen-binding domain of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or Fv fragments such as Fab fragments, F(ab')2 fragments, disulfide-linked Fv or scFv fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art) is not usually considered a single immunoglobulin variable domain, because in these cases, binding to each epitope of the antigen is usually not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains, such as light-chain and heavy-chain variable domains, i.e., the V of immunoglobulin domains that jointly bind to the epitopes of each antigen. H -V L Because they arise from pairs. In contrast, ISVD can specifically bind to antigen epitopes without pairing with additional immunoglobulin variable domains. The binding site of ISVD is a single V HH , V H or V L It is formed by domains. Therefore, the antigen-binding site of ISVD is formed by three or fewer CDRs.

[0064] Therefore, a single variable domain is a light chain variable domain sequence (for example, V L Sequence) or a suitable fragment thereof; or heavy chain variable domain sequence (e.g., V H Array or V HH It may be a sequence or a suitable fragment thereof, provided that it can form a single antigen-binding unit (i.e., a functional antigen-binding unit; which essentially consists of a single variable domain, and it is not necessary for a single antigen-binding domain to interact with another variable domain to form a functional antigen-binding unit). In one aspect of the present invention, ISVD is a heavy chain variable domain sequence (for example, V H The sequence is; more specifically, ISVD may be a heavy chain variable domain sequence derived from a conventional quadruple-chain antibody, or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0065] For example, ISVD may be a (single) domain antibody (or a peptide suitable for use as a (single) domain antibody), a "dAb" or a dAb (or a peptide suitable for use as a dAb), or a nanobody (as defined herein, including but not limited to VHH); another single variable domain, or a suitable fragment of any of these. In particular, ISVD may be Nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody® and Nanobodies® are registered trademarks of Ablynx NV.] A general description of nanobody is provided in the detailed description below and in the prior art cited herein, e.g., WO 08 / 020079 (page 16).

[0066] VHH, V H "V" is also known as the H domain, VHH antibody fragment, and VHH antibody. HHThe term "domain" has originally been described as the antigen-binding immunoglobulin (variable) domain of "heavy chain antibodies" (i.e., "antibodies lacking a light chain"; Hamers-Casterman et al. 1993 Nature 363: 446-448). HH The reason these variable domains were selected is that they are the heavy chain variable domains present in conventional quadruple-chain antibodies (referred to as "V" in this specification). H From the light chain variable domain (referred to as the "VH domain") and the light chain variable domain present in conventional quadruple-chain antibodies (referred to herein as "VH domain"), and from the light chain variable domain (referred to herein as "VH domain") LThis is to distinguish it from a "domain" or "VL domain." For details on VHH and nanobodies, see the review by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001) and the following patent applications mentioned as general background technology: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; WO of Vlaams Instituut voor Biotechnologie (VIB) 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531 by Algonomics NV and Ablynx NV; WO 01 / 90190 by National Research Council of Canada; WO 03 / 025020 (= EP 1433793) by Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO See also the further published patent applications by Ablynx NV, 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825. See also the further prior art referenced in these applications, particularly the list of references on pages 41-43 of international application WO 06 / 040153; that list and references are incorporated herein by reference. As described in these references, nanobodies (particularly VHH sequences and partially humanized nanobodies) are characterized in particular by the presence of one or more “Hallmark residues” in one or more framework sequences.Further descriptions of nanobodies, including humanization and / or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or “nanobody fusions,” multivalent constructs (including non-limiting examples of linker sequences), and different modifications and preparations for extending the half-life of nanobodies, can be found, for example, in WO 08 / 101985 and WO 08 / 142164. For a further general description of nanobodies, see the prior art cited herein, for example, in WO 08 / 020079 (page 16).

[0067] In particular, the framework sequences present in the MMP13 binders of the present invention, such as the ISVD and / or polypeptides of the present invention, may contain one or more hallmark residues (e.g., as defined in WO 08 / 020079 (Tables A-3 to A-8)) so that the MMP13 binders of the present invention are nanobodies. Some preferred but non-limiting examples of such framework sequences (or suitable combinations thereof) will become apparent from further disclosures herein (e.g., see Table A-2). In general, nanobodies (especially V HH Sequences and partially humanized nanobodies can be particularly characterized by the presence of one or more "Hallmark residues" in one or more framework sequences (as further described, e.g., in WO 08 / 020079, p. 61, line 24 to p. 98, line 3).

[0068] More specifically, the present invention provides an MMP13 conjugate comprising at least one immunoglobulin monovariable domain having the following (general) amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the ISVD is: i) At least one of the amino acid sequences of SEQ ID NOs. 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, or 8 (see Table A-1) has at least 80%, more preferably 90%, and even more preferably 95% amino acid identity, where amino acid residues forming CDR sequences are ignored for the purpose of determining the degree of amino acid identity. In this regard, also refer to Table A-2, which lists the framework 1 sequences (SEQ ID NOs. 67-79), framework 2 sequences (SEQ ID NOs. 80-87 and 108), and framework 3 sequences (SEQ ID NOs. 88-99 and 113-114) of the immunoglobulin monovariate domains of SEQ ID NOs. 1-22 and 109-112; or, ii) Combinations of framework arrays shown in Table A-2; And here: iii) Preferably, one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering are selected from hallmark residues, for example, those listed in Tables A-3 to A-8 of WO 08 / 020079.

[0069] The MMP13 conjugates of the present invention, such as the ISVD and / or polypeptide of the present invention, may also comprise specific mutant / amino acid residues described in the following concurrently pending U.S. provisional applications, all titled “Improved Immunoglobulin Variable Domains”: U.S. 61 / 994552 filed 16 May 2014; U.S. 61 / 014,015 filed 18 June 2014; U.S. 62 / 040,167 filed 21 August 2014; U.S. 62 / 047,560 filed 8 September 2014 (all assigned to Ablynx NV). In particular, the MMP13 binders of the present invention, such as the ISVD and / or polypeptide of the present invention, may preferably contain: (i) K or Q at position 112; or (ii) K or Q at position 110 in combination with V at position 11; or (iii) T at position 89; or (iv) L at position 89 and K or Q at position 110; or (v) V at position 11 and L at position 89; or an appropriate combination of (i) to (v).

[0070] As also described in the concurrently pending U.S. provisional application, if the MMP13 binder of the present invention, such as the ISVD and / or polypeptide of the present invention, contains a mutation according to one (or a suitable combination thereof) of (i) to (v) above: - The amino acid residue at position 11 is preferably selected from L, V, or K (and V is most preferred); and / or - The amino acid residue at position 14 is preferably appropriately selected from A or P; and / or - The amino acid residue at position 41 is preferably appropriately selected from A or P; and / or - The amino acid residue at position 89 is preferably appropriately selected from T, V, or L; and / or - The amino acid residue at position 108 is preferably appropriately selected from Q or L; and / or - The amino acid residue at position 110 is preferably appropriately selected from T, K, or Q; and / or - The amino acid residue at position 112 is preferably appropriately selected from S, K, or Q.

[0071] As stated in the aforementioned concurrently pending U.S. provisional application, the aforementioned mutation is effective in preventing or reducing the binding of so-called “existing antibodies” to the immunoglobulins and compounds of the present invention. For this purpose, the MMP13 conjugates of the present invention, such as the ISVD and / or polypeptide of the present invention, may also (optionally in combination with the aforementioned mutation) comprise a C-terminal extension (X)n (wherein n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)), for which the aforementioned U.S. provisional application and WO 12 / 175741 are again referenced. In particular, the MMP13 binders of the present invention, such as ISVD and / or polypeptides of the present invention, may include C-terminal elongation if they form the C-terminus of a protein, polypeptide, or other compound or construct containing these (again, further described in the aforementioned U.S. Provisional Application and WO 12 / 175741).

[0072] The MMP13 binder of the present invention may be an immunoglobulin derived from any suitable source in any suitable form, for example, an immunoglobulin monovariate domain, for example, naturally occurring V HH Sequences (i.e., derived from suitable species of camelid) or synthetic or semi-synthetic amino acid sequences including, but not limited to, “humanized” (as defined herein) nanobodies or VHH sequences, “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as nanobodies obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or native immunoglobulin sequences), CDR grafting, veneering, combination of fragments derived from different immunoglobulin sequences, PCR assembly using overlap primers, and similar techniques for manipulating immunoglobulin sequences known to those skilled in the art; or any suitable combination of any of the foregoing, further described herein. Also, immunoglobulins V HHIf the immunoglobulin includes a sequence, the immunoglobulin may be appropriately humanized as described further herein to provide one or more further (partially or completely) humanized immunoglobulins of the present invention. Similarly, if the immunoglobulin includes a synthetic or semi-synthetic sequence (such as a partially humanized sequence), the immunoglobulin may be optionally further appropriately humanized to provide one or more further (partially or completely) humanized immunoglobulins of the present invention, again as described herein.

[0073] "Domain antibodies" are also known as "Dab," "Domain Antibodies," and "dAbs" ("Domain Antibodies" and "dAbs" are trademarks used by the GlaxoSmithKline group), and 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 of Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammals, particularly human 4-chain antibodies. To bind an epitope as a single antigen-binding domain, i.e., without pairing it with a VL or VH domain, a specific selection of such antigen-binding properties is required, for example, by using a library of single human VH or VL domain sequences. Domain antibodies, like VHH, have a molecular weight of approximately 13 to 16 kDa and, if entirely derived from human sequences, do not require humanization for therapeutic use in humans, for example.

[0074] It should also be noted that single variable domains may originate from certain shark species, although this is less desirable in the context of the present invention because they are not of mammalian origin (e.g., the so-called "IgNAR domain," see, for example, WO 05 / 18629). The present invention relates in particular to ISVD, wherein the ISVD is selected from the group consisting of VHH, humanized VHH, and camelid VH. The numbering of amino acid residues in the VHH domain is based on Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) H Following the general numbering of domains, this is done, for example, as applied to the camel VHH domain, as shown in Figure 2 of Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999). H Alternative methods for numbering amino acid residues in a domain can be applied to VHH domains in a similar manner and are known in the art. However, unless otherwise specified, this specification, claims, and drawings follow the Kabat numbering applied to VHH domains as described above.

[0075] V H As is well known in the art with respect to domains and VHH domains, it should be noted that the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions indicated by Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This generally means that Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH domains and VHH domains will typically be in the range of 110-120, and often 112-115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this book.

[0076] With regard to CDRs, as is well known in the art, there are several conventions for defining and describing CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of the structural loop region). See, for example, the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and the claims, CDRs are defined most preferably based on the AbM definition (which is based on Oxford Molecular's AbM antibody modeling software), which is considered the best compromise between the Kabat and Chothia definitions (see: http: / / www.bioinf.org.uk / abs / ). As used herein, 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. In the sense of the present invention, the term “immunoglobulin monovariate domain” or “monovariate domain” includes polypeptides derived from a non-human source, preferably a camelid, preferably a heavy chain antibody of a camelid. As described herein, they may be humanized. Furthermore, the term includes polypeptides derived from a non-camelized source, such as mouse or human, which have been “camelized” as described herein.

[0077] Therefore, ISVDs such as domain antibodies and nanobodies (including VHH domains) may be subject to humanization. In particular, humanized ISVDs such as nanobodies (including VHH domains) may be ISVDs as generally defined herein, but where at least one amino acid residue (in particular, at least one of the framework residues) is present, which is a humanization substitution (as defined herein) and / or a corresponding one. Potentially useful humanization substitutions are naturally occurring VHH The sequence of the framework region of the sequence is one or more closely related human V H This can be confirmed by comparing the sequence with the corresponding framework sequence, and then one or more potentially useful humanization substitutions (or combinations thereof) thus determined are identified in V HH The sequence may be introduced (by any mode known in itself, as further described herein), resulting in the humanized V HH The sequences may be tested for affinity to the target, stability, ease and level of expression, and / or other desired properties. In this way, other suitable humanized substitutions (or suitable combinations thereof) can be determined by those skilled in the art based on the disclosure herein, through a limited degree of trial and error. Furthermore, based on the foregoing, ISVDs (or framework regions thereof), such as nanobodies (including VHH domains), may be partially or fully humanized.

[0078] Another particularly preferred class of ISVD in the present invention is naturally occurring V H It includes ISVDs that have amino acid sequences corresponding to the domain's amino acid sequence, but which are "camelized," meaning that the amino acid sequence is derived from naturally occurring V from conventional quadruple-chain antibodies. H One or more amino acid residues in the amino acid sequence of the domain are V of the heavy chain antibody HH This is done by substitution with one or more amino acid residues at corresponding positions within the domain. This can be done in a manner known in itself and will be apparent to those skilled in the art, for example, based on the description herein. Such “camelization” substitution is preferably performed as defined herein, V H -V L It is inserted at amino acid positions that form and / or are present at interfaces, and / or at so-called camelid hallmark residues (see also, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, it is used as a starting material or starting point for generating or designing camelid immunoglobulin monovariable domains. HThe sequence is preferably from mammals. H array, more preferably V H Human V sequences such as 3 H It is a sequence. However, such camelid immunoglobulin single variable domains of the present invention can be obtained in any suitable manner known by themselves, and therefore, natural V H It should be noted that this is not strictly limited to polypeptides obtained using polypeptides containing domains as starting materials. See, for example, 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).

[0079] For example, as further described herein, "humanization" and "camelization" can both be carried out as follows: naturally occurring V HH Domain or V H Provide a nucleotide sequence encoding each domain, and then, in a manner known by itself, modify one or more codons in the nucleotide sequence so that the new nucleotide sequence encodes the “humanized” or “camelized” ISVD of the present invention, respectively. This nucleic acid can then be expressed in a manner known by itself to provide the desired ISVD of the present invention. Alternatively, naturally occurring V HH Domain or V H Based on the amino acid sequence of each domain, the desired humanized or camelized ISVD of the present invention can be designed and synthesized de novo using self-known peptide synthesis techniques. HH Domain or V HBased on the amino acid sequence or nucleotide sequence of each domain, nucleotide sequences encoding the desired humanized or camelized ISVD of the present invention can be designed, and then synthesized de novo using nucleic acid synthesis techniques known to the present, and subsequently the nucleic acids thus obtained can be expressed in a manner known to the present to provide the desired ISVD of the present invention.

[0080] 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, which result in improved affinity for the resulting ISVD for each antigen compared to its respective parent molecule. The affinity-matured ISVD molecules of the present invention can be prepared by methods known in the art, for example, as described below: 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).

[0081] V H , V L , V HHThe process of designing / selecting and / or preparing a polypeptide, starting from an ISVD such as a domain antibody or nanobody, is also referred to herein as “formatting” the ISVD; and an ISVD that makes up part of a polypeptide is said to be a “formatted” or “that formatted” polypeptide. Examples of how an ISVD may be formatted and examples of such formats will be apparent to those skilled in the art based on the disclosure herein; and such formatted immunoglobulin single variable domains form a further aspect of the present invention. Preferred CDRs are shown in Table A-2.

[0082] In particular, the present invention relates to the ISVD described herein, wherein the ISVD is specifically coupled to MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where, (i) CDR1 is selected from the following group: (a) Sequence IDs 27, 28, 25, 26, 23, 29, 30, 31, 32, 33, 34, 35, 36 and 24; and (b) Amino acid sequences having one, two, or three amino acid differences from sequence numbers 27, 28, 25, 26, 23, 29, 30, 31, 32, 33, 34, 35, 36 and 24; (ii) CDR2 is selected from the following group: (c) Sequence IDs 42, 43, 40, 41, 37, 44, 45, 46, 47, 48, 49, 50, 51, 38 and 39; and (d) Amino acid sequences having one, two, or three amino acid differences from sequence numbers 42, 43, 40, 41, 37, 44, 45, 46, 47, 48, 49, 50, 51, 38 and 39, and (iii) CDR3 is selected from the following group: (e) Sequence IDs 56, 107, 57, 54, 106, 55, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 53; and (f) Amino acid sequences having 1, 2, 3 or 4 amino acid differences from sequence numbers 56, 107, 57, 54, 106, 55, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 53.

[0083] In particular, the present invention relates to the ISVD described herein, wherein the ISVD is specifically coupled to MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where, (i) CDR1 is selected from the following group: (a) Sequence ID 23; and (b) An amino acid sequence having one amino acid difference from sequence number 23, where Y is changed to R at position 7; (ii) CDR2 is selected from the following group: (c) Sequence ID 37; and (d) an amino acid sequence having one, two, or three amino acid differences from sequence number 37, where -At position 4, V is changed to T; -At position 5, G is changed to A; and / or -At position 9, N has been changed to H; (iii) CDR3 is selected from the following group: (e) Sequence ID 52; and (f) An amino acid sequence having one amino acid difference from sequence number 52, where Y is changed to S at position 6.

[0084] In particular, the present invention relates to the ISVD described herein, wherein the ISVD is specifically coupled to MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where, (i) CDR1 is sequence number 26; (ii) CDR2 is sequence number 41; and (iii) CDR3 is selected from the following group: (e) Sequence ID 55; and (f) an amino acid sequence having one or two amino acid differences from sequence number 55, where -At position 8, N is changed to Q or S, and / or -At position 19, N is changed to V or Q.

[0085] In particular, the present invention relates to the ISVD described herein, wherein the ISVD is specifically coupled to MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where, (i) CDR1 is sequence number 28; (ii) CDR2 is sequence number 43; and (iii) CDR3 is selected from the following group: (e) Sequence ID 57; and (f) an amino acid sequence having 1, 2, 3 or 4 amino acid differences from sequence number 57, where -At position 10, D is changed to E, G, A, P, T, R, M, W, or Y; -At position 16, M is changed to A, R, N, D, E, Q, Z, G, I, L, K, F, P, S, W, Y, or V; -At position 17, D is changed to A, R, N, C, E, Q, Z, G, H, I, L, K, M, S, T, W, Y, or V; and / or -At position 18, Y is changed to A, R, N, D, C, E, Q, Z, G, H, I, L, K, M, F, P, S, T, W, or V.

[0086] In particular, the present invention relates to the ISVD described herein, wherein the ISVD is specifically coupled to MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where, -CDR1 is selected from the group consisting of sequence numbers 27, 28, 25, 26, 23, 29, 30, 31, 32, 33, 34, 35, 36 and 24; -CDR2 is selected from the group consisting of Sequence IDs 42, 43, 40, 41, 37, 44, 45, 46, 47, 48, 49, 50, 51, 38 and 39; and -CDR3 is selected from the group consisting of sequence numbers 56, 107, 57, 54, 106, 55, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 53.

[0087] In particular, the present invention relates to the ISVD described herein, wherein the ISVD is specifically coupled to MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), wherein the ISVD is selected from the group of ISVDs, where: -CDR1 is sequence number 27, CDR2 is sequence number 42, and CDR3 is sequence number 56; -CDR1 is sequence number 28, CDR2 is sequence number 43, and CDR3 is sequence number 107; -CDR1 is sequence number 28, CDR2 is sequence number 43, and CDR3 is sequence number 57; -CDR1 is sequence number 25, CDR2 is sequence number 40, and CDR3 is sequence number 54; -CDR1 is sequence number 26, CDR2 is sequence number 41, and CDR3 is sequence number 106; -CDR1 is sequence number 26, CDR2 is sequence number 41, and CDR3 is sequence number 55; -CDR1 is sequence number 23, CDR2 is sequence number 37, and CDR3 is sequence number 52; -CDR1 is sequence number 26, CDR2 is sequence number 48, and CDR3 is sequence number 62; -CDR1 is sequence number 26, CDR2 is sequence number 41, and CDR3 is sequence number 63; -CDR1 is sequence number 29, CDR2 is sequence number 44, and CDR3 is sequence number 58; -CDR1 is sequence number 30, CDR2 is sequence number 45, and CDR3 is sequence number 58; -CDR1 is sequence number 31, CDR2 is sequence number 46, and CDR3 is sequence number 59; -CDR1 is sequence number 32, CDR2 is sequence number 47, and CDR3 is sequence number 60; -CDR1 is sequence number 33, CDR2 is sequence number 41, and CDR3 is sequence number 61; -CDR1 is sequence number 34, CDR2 is sequence number 49, and CDR3 is sequence number 64; -CDR1 is sequence number 35, CDR2 is sequence number 50, and CDR3 is sequence number 65; -CDR1 is sequence number 36, CDR2 is sequence number 51, and CDR3 is sequence number 66; -CDR1 is sequence number 23, CDR2 is sequence number 39, CDR3 is sequence number 53; and -CDR1 is sequence number 24, CDR2 is sequence number 38, and CDR3 is sequence number 52.

[0088] In particular, the present invention relates to the ISVD described herein, wherein the ISVD is specifically coupled to MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), where CDR1 is sequence number 27, CDR2 is sequence number 42, and CDR3 is sequence number 56. In particular, the present invention relates to an ISVD as described herein, wherein the ISVD is specifically coupled to an MMP13 and essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), wherein the ISVD is selected from the group consisting of sequence numbers 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, and 8.

[0089] Without limitation, the immunoglobulin monovariable domains of the present invention can be used as “building blocks” for polypeptide preparation, and it will be understood that these may optionally include one or more further immunoglobulin monovariable domains that can function as building blocks (i.e., for the same or a different epitope on MMP13 and / or for one or more other antigens, proteins, or targets other than MMP13).

[0090] polypeptide The polypeptide of the present invention comprises at least one ISVD bound to an MMP, preferably an MMP13, for example, two ISVDs bound to an MMP13, and preferably at least one ISVD bound to aggrecan, more preferably two ISVDs bound to aggrecan. In the polypeptide of the present invention, the ISVDs may be directly linked or linked via a linker. Even more preferably, the polypeptide of the present invention comprises a C-terminal elongation. As detailed below, the C-terminal elongation essentially prevents / removes the binding of existing antibodies / factors in most samples of human subjects / patients. The C-terminal elongation is located at the C-terminus of the last amino acid residue (usually a serine residue) of the last (most C-terminal) ISVD.

[0091] As will be further detailed herein, ISVD is V HH , V H or V L It may originate from the domain, however ISVD is V in the polypeptide of the present invention. H and V L They are selected so as not to form complementary pairs of domains. Nanobody, V HH , and humanized V HH It is unusual in that it originates from natural camel antibodies that lack light chains, and in fact these domains associate with the light chains of camelids to form complementary V HH and V L It is not possible to form a pair. Therefore, the polypeptide of the present invention does not contain complementary ISVD and / or complementary V, for example. H / V LThey do not form complementary ISVD pairs such as pairs. Generally, polypeptides or constructs containing or essentially consisting of a single building block, such as 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 herein as “polyvalent” polypeptides or constructs, and the building blocks / ISVDs present in such polypeptides or constructs are referred herein as “polyvalent format.” For example, a “divalent” polypeptide may contain two ISVDs optionally linked via linker sequences, 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.

[0092] In a polyvalent polypeptide, two or more ISVDs may be identical or different, may be directed to the same antigen or antigenic determinant (e.g., to the same portion of an epitope or to different portions of an epitope), or alternatively, may be directed to different antigens or antigenic determinants, or any suitable combination thereof. Polypeptides and constructs comprising at least two building blocks (e.g., ISVDs), wherein at least one building block is directed to a first antigen (i.e., MMP13) and at least one building block is directed to a second antigen (i.e., one different from MMP13), are also called “multispecific” polypeptides and constructs, and the building blocks present in such polypeptides and constructs (e.g., ISVDs) are also referred to herein as “multispecific format.” Therefore, for example, the “bispecific” polypeptide of the present invention is a polypeptide comprising at least one ISVD directed to a first antigen (i.e., MMP13) and at least one ISVD directed to a second antigen (i.e., different from MMP13), while the “triplespecific” polypeptide of the present invention is a polypeptide comprising at least one ISVD directed to a first antigen (i.e., MMP13), at least one further ISVD directed to a second antigen (i.e., different from MMP13), and at least one further ISVD directed to at least a third antigen (i.e., different from both MMP13 and the second antigen), and so on.

[0093] In one aspect, the present invention relates to a polypeptide comprising two or more ISVDs that specifically bind to MMP13, wherein: a) At least the "first" ISVD specifically binds to a first antigenic determinant, epitope, portion, domain, subunit or conformation of MMP13; preferably, the "first" ISVD that specifically binds to MMP13 is selected from the group consisting of SEQ ID NOs: 111, 11, 110, 10, 112, 12, 109, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22; and here, b) At least the “second” ISVD specifically binds to a second antigenic determinant, epitope, partial, domain, subunit, or conformation of MMP13, which is distinct from the first antigenic determinant epitope, partial, domain, subunit, or conformation, and preferably specifically binds to MMP13. The “second” ISVD is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8. In one aspect, the present invention relates to a polypeptide comprising two or more ISVDs that specifically bind to MMP13, selected from the group consisting of SEQ ID NOs. 160 to 165, preferably SEQ ID NO. 160 (see Table A-3).

[0094] "Multiple paratopic" polypeptides and "multiple paratopic" constructs, such as "dual paratopic" polypeptides or constructs and "triple paratopic" polypeptides or constructs, each contain or consist essentially of two or more building blocks, each having a different paratope.

[0095] Accordingly, the ISVD of the present invention that binds to MMP13 may be in an essentially isolated form (as defined herein), or may form part of a construct or polypeptide comprising or essentially consisting of one or more ISVDs that bind to MMP13, and optionally comprising one or more further amino acid sequences (all optionally linked via one or more suitable linkers). The present invention relates to a polypeptide or construct comprising or essentially consisting of at least one ISVD according to the present invention, such as one or more ISVDs of the present invention (or suitable fragments thereof) that bind to MMP13. One or more ISVDs of the present invention are used as building blocks in such polypeptides or constructs, providing monovalent, polyvalent, or multiple paratopic polypeptides or constructs of the present invention, all as described herein. Thus, the present invention also relates to polypeptides that are monovalent constructs comprising or essentially comprising the monovalent polypeptides or ISVDs of the present invention.

[0096] Accordingly, the present invention also relates to polypeptides or constructs that are polyvalent polypeptides or polyvalent constructs, such as divalent or trivalent polypeptides or constructs that contain or essentially consist of two or more ISVDs of the present invention (for polyvalent and multispecific polypeptides containing one or more VHH domains and their preparation, see, for example, Conrath et al. (J. Biol. Chem. 276: 7346-7350, 2001), as well as, for example, WO96 / 34103, WO99 / 23221 and WO 2010 / 115998).

[0097] In one aspect, in its simplest form, the polyvalent polypeptide or construct of the present invention is a divalent polypeptide or construct of the present invention comprising a first ISVD such as a nanobody directed to MMP13, and the same second ISVD such as a nanobody directed to MMP13, wherein the first and second ISVDs such as nanobody may be optionally linked via linker sequences (as defined herein). In its simplest form, the polyvalent polypeptide or construct of the present invention is a trivalent polypeptide or construct of the present invention comprising a first ISVD such as a nanobody directed to MMP13, the same second ISVD such as a nanobody directed to MMP13, and the same third ISVD such as a nanobody directed to MMP13, wherein the first, second and third ISVDs such as nanobody may be optionally linked via one or more, in particular two, linker sequences. In one aspect, the present invention relates to a polypeptide or construct comprising, or essentially comprising, at least two ISVDs according to the present invention, such as two, three, or four ISVDs (or suitable fragments thereof) bound to MMP13. The two or more ISVDs may optionally be linked via one or more peptide linkers.

[0098] In another aspect, the polyvalent polypeptide or construct of the present invention may be a bispecific polypeptide or construct of the present invention comprising a first ISVD, such as a nanobody directed to MMP13, and a second ISVD, such as a nanobody directed to a second antigen, such as aggrecan, wherein the first and second ISVDs, such as nanobodies, may optionally be linked via linker sequences (as defined herein); on the other hand, the polyvalent polypeptide or construct of the present invention may also be a triplicate polypeptide or construct of the present invention comprising a first ISVD, such as a nanobody directed to MMP13, a second ISVD, such as a nanobody directed to a second antigen, such as aggrecan, and a third ISVD, such as a nanobody directed to a third antigen, wherein the first, second and third ISVDs, such as nanobodies, may optionally be linked via one or more, particularly two, linker sequences. The present invention further relates to a polyvalent polypeptide comprising, or (essentially) comprising, at least one ISVD (or a suitable fragment thereof) that binds to MMP13, preferably human MMP13, and at least one additional ISVD, such as an ISVD that binds to aggrecan.

[0099] Particularly preferred trivalent, bispecific polypeptides or constructs according to the present invention are those shown in the examples and Table A-3 described herein. In a preferred aspect, the polypeptide or construct of the present invention comprises or essentially consists of at least two ISVDs, wherein the at least two ISVDs may be identical or different, but at least one of the ISVDs is directed toward MMP13.

[0100] Two or more ISVDs present in the polyvalent polypeptide or construct of the present invention are light chain variable domain sequences (e.g., V L (array) or heavy chain variable domain sequence (e.g., V HThey may consist of a heavy chain variable domain sequence derived from a conventional quadruple-chain antibody, or a heavy chain variable domain sequence derived from a heavy chain antibody. Preferably they may be a domain antibody (or a peptide suitable for use as a domain antibody), a single-domain antibody (or a peptide suitable for use as a single-domain antibody), or a "dAb" (or a peptide suitable for use as a dAb), Nanobody® (V HH (including but not limited to), humanized V HH Sequence, camelization V H V obtained by sequence; or affinity maturation HH It consists of a sequence. Two or more immunoglobulin monovariable domains may consist of partially or fully humanized nanobodies or partially or fully humanized VHHs.

[0101] In one aspect of the present invention, the first and second ISVDs present in the multiple paratopic (preferably double or triple paratopic) polypeptide or construct of the present invention do not (cross-)compete with each other for binding to MMP13 and therefore belong to different families. Accordingly, the present invention relates to a multiple paratopic (preferably double paratopic) polypeptide or construct comprising two or more ISVDs, each belonging to a different family. In one aspect, the first ISVD of this multiple paratopic (preferably double paratopic) polypeptide or construct of the present invention does not cross-block the binding of the second ISVD of this multiple paratopic (preferably double paratopic) polypeptide or construct of the present invention to MMP13, and / or the first ISVD is not cross-blocked for binding to MMP13 by the second ISVD. In another aspect, the first ISVD of the multiple paratopic (preferably double paratopic) polypeptide or construct of the present invention cross-blocks the binding of the second ISVD of this multiple paratopic (preferably double paratopic) polypeptide or construct of the present invention to MMP13, and / or the first ISVD is cross-blocked by the second ISVD for binding to MMP13.

[0102] In a particularly preferred aspect, the polypeptide or construct of the present invention comprises or essentially consists of three or more ISVDs, of which at least two ISVDs are directed to MMP13. It will be understood that the at least two ISVDs directed to MMP13 may be identical or different, may be directed to the same or different epitopes of MMP13, may belong to the same or different epitope bins, and / or may bind to the same or different domains of MMP13.

[0103] In a preferred aspect, the polypeptide or construct of the present invention comprises or essentially consists of at least two ISVDs bound to MMP13, wherein the at least two ISVDs may be identical or different, and are independently selected from the group consisting of SEQ ID NOs: 111, 11, 110, 10, 112, 12, 109, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8, and more preferably... Alternatively, at least two ISVDs are independently selected from the group consisting of sequence numbers 111, 11, 110, 10, 112, 12, 109, 9, and 1, and / or at least one ISVD is selected from the group consisting of sequence numbers 111, 11, 110, 10, 112, 12, 109, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, and at least one ISVD is selected from the group consisting of sequence numbers 1, 2, 3, 4, 5, 6, 7, and 8.

[0104] In a further embodiment, the present invention relates to a multiple paratopic (preferably double paratopic) polypeptide or construct comprising two or more ISVDs directed to MMP13, wherein the ISVDs are conjugated by any one of SEQ ID NOs: 111, 11, 110, 10, 112, 12, 109, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, or conjugated by any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8. In a further aspect, the present invention relates to a polypeptide described herein, wherein the polypeptide has sequence identity of at least 80%, 90%, 95%, or 100% (more preferably at least 95%, and most preferably 100%) with any one of SEQ ID NOs. 160-165 (i.e., 160, 161, 162, 163, 164, or 165) and 176-192 (i.e., 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, or 192), and preferably SEQ ID NO. 192.

[0105] retention This technology addresses the need for more effective treatments for disorders affecting articular cartilage, such as osteoarthritis. Even when administered intraarticularly, the residence time of most drugs to treat affected cartilage is insufficient. The inventors hypothesized that the efficacy of therapeutic agents such as the constructs, polypeptides, and ISVD of the present invention can be significantly increased by binding to a portion that "fixes" the drug within the joint, thereby increasing drug retention, while not interfering with the efficacy of the therapeutic agent (this portion is also referred to herein as the "cartilage fixation protein" or "CAP"). This fixation concept can increase not only the efficacy of the drug but also the operational specificity of the affected joint by reducing toxicity and side effects, thereby increasing the number of potentially useful drugs.

[0106] The final molecular format for clinical use was expected to include one or two building blocks, such as ISVD, that bind to MMP13, and one or more building blocks, such as ISVD, that have a retention mode of action, and possibly further parts. In the Examples section, such a format has been demonstrated to retain both MMP13 binding and therapeutic effects, such as inhibitory activity, as well as retention properties. One or more building blocks, such as ISVD, that have a retention mode of action are any building blocks that have a retention effect in MMP13-related diseases such as ("CAP building blocks"): arthritis, osteoarthritis, spondyloepiphyseal dysplasia, lumbar disc degenerative disease, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans and agricanopathies and metastatic malignancies. "CAP building blocks" are used to guide, fix, and / or retain other building blocks, such as therapeutic building blocks, such as ISVDs that bind to MMP13, in a desired location, such as a joint, where the other therapeutic building blocks exert their effects, such as inhibiting the binding and / or activity of MMP13.

[0107] The inventors further hypothesized that aggrecan binders such as ISVD, which bind to aggrecan, could potentially function as anchors, even though aggrecan is severely glycosylated and degraded in various disorders affecting articular cartilage. Furthermore, considering the costs and extensive testing required in various animal models before a drug enters clinical practice, such aggrecan binders should preferentially exhibit broad species cross-reactivity; for example, the aggrecan binder should bind to aggrecans of various species. Using a variety of innovative immunization, screening, and characterization methods, we were able to identify a range of aggrecan conjugates with excellent selectivity, stability, and specificity that enable extended retention time and activity within the joint. In one aspect, the present invention relates to a method for reducing and / or inhibiting the outflow of a composition, polypeptide, or construct from a joint, the method comprising administering a pharmaceutically active amount of at least one polypeptide, construct, or composition according to the present invention to a person in need thereof.

[0108] In the present invention, the term “reduction and / or inhibition of efflux” means reducing and / or inhibiting the outward flow of a composition, polypeptide, or construct from the joint to the outside. Preferably, efflux is the efflux of the aforementioned composition, polypeptide, or construct within the joint, which is reduced and / or inhibited by at least 10%, for example, at least 20%, 30%, 40%, or 50%, or even more, for example, at least 60%, 70%, 80%, 90%, or 100%, compared to efflux without the presence of the aggrecan binder of the present invention, for example, ISVD bound to aggrecan, under the same conditions. In addition to diseases involving MMP13, such as arthritis, osteoarthritis, spondyloepiphysis, lumbar disc degenerative disease, degenerative joint disease, rheumatoid arthritis, osteochondritis dissecans, aggrecanopathy, and metastasis of malignant tumors, the aggrecan conjugate of the present invention is also expected to be used in a variety of other diseases affecting cartilage, such as arthropathy and chondrodysplasia, arthritis (e.g., osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment), chondrodysplasia, costochondritis, spondyloepiphysis, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis (collectively referred to herein as “agrecan-related diseases”).

[0109] The CAP building block, for example, ISVD that binds to aggrecan, preferably binds to cartilage and / or cartilaginous tissue such as the meniscus. Preferably, the CAP building block is cross-reactive to other species and specifically binds to one or more of the following: human aggrecan (SEQ ID NO: 105), canine aggrecan, bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus aggrecan and / or rhesus aggrecan. The relevant structural information of aggrecan is listed by its (UniProt) accession number, as shown in Table 2 below, for example. A preferred CAP building block is an ISVD-conjugated aggrecan, preferably a human aggrecan, and preferably represented by Sequence ID No. 105 as shown in Table B. Table 2 [Table 2]

[0110] Therefore, the present invention relates to a polypeptide or construct according to the present invention, further comprising at least one CAP building block. Accordingly, the present invention relates to polypeptides or constructs according to the present invention, further comprising at least one ISVD that specifically binds to aggrecan, such as those shown in Table E, preferably selected from ISVDs represented by Sequence IDs 166-168. In particular, the present invention relates to an ISVD that specifically binds to aggrecan, wherein the ISVD essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity determination regions (CDR1 to CDR3, respectively), wherein the ISVD is selected from the group of ISVDs consisting of: (a) CDR1 is sequence number 169, CDR2 is sequence number 170, and CDR3 is sequence number 171; and (b) CDR1 is sequence number 172, CDR2 is sequence number 173, and CDR3 is sequence number 174.

[0111] In one aspect, the present invention relates to a polypeptide described herein, comprising at least two ISVDs that specifically bind to aggrecan. In one aspect, the present invention relates to a polypeptide according to this specification comprising at least two ISVDs that specifically bind to aggrecan, wherein the at least two ISVDs that specifically bind to aggrecan may be the same or different. In one aspect, the present invention relates to a polypeptide described herein, comprising at least two ISVDs that specifically bind to aggrecan, wherein the at least two ISVDs that specifically bind to aggrecan are independently selected from the group consisting of SEQ ID NOs: 166 to 168. In one aspect, the present invention relates to a polypeptide described herein, comprising at least two ISVDs that specifically bind to aggrecan, wherein the at least two ISVDs that specifically bind to aggrecan are represented by SEQ ID NOs: 166-168. In one aspect, the present invention relates to a polypeptide according to this specification comprising an ISVD that specifically binds to aggrecan, wherein the ISVD that specifically binds to aggrecan specifically binds to human aggrecan [SEQ ID NO: 105].

[0112] In one aspect, the present invention relates to the polypeptide described herein, wherein the ISVD that specifically binds to aggrecan specifically binds to human aggrecan (SEQ ID NO: 105), canine aggrecan, bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and / or rhesus monkey aggrecan. In one aspect, the present invention relates to the polypeptide described herein, wherein the ISVD that specifically binds to aggrecan preferably binds to cartilage and / or cartilaginous tissue such as the meniscus. It will be understood that the ISVDs, polypeptides, and constructs of the present invention are preferably stable. The stability of the polypeptides, constructs, or ISVDs of the present invention can be measured by routine assays known to those skilled in the art. Typical assays include, for example, those detailed in the Examples section, in which the activity of the polypeptide, construct, or ISVD is measured, followed by incubation in synovial fluid for a desired period, and then the activity is measured again.

[0113] In one aspect, the present invention relates to ISVDs, polypeptides, or constructs that are stable in synovial fluid (SF) at 37°C for at least 7 days, for example, at least 14 days, 21 days, 1 month, 2 months, or 3 months. In one aspect, the present invention relates to an ISVD, polypeptide, or construct of the present invention that penetrates cartilage to a depth of at least 5 μm, for example, at least 10 μm, 20 μm, 30 μm, 40 μm, 50 μm or more. The desired activity of therapeutic building blocks, such as ISVD bound to MMP13 in the polyvalent polypeptide or construct of the present invention, can be measured by routine assays known to those skilled in the art. Typical assays include (but are not limited to) the GAG ​​release assay detailed in the Examples section.

[0114] Relative affinity may depend on the position of the ISVD within the polypeptide. It will be understood that the order (orientation) of the ISVDs in the polypeptide of the present invention may be selected according to the needs of those skilled in the art. The order of individual ISVDs, and whether the polypeptide contains a linker, is a matter of design choice. With or without a linker, some orientations may provide preferred binding properties compared to others. For example, the order of the first ISVD (e.g., ISVD1) and the second ISVD (e.g., ISVD2) in the polypeptide of the present invention is (N-terminus to C-terminus): (i) ISVD1 (e.g., nanobody 1) - [linker] - ISVD2 (e.g., nanobody 2) - [C-terminus extension]; or (ii) ISVD2 (e.g., nanobody 2) - [linker] - ISVD1 (e.g., nanobody 1) - [C-terminus extension]; (where the parts in square brackets, i.e., the linker and C-terminus extension, are optional). All orientations are encompassed in the present invention. Polypeptides containing ISVD orientations that provide desired binding properties can be readily identified by routine screening, for example, as illustrated in the Examples section. A preferred order is N-terminus to C-terminus: ISVD-[linker]-agrecan-bound to MMP13-ISVD-[C-terminal extension], where the part in square brackets is optional. A further preferred order is N-terminus to C-terminus: ISVD-[linker]-agrecan-bound to MMP13-ISVD-[linker]-agrecan-bound to ISVD-[C-terminal extension], where the part in square brackets is optional. See, for example, Table F.

[0115] Half-life In certain aspects of the present invention, a construct or polypeptide of the present invention may have a portion that confers an increased half-life compared to a corresponding construct or polypeptide of the present invention without it. Some preferred but non-limiting examples of such constructs and polypeptides of the present invention will become apparent to those skilled in the art based on further disclosures herein, and these include, for example: an ISVD or polypeptide of the present invention that is chemically modified to increase its half-life (e.g., by pegylation); an MMP13 binder of the present invention, such as an ISVD and / or polypeptide of the present invention, comprising at least one additional binding site for binding to a serum protein (e.g., serum albumin); or a polypeptide of the present invention comprising at least one ISVD of the present invention linked to at least one portion (in particular at least one amino acid sequence) that extends the half-life of an amino acid sequence of the present invention.Examples of constructs of the present invention, such as polypeptides of the present invention containing such half-life extension portions or ISVDs, will become apparent to those skilled in the art based on further disclosures herein; examples, but not limited to, the following polypeptides: polypeptides in which one or more ISVDs of the present invention are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof), or polypeptides suitably linked to one or more binding units that can bind to serum proteins (examples of serum proteins include, for example, domain antibodies, immunoglobulin monovariate domains suitable for use as domain antibodies, single domain antibodies, immunoglobulins suitable for use as single domain antibodies). The immunoglobulin monovariate domains are immunoglobulin monovariate domains suitable for use as dAbs, or nanobodies capable of binding to serum proteins such as serum albumin (e.g., human serum albumin), serum immunoglobulins such as IgG, or transferrin; see further descriptions and references provided herein; polypeptides in which the amino acid sequence of the present invention is linked to an Fc portion (e.g., human Fc) or a suitable portion or fragment thereof; or polypeptides in which one or more immunoglobulin monovariate domains of the present invention are appropriately linked to one or more small proteins or peptides capable of binding to serum proteins, e.g., the proteins and peptides described below: WO 91 / 01743, WO 01 / 45746, WO 02 / 076489, WO2008 / 068280, WO2009 / 127691 and PCT / EP2011 / 051559.

[0116] In one aspect, the present invention provides constructs or polypeptides, wherein the construct or polypeptide further comprises a serum protein-binding moiety or a serum protein. Preferably, the serum protein-binding moiety binds to serum albumin, such as human serum albumin. In one aspect, the present invention relates to polypeptides described herein, comprising ISVD that binds to serum albumin. In general, a construct or polypeptide of the present invention with an increased half-life preferably has a half-life that is longer than the corresponding construct or polypeptide of the present invention itself, i.e., the half-life without the portion resulting in the increased half-life, at least 1.5 times, preferably at least 2 times, for example at least 5 times, for example at least 10 times or 20 times longer. For example, a construct or polypeptide of the present invention with an increased half-life may have an increased half-life of 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 corresponding construct or polypeptide of the present invention itself, i.e., compared to the case without the portion resulting in the increased half-life, in humans, for example.

[0117] In preferred but non-limiting aspects of the present invention, the constructs and polypeptides of the present invention have, for example in humans, a serum half-life that is increased by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example more than 12 hours, and even more than 24, 48, or 72 hours, compared to the corresponding construct or polypeptide of the present invention itself, i.e., without the portion resulting in an increased half-life. In another preferred but non-limiting aspect of the present invention, such constructs of the present invention, such as polypeptides of the present invention, exhibit a serum half-life in humans of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 72 hours or more. For example, constructs or polypeptides of the present invention may have a serum half-life of at least 5 days (e.g., about 5 to 10 days), preferably at least 9 days (e.g., about 9 to 14 days), more preferably at least about 10 days (e.g., about 10 to 15 days), or at least about 11 days (e.g., about 11 to 16 days), more preferably at least about 12 days (e.g., about 12 to 18 days or more), or 14 days or more (e.g., about 14 to 19 days).

[0118] In a particularly preferred but non-limiting aspect of the present invention, the present invention provides constructs and polypeptides of the present invention comprising, in addition to one or more building blocks that bind to MMP13 and one or more CAP building blocks that potentially bind to aggrecan, for example, ISVDs that bind to serum albumin, in addition to at least one building block that binds to serum albumin, for example, ISVDs that bind to serum albumin, such as human serum albumin as described herein. Preferably, the ISVDs that bind to serum albumin comprise or consist of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), where CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG, and CDR3 is GGSLSR. Preferably, the ISVD that binds to serum albumin 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, Alb135, or Alb223 (see Table D). In one aspect, the present invention relates to constructs of the present invention such as polypeptides containing a serum protein-binding moiety, wherein the serum protein-binding moiety is a non-antibody-based polypeptide.

[0119] Other parts In one aspect, the present invention relates to constructs described herein comprising at least one ISVD or polypeptide and one or more other groups, residues, moieties or binding units. The one or more other groups, residues, moieties or binding units are preferably selected from the group consisting of: polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins, further amino acid residues, tags or other functional parts such as toxins, labels, radiochemicals, etc. In one aspect, as will be discussed below, the present invention relates to constructs of the present invention, such as polypeptides containing a portion that provides an extension of half-life, wherein the portion is PEG. Thus, the present invention also relates to constructs or polypeptides of the present invention that contain PEG.

[0120] Further amino acid residues may or may not alter, modify, or otherwise affect other (biological) properties of the polypeptide of the present invention, and may or may not add further functionality to the polypeptide of the present invention. For example, such amino acid residues may include: a) The N-terminal Met residue may be included, for example, as a result of expression in a heterologous host cell or host organism; b) A signal sequence or leader sequence may be formed during synthesis to direct the secretion of the polypeptide from a host cell (for example, to provide a pre, pro, or prepro form of the polypeptide of the present invention, depending on the host cell used to express the polypeptide of the present invention). Suitable secretion leader peptides will be obvious to those skilled in the art and may be further described herein. Typically, such a leader sequence is ligated to the N-terminus of the polypeptide, but the present invention is not limited thereto in the broadest sense;

[0121] c) A “tag,” i.e., an amino acid sequence or residue that enables or facilitates the purification of a polypeptide, can be formed, for example, using affinity techniques directed to the sequence or residue. The sequence or residue can then be removed (e.g., by chemical or enzymatic cleavage) to provide the polypeptide (for this purpose, the tag may optionally be linked to the amino acid sequence or polypeptide sequence via a cleavable linker sequence, or may contain a cleavable motif). Some preferred but non-limiting examples of such residues are multiple histidine residues, glutathione residues, and myc tags such as AAAEQKLISEEDLNGAA (SEQ ID NO: 175), MYC-HIS tags (SEQ ID NO: 123), or FLAG-HIS6 tags (SEQ ID NO: 124) (see Table B); d) One or more amino acid residues that are functionalized and / or can function as binding sites for functional groups. Suitable amino acid residues and functional groups will be obvious to those skilled in the art and include, but are not limited to, the amino acid residues and functional groups referred herein with respect to the polypeptide derivatives of the present invention.

[0122] Further encompassed in the present invention are constructs and / or polypeptides comprising the ISVD of the present invention, and further comprising other functional components, such as toxins, labels, radiochemicals, etc. Other groups, residues, moieties, or binding units may be, for example, chemical groups, residues, or moieties, which may or may not be biologically and / or pharmacologically active on their own. For example, but not limited to, such groups may be linked to one or more ISVDs or polypeptides of the present invention to provide “derivatives” of the polypeptides or constructs of the present invention. Therefore, in its broadest sense, the present invention also includes constructs and / or polypeptides, which are derivatives of the constructs and / or polypeptides of the present invention. Such derivatives can generally be obtained by modification, and in particular by chemical and / or biological (e.g., enzymatic) modification, of one or more amino acid residues forming the constructs and / or polypeptides of the present invention and / or polypeptides of the present invention.

[0123] Examples of such modifications, as well as examples of amino acid residues in polypeptide sequences that can be modified in this manner (i.e., on the protein backbone, or preferably on the side chains), methods and techniques that can be used for such modifications, and the potential uses and benefits of such modifications will be apparent to those skilled in the art (see also Zangi et al., Nat Biotechnol 31(10):898-907, 2013). For example, such modifications may include the introduction of one or more (functional) groups, residues, or moieties into or on the polypeptide of the present invention (e.g., by covalent bonding or in any other suitable manner), and in particular the introduction of one or more (functional) groups, residues, or moieties that confer one or more desired properties or functionalities to the construct and / or polypeptide of the present invention. Examples of such functional groups will be obvious to those skilled in the art.

[0124] For example, such modifications may include the introduction (e.g., by covalent bonding or in any other suitable manner) of one or more functional moieties; or any combination of two or more of the aforementioned functional moieties, which increase the half-life, solubility and / or absorption of the construct or polypeptide of the present invention, reduce the immunogenicity and / or toxicity of the construct or polypeptide of the present invention, eliminate or reduce undesirable side effects of the construct or polypeptide of the present invention, and / or confer other advantageous properties to the construct or polypeptide of the present invention and / or reduce undesirable properties. Examples of such functional moieties and techniques for introducing them will be apparent to those skilled in the art and generally include all functional moieties and techniques mentioned in the general background art cited above herein, as well as functional moieties and techniques known in themselves for the modification of pharmaceutical proteins; and in particular, for the modification of antibodies or antibody fragments (including ScFv and single-domain antibodies), see, for example, Remington (Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA, 1980). Such functional parts can be linked, for example, directly (e.g., by covalent bonds) to the polypeptide of the present invention, or optionally via appropriate linkers or spacers, which will again be apparent to those skilled in the art.

[0125] One specific example is a derivative polypeptide or construct of the present invention, in which the polypeptide or construct of the present invention is chemically modified to increase its half-life (e.g., by pegylation). This is one of the most widely used methods for extending the half-life and / or reducing the immunogenicity of pharmaceutical proteins and involves the bonding of a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or its derivatives (methoxypoly(ethylene glycol) or mPEG). In general, any suitable form of pegylation, such as the pegylation used in the art for antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv); see, for example, Chapman (Nat. Biotechnol. 54: 531-545, 2002), Veronese and Harris (Adv. Drug Deliv. Rev. 54: 453-456, 2003), Harris and Chess (Nat. Rev. Drug. Discov. 2: 214-221, 2003), and WO 04 / 060965. Various reagents for protein pegylation are also commercially available, for example, from Nektar Therapeutics, USA.

[0126] Preferably, site-directed pegylation via cysteine ​​residues is used (see, for example, Yang et al. (Protein Engineering 16: 761-770, 2003)). For this purpose, for example, PEG can be conjugated to a naturally occurring cysteine ​​residue in the polypeptide of the present invention, the construct or polypeptide of the present invention may be modified to appropriately introduce one or more cysteine ​​residues for PEG conjugation, or an amino acid sequence containing one or more cysteine ​​residues for PEG conjugation may be fused to the N-terminus and / or C-terminus of the construct or polypeptide of the present invention, all of which utilize protein engineering techniques known to those skilled in the art. Preferably, the construct or polypeptide of the present invention has a molecular weight greater than 5,000, for example greater than 10,000 and less than 200,000, for example less than 100,000; for example, PEG having a molecular weight in the range of 20,000 to 80,000 is used.

[0127] Another, and generally less desirable, modification involves N-linked or O-linked glycosylation, typically as part of co-translation and / or post-translational modification, depending on the host cell used for the expression of the polypeptide of the present invention. Further modifications may include the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the polypeptide or construct of the present invention. Suitable labels and techniques for binding, using, and detecting them will be apparent to those skilled in the art and include, but are not limited to, fluorescent labels (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescein) and 152 Fluorescent metals such as Eu, or other lanthanide metals), phosphorescent labels, chemiluminescent labels or bioluminescent labels (e.g., luminal, isoluminol, theromatic acridinium esters, imidazole, acridinium salts, oxalate esters, dioxetane or GFP and its analogues), radioisotopes (e.g., 3 H, 125 I, 32 P, 35 S, 14 C, 51 Cr, 36 Cl, 57 Co, 58 Co, 59 Fe, and 75 (e.g., Se), metals, metal chelates, or metal cations (e.g., metal cations, e.g., 99m Tc, 123 I, 111 In, 131 I, 97 Ru, 67 Cu, 67 Ga, and 68Ga, or other metals or metal cations particularly suitable for use in vivo, in vitro, or in situ diagnostics and imaging, for example ( 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe), as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase). Other suitable labels are obvious to those skilled in the art and include, for example, portions detectable by NMR or ESR spectroscopy.

[0128] Such labeled polypeptides and constructs of the present invention can be used, for example, in in vitro, in vivo, or in situ assays (including immunoassays known in themselves, such as ELISA, RIA, EIA, and other “sandwich assays”), as well as for in vivo diagnostic and imaging purposes, depending on the selection of a specific label. As will be apparent to those skilled in the art, other modifications may involve the introduction of a chelating group to chelate one of the above-mentioned metals or metal cations. Suitable chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0129] Further modifications may include the introduction of a functional moiety, which is part of a specific binding pair, such as a biotin-(strept)avidin binding pair. Using such a functional moiety, the polypeptide of the present invention can be ligated to another protein, polypeptide, or chemical compound that binds to the other half of the binding pair, for example, by the formation of a binding pair. For example, the construct or polypeptide of the present invention can be conjugated to biotin and ligated to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such a conjugated construct or polypeptide of the present invention may be used as a reporter, for example, in a diagnostic system in which a detectable signal generator is conjugated to avidin or streptavidin. Such a binding pair can also be used to ligate, for example, the construct or polypeptide of the present invention to a carrier containing a carrier suitable for a pharmaceutical purpose. One non-limiting example is the liposomal formulation described in Cao and Suresh (Journal of Drug Targeting 8: 257, 2000). Such binding pairs can also be used to link therapeutically active agents to the polypeptides of the present invention.

[0130] Other possible chemical and enzymatic modifications will be obvious to those skilled in the art. Such modifications may also be introduced for research purposes (e.g., to study function-activity relationships). See, for example, Lundblad and Bradshaw (Biotechnol. Appl. Biochem. 26: 143-151, 1997). Preferably, the construct, polypeptide and / or derivative has affinity to MMP13 as defined herein (as further described herein, K D Value (actual or apparent value), K A Value (actual or apparent value), k on Rate or k off Rate, or alternatively IC 50 The values ​​are bound together (i.e., as defined for the polypeptide of the present invention) (appropriately measured and / or displayed as values). Such constructs and / or polypeptides of the present invention, as well as their derivatives, may also be in essentially isolated forms (as defined herein).

[0131] In one aspect, the present invention relates to constructs comprising or essentially comprising the ISVD of the present invention or the polypeptide of the present invention, and further comprising one or more other groups, residues, parts or binding units optionally linked via one or more peptide linkers. In one aspect, the present invention relates to constructs of the present invention in which one or more other groups, residues, moieties or binding units are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins.

[0132] Linker In constructs of the present invention, such as polypeptides of the present invention, two or more building blocks, such as ISVD, and optionally one or more other groups, drugs, agents, residues, moieties, or binding units may be directly linked to one another (e.g., as described in WO 99 / 23221), and / or linked to one or more suitable spacers or linkers, or any combination thereof. Spacers or linkers suitable for use in polyvalent and multispecific polypeptides will be obvious to those skilled in the art and may generally be any linkers or spacers used in the art to link amino acid sequences. Preferably, the linkers or spacers are suitable for use in constructing constructs, proteins, or polypeptides intended for pharmaceutically active use.

[0133] For example, the polypeptide of the present invention is a trivalent, triple-specific polypeptide comprising, for example, one building block such as an ISVD bound to MMP13, a CAP building block such as an ISVD bound to aggrecan, and potentially another building block such as a third ISVD, wherein the first, second, and third building blocks such as ISVD may be optionally linked via one or more, particularly two, linker sequences. The present invention also provides a construct or polypeptide of the present invention comprising a first ISVD bound to MMP13, optionally a second ISVD bound to aggrecan, and / or optionally a third ISVD, and / or optionally a fourth ISVD, wherein the first ISVD and / or optionally the second ISVD and / or optionally the third ISVD and / or optionally the fourth ISVD are linked via linkers, particularly three linkers.

[0134] Some particularly preferred linkers include those used in the art to ligate antibody fragments or antibody domains. These include the linkers mentioned in the general background art above, as well as those used in the art to construct, for example, diabodies or ScFv fragments (however in this regard, in diabodies and ScFv fragments, the linker sequence used is appropriate V H and V L The domains should have length, some flexibility, and other properties that allow them to come together to form a complete antigen-binding site, and there are no particular restrictions on the length or flexibility of the linker used in the polypeptide of the present invention, since each ISVD, such as a nanobody, forms a complete antigen-binding site on its own.

[0135] For example, the linker may be a suitable amino acid sequence, and may be an amino acid sequence of 1 to 50, preferably 1 to 30, for example, 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include, for example, (glyx ser y ) Two types of gly-ser linkers, such as (gly4ser)3 or (gly3ser2)3 as described in WO 99 / 42077, and the GS30, GS15, GS9 and GS7 linkers described in the Ablynx applications described herein (see, for example, WO 06 / 040153 and WO 06 / 122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (described in WO 94 / 04678) are included. Preferred linkers are shown in Table C. Some other particularly preferred linkers are polyalanine (such as AAA), as well as linker GS30 (SEQ ID NO: 85 of WO 06 / 122825) and GS9 (SEQ ID NO: 84 of WO 06 / 122825). Other suitable linkers generally include organic compounds or polymers, particularly those suitable for use in proteins for pharmaceutical applications. For example, poly(ethylene glycol) moieties have been used to link antibody domains; see, for example, WO 04 / 081026.

[0136] It is within the scope of the present invention that the length, degree of flexibility, and / or other properties of the linker used (although usually not as critical as in the case of linkers used with ScFv fragments) can have some effect on properties (including but not limited to) the affinity, specificity or binding strength of the final constructs of the present invention, such as the polypeptides of the present invention, for MMP13 or one or more other antigens. Based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linker for use in a particular construct of the present invention, such as a polypeptide of the present invention, optionally after some limited routine experimentation. For example, in the multivalent polypeptides of the invention comprising building blocks, ISVDs or nanobodies directed against MMP13 and another target, the length and flexibility of the linker are preferably such that each building block such as an ISVD of the invention in the polypeptide is able to bind to its cognate target, for example an epitope of each target. Again, based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linker for use in a particular construct of the invention, such as a polypeptide of the invention, optionally after some limited routine experimentation.

[0137] Also within the scope of the invention is that the linker used imparts one or more other desirable properties or functionalities to a construct of the invention, such as a polypeptide of the invention, and / or provides one or more sites for formation of derivatives and / or attachment of functional groups (e.g., as described herein for derivatives of an ISVD of the invention). For example, a linker comprising one or more charged amino acid residues can provide improved hydrophilicity, while a linker that forms or contains a small epitope or tag can be used for detection, identification, and / or purification purposes. Again, based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linker for use in a particular polypeptide of the invention, optionally after some limited routine experimentation.

[0138] Finally, if two or more linkers are used in a construct such as a polypeptide of the invention, these linkers may be the same or different. Again, based on the disclosure herein, one of ordinary skill in the art will be able to determine the optimal linker for use in a particular construct or polypeptide of the invention, optionally after some limited routine experimentation. Typically, to facilitate expression and production, the constructs of the present invention, such as the polypeptides of the present invention, are linear polypeptides. However, the present invention is not limited to this in its broadest sense. For example, if the constructs of the present invention, such as the polypeptides of the present invention, comprise three or more building blocks, ISVDs, or nanobodies, they can be linked together by the use of a linker having three or more "arms," ​​each "arm" being linked to a building block, ISVD, or nanobodies to provide a "star-shaped" construct. It is also possible to use circular constructs, although this is generally less preferred.

[0139] Therefore, the present invention relates to constructs of the present invention, such as polypeptides of the present invention, wherein the ISVDs are linked to each other directly or via linkers. Accordingly, the present invention relates to constructs of the present invention, such as polypeptides of the present invention, wherein a first ISVD and / or a second ISVD and / or possibly a serum albumin-bound ISVD are linked via a linker. Accordingly, the present invention relates to constructs of the present invention such as polypeptides of the present invention, wherein the linker is selected from the group consisting of linkers A3, 5GS, 7GS, 8GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, 35GS, 40GS, G1 hinge, 9GS-G1 hinge, long hinge region on the upper part of the rama, and G3 hinge, which are shown, for example, in Table C. Accordingly, the present invention relates to constructs of the present invention, such as polypeptides, wherein the polypeptide is selected from the group shown in Tables A-3 and F, for example, from the group consisting of SEQ ID NOs: 164-165, 160, 161, 162, 163, and SEQ ID NOs: 176, 192, and 175-191 (i.e., 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, or 191).

[0140] preparation The present invention further relates to methods for preparing constructs, polypeptides, ISVDs, nucleic acids, host cells, and compositions described herein. The polyvalent polypeptides of the present invention can generally be prepared by a method comprising the step of appropriately linking at least the ISVD and / or monovalent polypeptide of the present invention to one or more further ISVDs, optionally via one or more suitable linkers, thereby providing the polyvalent polypeptide of the present invention. The polypeptides of the present invention can also be prepared by a method comprising at least the steps of providing a nucleic acid encoding the polypeptide of the present invention, expressing the nucleic acid in a suitable manner, and recovering the expressed polypeptide of the present invention. Such methods can be carried out in ways known by themselves, which will be apparent to those skilled in the art, for example, based on the methods and techniques further described herein.

[0141] A method for preparing the polyvalent polypeptide of the present invention may include the step of linking together at least two or more ISVDs and, for example, one or more linkers of the present invention in an appropriate manner. The ISVDs (and linkers) of the present invention can be linked by any method known in the art and further described herein. Preferred techniques include linking nucleic acid sequences encoding the ISVDs (and linkers) of the present invention to prepare a gene construct that expresses the polyvalent polypeptide. Techniques for linking amino acids or nucleic acids are obvious to those skilled in the art, and refer again to standard handbooks, such as Sambrook et al. and Ausubel et al. above, as well as the following examples.

[0142] Accordingly, the present invention also relates to the use of the ISVD of the present invention in the preparation of the polyvalent polypeptide of the present invention. A method for preparing a polyvalent polypeptide includes linking the ISVD of the present invention to at least one further ISVD of the present invention via optionally one or more linkers. The ISVD of the present invention is then used as a binding domain or building block when providing and / or preparing a polyvalent polypeptide comprising two (e.g., in a divalent polypeptide), three (e.g., in a trivalent polypeptide), four (e.g., in a tetravalent polypeptide), or more (e.g., in a polyvalent polypeptide) building blocks. In this regard, the ISVD of the present invention can be used as a binding domain or binding unit when providing and / or preparing a polyvalent polypeptide such as a divalent, trivalent or tetravalent of the present invention comprising two, three, four, or more building blocks.

[0143] Accordingly, the present invention also relates to the use of the ISVD polypeptide of the present invention (as described herein) in the preparation of polyvalent polypeptides. The method for preparing polyvalent polypeptides includes linking the ISVD of the present invention to at least one further ISVD of the present invention via optionally one or more linkers. The polypeptides and nucleic acids of the present invention can be prepared in ways known by themselves, as will be apparent to those skilled in the art from further description herein. For example, the polypeptides of the present invention can be prepared in any way known by themselves for the preparation of antibodies, particularly antibody fragments (including, but not limited to, (single)-domain antibodies and ScFv fragments). Some preferred but non-limiting methods for preparing polypeptides and nucleic acids include the methods and techniques described herein.

[0144] A method for producing the polypeptide of the present invention may include the following steps: - Expression of the nucleic acid encoding the polypeptide of the present invention (also referred to herein as "the nucleic acid of the present invention") in a suitable host cell or host organism (also referred to herein as "the host of the present invention"), or in another suitable expression system; subsequently, optionally, - Isolating and / or purifying the polypeptide obtained in this manner. In particular, such a method may include the following steps: -Culturing and / or maintaining the host of the present invention under conditions such that the host of the present invention expresses and / or produces at least one polypeptide of the present invention; then optionally, -Isolate and / or purify the polypeptide obtained in this manner.

[0145] Therefore, the present invention also relates to nucleic acids or nucleotide sequences (also referred to as "nucleic acids of the present invention") that encode the polypeptides, ISVDs, or constructs of the present invention. The nucleic acids of the present invention may be in the form of single-stranded or double-stranded DNA or RNA. According to one aspect of the present invention, the nucleic acids of the present invention are essentially isolated, as defined herein. The nucleic acids of the present invention may also be in the form of a vector, such as an expression vector, for example, a plasmid, cosmid or YAC (which may also be in an essentially isolated form), present in it, and / or as part of a vector. Thus, the present invention also relates to an expression vector comprising the nucleic acids or nucleotide sequences of the present invention.

[0146] The nucleic acids of the present invention can be prepared or obtained in a manner known by itself, and / or isolated from a suitable natural source, based on the information relating to the polypeptides of the present invention described herein. Also, as will be apparent to those skilled in the art, several nucleotide sequences, e.g., at least two nucleic acids encoding the ISVD of the present invention, and, e.g., one or more nucleic acids encoding a linker, etc., can be linked together in a suitable manner to prepare the nucleic acids of the present invention. Techniques for producing the nucleic acids of the present invention will be apparent to those skilled in the art and may include, for example, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring sequences and / or synthetic sequences (or two or more parts thereof); introducing mutations resulting in the expression of a cleaved expression product; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be readily digested and / or ligated using a suitable restriction enzyme); and / or introducing mutations by PCR reaction using one or more "mismatched" primers. These and other techniques will be apparent to those skilled in the art and again refer to standard handbooks, e.g., Sambrook et al. and Ausubel et al. above, as well as the following examples.

[0147] In a preferred but non-limiting embodiment, the gene constructs of the present invention include: a) At least one nucleic acid of the present invention; b) One or more modulators operably connected to, for example, a promoter, and optionally a suitable terminator; and optionally also c) One or more further elements of a gene construct that are known in themselves; Here, the terms “regulatory element,” “promoter,” “terminator,” and “operably connected” have their usual meanings in the art. The gene constructs of the present invention can generally be provided by appropriately ligating the nucleotide sequences of the present invention to one or more of the above-mentioned further elements using techniques described in general handbooks, such as those by Sambrook et al. and Ausubel et al.

[0148] The nucleic acids and / or gene constructs of the present invention may be used to transform host cells or host organisms, i.e., for the expression and / or production of polypeptides of the present invention. Suitable hosts or host cells will be obvious to those skilled in the art and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or (non-human) eukaryotic organism, as well as host cells or (non-human) hosts known in themselves for the expression and production of antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments), which will be obvious to those skilled in the art. See also the general background art cited above, as well as, for example, WO 94 / 29457; WO 96 / 34103; WO 99 / 42077; Frenken et al. (Res Immunol. 149: 589-99, 1998); Riechmann and Muyldermans (1999) above; van der Linden (J. Biotechnol. 80: 261-70, 2000); Joosten et al. (Microb. Cell Fact. 2: 1, 2003); Joosten et al. (Appl. Microbiol. Biotechnol. 66: 384-92, 2005); and further references cited herein. Furthermore, the polypeptides of the present invention can also be expressed and / or produced in cell-free expression systems, and suitable examples of such systems will be apparent to those skilled in the art. Appropriate techniques for transforming the host or host cells of the present invention will be apparent to those skilled in the art and may depend on the intended host cell / host organism and the gene construct used. Again, refer to the handbook and patent applications mentioned above. Transformed host cells (which may be in the form of a stable cell line) or host organism (which may be in the form of a stable mutant line or strain) form further aspects of the present invention. Thus, the present invention relates to a host or host cell comprising nucleic acids according to the present invention, or expression vectors according to the present invention.Preferably, these host cells or host organisms express or are capable of expressing (at least) the polypeptide of the present invention (and in the case of a host organism: in at least one of its cells, parts, tissues or organs). The present invention also includes further generations, progeny and / or offspring of the host cells or host organism of the present invention, which can be obtained, for example, by cell division or sexual or asexual reproduction.

[0149] To generate / obtain expression of the polypeptide of the present invention, transformed host cells or transformed host organisms can generally be preserved, maintained, and / or cultured under conditions such that the (desired) polypeptide of the present invention is expressed / produced. Suitable conditions will be apparent to those skilled in the art and typically depend on the host cells / host organisms used, as well as the regulatory factors controlling the expression of the (relevant) nucleotide sequence of the present invention. Again, refer to the handbook and patent applications mentioned above in the paragraph relating to the gene constructs of the present invention.

[0150] Subsequently, the polypeptide of the present invention can be isolated from host cells / host organisms and / or the culture medium in which the host cells or host organisms are cultured using isolation and / or purification techniques for proteins known in themselves, such as (preparative) chromatography and / or electrophoresis, differential precipitation, affinity techniques (e.g., using a specific cleavable amino acid sequence fused with the polypeptide of the present invention) and / or preparative immunological techniques (i.e., using an antibody against the polypeptide to be isolated). In one aspect, the present invention relates to a method for producing constructs, polypeptides, or ISVDs according to the present invention, comprising at least the following steps: (a) expressing a nucleic acid sequence according to the present invention in a suitable host cell or host organism or another suitable expression system; optionally thereafter (b) isolating and / or purifying a construct, polypeptide, or ISVD according to the present invention.

[0151] Pharmaceuticals (use of ISVD, polypeptides, and constructs of the present invention) As described herein, there remains a need for safe and effective OA pharmaceuticals. Based on non-conventional screening, characterization, and combination strategies, the inventors have identified ISVDs that bind to and inhibit MMP13. These MMP13 binders demonstrated very good performance in in vitro and in vivo experiments. Furthermore, the ISVDs of the present invention were also demonstrated to be significantly more effective than comparative molecules. Thus, the present invention provides ISVDs and polypeptides that antagonize MMP, particularly MMP13, and that have improved prophylactic, therapeutic, and / or pharmacological properties, including a safer profile compared to comparative molecules. Further, when these MMP13 binders were linked to CAP building blocks, retention in joints increased while activity was retained.

[0152] In one aspect, the present invention relates to a composition according to the present invention, an ISVD according to the present invention, a polypeptide according to the present invention, and / or a construct according to the present invention for use as a medicament. In another aspect, the present invention relates to the use of an ISVD, polypeptide, and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least MMP13-related diseases and / or for use in one or more of the treatment methods described herein.

[0153] The present invention also relates to the use of an ISVD, polypeptide, compound, and / or construct of the present invention for the preparation of a pharmaceutical composition for the prevention and / or treatment of at least one disease or disorder that can be prevented and / or treated by modulating the activity of MMP, preferably MMP13, for example by inhibiting the degradation of aggrecan and / or collagen. The present invention also relates to the use of the ISVD, polypeptide, compound and / or construct of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of at least one disease, disorder or condition that can be prevented and / or treated by administering the ISVD, polypeptide, compound and / or construct of the present invention to a patient.

[0154] The present invention further relates to ISVDs, polypeptides, compounds and / or constructs of the present invention, or pharmaceutical compositions comprising them, for use in the prevention and / or treatment of at least one MMP13-related disease. The MMP13 binder of the present invention is expected to be usable in a variety of diseases affecting cartilage, such as arthropathy and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and agricanopathies (generally referred to herein as "MMP13-related diseases").

[0155] In one aspect, the present invention relates to compositions, ISVDs, polypeptides and / or constructs according to the present invention for use in treating or preventing symptoms of MMP13-related diseases, the diseases being, for example, arthritis and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and agricanopathies. More preferably, the disease or disorder is arthritis, most preferably osteoarthritis. In one aspect, the present invention relates to methods for preventing or treating arthritis and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, wherein the method comprises administering to a subject in need at least a pharmaceutically active amount of a composition, immunoglobulin, polypeptide, or construct according to the present invention. More preferably, the disease is arthritis, most preferably osteoarthritis.

[0156] In one aspect, the present invention relates to the use of ISVD, polypeptides, compositions or constructs according to the present invention in the preparation of pharmaceutical compositions for treating or preventing diseases or disorders such as arthritis and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and agricanopathies. More preferably, the disease or disorder is arthritis, most preferably osteoarthritis. By binding to aggrecan, the constructs and / or polypeptides of the present invention may reduce or inhibit the activity of members of the serine protease family, cathepsins, matrix metalloproteinase (MMP) / matrixin, or A disintegrin and metalloproteinase (ADAMTS) having a thrombospondin motif, MMP20, ADAMTS5 (agrecanase 2), ADAMTS4 (agrecanase 1), and / or ADAMTS11 in the degradation of aggrecan.

[0157] In the context of the present invention, the term “prevention and / or treatment” includes not only the prevention and / or treatment of a disease, but also generally the following: prevention of the onset of a disease, delay or reversal of the progression of a disease, prevention or delay of a disease, prevention or delay of the onset of one or more symptoms associated with a disease, reduction and / or mitigation of one or more symptoms associated with a disease, reduction of the severity and / or duration of a disease and / or associated symptoms, and / or prevention of further increase of the severity and / or any associated symptoms of a disease, prevention, reduction or reversal of any physiological damage caused by a disease, and any generally beneficial pharmacological effect to the patient being treated.

[0158] The administration plan is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any given patient depends on many factors, including, for example, the patient's size, weight, body surface area, age, the specific compound being administered, the activity of the polypeptide (including antibodies) used, the time and route of administration, general health status, and combinations with other therapies or treatments. Protein-based pharmaceutically active substances may be present in amounts of 1 g to 100 mg / kg body weight per dose; however, doses below or above this exemplary range are also conceivable. If the plan is for continuous infusion, the range may be 1 pg to 100 mg per kilogram of body weight per minute.

[0159] The ISVD, polypeptide, or construct of the present invention may be used at concentrations of, for example, 0.01, 0.1, 0.5, 1, 2, 5, 10, 20, or 50 pg / ml to inhibit and / or neutralize at least about 50%, preferably 75%, more preferably 90%, 95%, or up to 99%, and most preferably about 100% (essentially completely) when the biological function of MMP13 is assayed by methods well known in the art. The ISVD, polypeptide, or construct of the present invention may be used at concentrations of, for example, 1, 2, 5, 10, 20, 25, 30, 40, 50, 75, 100, 200, 250 or 500 ng / mg of cartilage to inhibit and / or neutralize at least about 50%, preferably 75%, more preferably 90%, 95%, or up to 99%, and most preferably about 100% (essentially completely) when the biological function of MMP13 is assayed by methods well known in the art.

[0160] Generally, the treatment plan involves administering one or more ISVDs, polypeptides, and / or constructs of the present invention, or one or more compositions comprising them, in one or more pharmaceutically effective amounts or doses. The specific amount or dose to be administered can be determined by the clinician, again based on the factors described above. Useful doses of the constructs, polypeptides, and / or ISVDs of the present invention can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art; see, for example, US 4,938,949.

[0161] In general, depending on the specific disease, disorder, or condition being treated, the potency of the specific ISVD, polypeptide, and / or construct of the present invention used, the specific route of administration, and the specific pharmaceutical formulation or composition used, a clinician may determine an appropriate daily dose. The amount of the constructs, polypeptides, and / or ISVDs of the present invention required for use in treatment will vary depending not only on the specific immunoglobulin, polypeptide, compound, and / or construct selected, but also on the route of administration, the nature of the condition being treated, the patient's age and condition, and ultimately on the discretion of the attending physician or clinician. Furthermore, the dose of the constructs, polypeptides, and / or ISVDs of the present invention will vary depending on the target cells, tissues, grafts, joints, or organs.

[0162] The desired dose can be conveniently presented as a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more subdoses per day. The subdose itself may be further divided into several separate, roughly spaced doses. Preferably, the dose may be administered once a week or less frequently, for example, once every two weeks, once every three weeks, once a month, or once every two months. The treatment plan may include long-term treatment. “Long-term” means a period of at least two weeks, preferably several weeks, months, or years. Any necessary modifications to this dose range can be determined by those skilled in the art using only the teachings herein and standard experimentation. See Remington's Pharmaceutical Sciences (Martin, EW, ed. 4), Mack Publishing Co., Easton, PA. Dosage may also be adjusted by the individual physician in the event of any complications.

[0163] Typically, the ISVDs, polypeptides, and / or constructs of the present invention are used in the methods described above. However, the use of two or more ISVDs, polypeptides, and / or constructs of the present invention in combination is within the scope of the present invention. The ISVD, polypeptides, and / or constructs of the present invention can be used in combination with one or more further pharmaceutically active compounds or components, i.e., as a combination therapy plan, which may or may not result in a synergistic effect. The pharmaceutical composition may also include at least one further activator, such as one or more further antibodies or their antigen-binding fragments, peptides, proteins, nucleic acids, organic and inorganic molecules.

[0164] Again, clinicians may select such additional compounds or components, as well as appropriate combination therapy plans, based on the factors described above and their own expert judgment. In particular, the ISVD, polypeptides and / or constructs of the present invention may or may not be used in combination with other pharmaceutically active compounds or components used or usable for the prevention and / or treatment of diseases, disorders and conditions cited herein, resulting in synergistic effects. Examples of such compounds and components, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to clinicians.

[0165] When two or more substances or components are used as part of a combination therapy plan, they may be administered via the same or different routes of administration, essentially simultaneously or at different times (e.g., essentially simultaneously, sequentially, or alternately). When substances or components are administered simultaneously via the same route of administration, they may be administered as part of different pharmaceutical formulations or compositions, or as part of a combined pharmaceutical formulation or composition, as will be apparent to those skilled in the art.

[0166] Furthermore, when two or more active substances or components are used as part of a combined treatment plan, each substance or component may be administered in the same amount and according to the same plan as when the compound or component is used alone, and such combination may or may not produce a synergistic effect. However, if the combined use of two or more active substances or components produces a synergistic effect, it may be possible to reduce the amount of one, more, or all of the administered substances or components while still achieving the desired therapeutic effect. This may, for example, help to avoid, limit, or reduce any undesirable side effects associated with the use of one or more substances or components when used in normal amounts, while still achieving the desired pharmaceutical or therapeutic effect.

[0167] The effectiveness of the treatment plans used in accordance with the present invention can be determined and / or followed in any manner known in itself for the disease, disorder or condition involved, as will be apparent to the clinician. The clinician can also modify or alter specific treatment plans as needed and on a case-by-case basis to achieve desired therapeutic effects and avoid, limit, or mitigate undesirable side effects, and / or to achieve an appropriate balance between achieving desired therapeutic effects on the one hand and avoiding, limiting, or mitigate undesirable side effects on the other hand.

[0168] Generally, the treatment plan will continue until the desired therapeutic effect is achieved and / or maintained. Again, this is at the discretion of the clinician. Accordingly, in a further aspect, the present invention relates to a pharmaceutical composition comprising at least one construct of the present invention, at least one polypeptide of the present invention, at least one ISVD of the present invention, or at least one nucleic acid of the present invention, and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances. In a particular aspect, the present invention relates to constructs, polypeptides, ISVDs or nucleic acids according to the present invention, preferably SEQ ID NOs: 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, 8, 160-165 (i.e., SEQ ID NOs: 160, 161, 162, 163, 164 or 165) and 1 The present invention relates to a pharmaceutical composition comprising at least one of 76-192 (i.e., SEQ ID NOs: 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, or 192), at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances.

[0169] The subject of treatment may be any warm-blooded animal, but more specifically mammals, and more specifically humans. In veterinary applications, the subject of treatment includes any animal that is commercially bred or kept as a pet. As will be apparent to those skilled in the art, the subject of treatment is particularly a person suffering from or at risk of suffering from any of the diseases, disorders and conditions referred to herein. Thus, in a preferred aspect of the present invention, the pharmaceutical compositions comprising the polypeptides of the present invention are intended for use in medicine or diagnosis. Preferably, the pharmaceutical compositions are intended for use in human medicine, but may also be used for veterinary purposes.

[0170] Again, in such pharmaceutical compositions, a pharmaceutical composition comprising one or more immunoglobulins, polypeptides, compounds and / or constructs of the present invention, or nucleotides encoding them, and / or thereof, can be appropriately combined with one or more other active ingredients, such as those described herein. The present invention also relates to compositions (e.g., pharmaceutical compositions or formulations further described herein, without limitation) for use either in vitro (e.g., in vitro or cell assays) or in vivo (e.g., in single-cell or multicellular organisms, and in particular mammals, more specifically humans, e.g., humans who are at risk of or suffering from the disease, disorder or condition of the present invention).

[0171] Unless otherwise specified, please understand that references to treatment include both treatment for established symptoms and preventative measures. In general, for pharmaceutical applications, the constructs, polypeptides, and / or ISVDs of the present invention can be formulated as pharmaceutical formulations or compositions comprising at least one construct, polypeptide, and / or ISVD of the present invention and at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally one or more pharmaceutically active polypeptides and / or compounds. In non-limiting examples, such formulations may be in forms suitable for oral administration, parenteral administration (such as intravenous, intramuscular, or subcutaneous injection or intravenous infusion), topical administration (e.g., intra-articular administration), and inhalation administration, such as skin patches, implants, suppositories, where intra-articular administration is preferred. Such suitable forms of administration—which may be solid, semi-solid, or liquid depending on the mode of administration—and methods and carriers for their preparation will be apparent to those skilled in the art and will be further described herein. Such pharmaceutical formulations or compositions are generally referred to herein as “pharmaceutical compositions.”

[0172] Exemplary excipients may include disintegrants, binders, fillers, and lubricants. Examples of disintegrants include agar, algin, calcium carbonate, cellulose, colloidal silicon dioxide, rubber, 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, carboxymethylcellulose calcium, cellulose, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrin, maltose, sorbitol, starch, sucrose, sugars, and xylitol. Examples of lubricants include agar, ethyl oleate, ethyl laurate, glycerin, glyceryl palmitostearate, hydrogenated vegetable oil, magnesium oxide, stearate, mannitol, poloxamer, glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc. Common stabilizers, preservatives, humectants and emulsifiers, viscosity modifiers, flavor modifiers, osmotic salts, buffers, solubilizers, diluents, emollients, colorants, and masking agents, and antioxidants are considered pharmaceutical adjuvants.

[0173] Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugars, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point waxes, cocoa butter, water, alcohol, polyols, glycerol, and vegetable oils. In general, the constructs, polypeptides, and / or ISVDs of the present invention can be formulated and administered in any suitable form known to the present. For example, the general background art cited above (and in particular WO 04 / 041862, WO 04 / 041863, WO 04 / 041865, WO 04 / 041867 and WO 08 / 020079), as well as standard handbooks, e.g., Remington's Pharmaceutical Sciences, 18 th See Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see, for example, pages 252-255).

[0174] In certain aspects, the present invention relates to a pharmaceutical composition comprising a construct, polypeptide, ISVD or nucleic acid according to the present invention, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more further pharmaceutically active polypeptides and / or compounds. The constructs, polypeptides, and / or ISVDs of the present invention can be formulated and administered in any form known to the present invention for conventional antibodies and antibody fragments (including ScFv and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing them will be apparent to those skilled in the art and include, for example, formulations preferably suited for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial, intraspinal, intranasal, or intrabronchial administration), and further, topical administration (e.g., intra-articular, transdermal, or intradermal).

[0175] Formulations for topical or parenteral administration may be, for example, sterile solutions, suspensions, dispersions, or emulsions suitable for infusion or injection. Suitable carriers or diluents for such formulations include, for example, those described on page 143 of WO 08 / 020079. Aqueous solutions or suspensions are usually preferred. The constructs, polypeptides, and / or ISVDs of the present invention may also be administered using delivery methods known from gene therapy, see, for example, U.S. Patent No. 5,399,346, incorporated by reference with respect to such gene therapy delivery methods. Using gene therapy delivery methods, primary cells transfected with the genes encoding the constructs, polypeptides, and / or ISVDs of the present invention may be further transfected with tissue-specific promoters targeting specific organs, tissues, grafts, joints, or cells, and may be further transfected with signaling and stabilizing sequences for intracellular localization expression.

[0176] According to further aspects of the present invention, the polypeptides of the present invention may be used for further in vivo and in vitro applications. For example, the polypeptides of the present invention may be used for diagnostic purposes, for example, in assays designed to detect and / or quantify the presence of MMP13 and / or purify MMP13. The polypeptides may also be tested in animal models of specific diseases to conduct toxicological, safety, and dosage studies. Finally, the present invention relates to a kit comprising at least one polypeptide according to the present invention, at least one nucleic acid sequence encoding the component, a vector or vector system according to the present invention, and / or a host cell according to the present invention. The kit is intended to be provided in different forms, for example, as a diagnostic kit.

[0177] The present invention will be further described by the following non-limiting preferred aspects, examples, and figures. The entire contents of all references cited throughout this application (including references to documents, issued patents, published patent applications, and concurrently pending patent applications) are expressly incorporated herein by reference, in particular with respect to the teachings referenced above.

[0178] Sequences are described in the text of the specification and in a separate sequence list in accordance with WIPO standard ST.25. Sequences designated by a specific number should be identical in the text of the specification and in the separate sequence list. For example, sequence number 1 should define the same sequence in both the text of the specification and the separate sequence list. If there is a discrepancy between the sequence definitions in the text of the specification and the separate sequence list (for example, sequence number 1 in the text of the specification incorrectly corresponds to sequence number 2 in the separate sequence list), references to a particular sequence in this application, especially references in a particular aspect, should be understood as references to the sequence in the text of this application, rather than references to the separate sequence list. In other words, any discrepancies in sequence definitions / designations between the text of the specification and the separate sequence list are resolved by modifying the separate sequence list to reflect the sequences and their designations disclosed in the text of the application, including the specification, examples, figures, and claims. [Examples]

[0179] 6. Example The following examples illustrate the methods and products of the present invention. Appropriate modifications and adaptations of the described conditions and parameters, which are common to those skilled in the art and commonly encountered in the fields of molecular biology and cell biology, are within the spirit and scope of the present invention.

[0180] 6.1 Method 6.1.1 Immune development of llamas The lamara immunization was performed following standard procedures, with variations in the amount of antigen used, the type of adjuvant, and the injection method. These modifications are described in detail in the following sections. All immunization experiments were approved by the local ethics committee.

[0181] 6.1.2 Building the Library cDNA was prepared using total RNA extracted from blood samples of all llamas / alpacas immunized with MMP13. The nucleotide sequences encoding the nanobodies were amplified from the cDNA by a one-step RT-PCR reaction and ligated to the corresponding restriction sites of the phagemide vector pAX212. Subsequently, transformation of E. coli strain TG-1 was carried out via electroporation using the ligation product. The NNK library was generated by overlap extension PCR using denaturing primers. The PCR product was cloned into an expression vector (pAX129) and used to transform E. coli TG-1 competent cells. Within the frame of nanobody coding sequences, the vector encodes the C-terminal FLAG3- and His6- tags. The sequence of the target clone was verified.

[0182] 6.1.3 Selection The phage display library was investigated using recombinant MMP13. Different antigens were used in the selection of different rounds, as detailed in the Results section, i.e., Example 6.2 onward. Selection consisted of incubating the antigens with library phage particles for 2 hours (in PBS supplemented with 2% Marvel and 0.05% Tween 20). Biotinylated antigens were captured using streptavidin-coated magnetic beads (Invitrogen, 112-05D). Unbiotinylated antigens were coated onto MaxiSorp plates (Nunc, 430341). Unbound phages were washed away (in PBS supplemented with 0.05% Tween 20); bound phages were eluted for 15 minutes with the addition of trypsin (1 mg / mL in PBS). The eluted phages were used to infect exponentially growing E. coli TG-1 cells for phage rescue. Phages prepared from the selected output were used as input in subsequent selection rounds.

[0183] 6.1.4 ELISA direct coating antigen MaxiSorp plates (Nunc, 430341) were coated with human proMMP13 overnight at 4°C, followed by 1 hour of blocking in RT (PBS, 1% casein). After a washing step with PBS + 0.05% Tween 20, a 10-fold dilution of periplasm extract in PBS, 0.1% casein, and 0.05% Tween 20 was added for 1 hour in RT. Binding nanobodies were detected by mouse anti-FLAG-HRP (Sigma (A8592)). Captured antigen - Activated Human MMP13 ELISA - Activated rat MMP13 ELISA - Activated Canine MMP13 ELISA MaxiSorp plates (Nunc, 430341) were coated overnight at 4°C with human MMP13 antibody mAb511, followed by 1 hour of blocking at RT (PBS, 1% casein). After a washing step with PBS + 0.05% Tween 20, 20 nM activated human, rat, or canine MMP13 was added at RT for 1 hour. After a second washing step, a 10-fold dilution of periplasm extract or a dilution series of purified nanobodies in PBS, 0.1% casein, and 0.05% Tween 20 was added at RT for 1 hour. Binding nanobodies were detected by mouse anti-FLAG-HRP (Sigma (A8592)).

[0184] 6.1.5 Fluorescence-generating peptide assay The setup for the MMP13 fluorescence peptide assays in humans, cynomolgus monkeys, rats, dogs, and cattle, as well as the human MMP1 and MMP14 fluorescence peptide assays, is outlined below: Activated MMPs were incubated at 37°C for 2 hours using the following: fluorescent peptide substrate Mca-PLGL-Dpa-AR-NH2 (R&D Systems #ES001), and a 1 / 5 dilution of periplasm extract or a dilution series of purified nanobodies / positive control (total volume = 20 μl in assay buffer: 50 mM Tris, pH 7.5, 100 mM NaCl, 10 mM CaCl2, and 0.01% Tween20). A linear increase in fluorescence (v0-15 ~ 45 minutes incubation) was used as a measure of enzyme activity, and % inhibition was calculated using the formula: 100-100 (v0 in the presence of test nanobodies / v0 in the presence of negative control nanobodies (Cablys)).

[0185] 6.1.6 Collagen degradation assay The setup for this assay was outlined as follows: 250 ng / ml immunograde human collagen II (Chondrex #20052) was incubated with 5 nM activated MMP in 100 μl of assay buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM CaCl2, 0.01% Tween-20). After incubation at 35°C for 1.5 hours, the reaction was neutralized with EDTA (10 μl of 30 mM stock). The collagen cleaved by MMP13 was further degraded with elastase at 38°C for 20 minutes to avoid re-annealing of the degraded collagen II (10 μl of 1 / 3 diluted stock provided in the Type II Collagen Detection Kit (Chondrex #6009)). The remaining undiluted collagen was detected via ELISA (reagent provided in the Type II Collagen Detection Kit (Chondrex #6009)).

[0186] 6.1.7 Fluorescence-generating collagen assay In short, the assay setup was as follows: 100 μg / ml of DQ® type I collagen (fluorescein conjugate, Molecular Probes #D-12060 lot 1149062) from bovine skin was incubated with 10 nM activated MMP13 and a dilution series of purified nanobodies / positive control in 40 μl assay buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM CaCl2, 0.01% Tween-20) at 37°C for 2 hours. A linear increase in fluorescence (v0-15 to 45 minutes of incubation) was used as a measure of enzyme activity, and % inhibition was calculated using the formula: 100-100 (v0 in the presence of test nanobodies / v0 in the presence of negative control nanobodies (Cablys)).

[0187] 6.1.8 TIMP-2 competitive assay 50 μl of TIMP-2 (0.63 nM; R&D Systems #971-TM) was captured on a plate coated with anti-human TIMP-2 antibody (R&D Systems #MAB9711) (2 μg / ml in PBS; overnight) (1 hour at RT). During this capture, 1.26 nM activated MMP13-biotin was pre-incubated with a dilution series of nanobody / TIMP-3 / MSC2392891A in 70 μl of assay buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM CaCl2, 0.01% Tween-20). 50 μl of this mixture was added to the captured TIMP-2 and incubated at room temperature for 1 hour. MMP13-biotin was detected using 50 μl of streptavidin-HRP (1:5000, DakoCytomation #P0397).

[0188] 6.1.9 Thermal Shift Assay (TSA) TSA was performed using 5 μl of purified monovalent nanobodies, basically following Ericsson et al. (2006 Anals of Biochemistry, 357: 289-298).

[0189] 6.1.10 Analytical Size Exclusion Chromatography (Analytical SEC) The analytical SEC experiment was performed using an Ultimate 3000 machine (Dionex) in combination with a Biosep-SEC-3 (Agilent) column.

[0190] 6.1.11 Forced oxidation Nanobody samples (1 mg / ml) were exposed to 10 mM H2O2 in PBS in the dark for 4 hours at RT, in parallel with a control sample without H2O2. The buffer was then switched back to PBS using a Zeba desalting spin column (0.5 ml) (Thermo Scientific). Subsequently, the stressed samples and control samples were analyzed by RPC at 70°C using a Zorbax 300SB-C3 column (Agilent Technologies) on a Series 1200 or 1290 machine (Agilent Technologies). Nanobody oxidation was quantified by determining the peak area % of the pre-peak resulting from oxidative stress compared to the major protein peak.

[0191] 6.1.12 Temperature stress Nanobody samples (1–2 mg / ml) were stored in PBS at -20°C (negative control), 25°C, and 40°C for 4 weeks. After this incubation period, the nanobodies were digested with trypsin or LysC. Next, peptides from the stressed and control samples were analyzed by RPC using an Acquity UPLC BEH300-C18 column (Agilent Technologies) connected to a Q-TOF mass spectrometer (6530 Accurate Mass Q-TOF (Agilent)) at 60°C on a Series 1200 machine (Agilent Technologies).

[0192] 6.2 Immunization MMP13 is secreted as an inactive pro-form (proMMP13). When the pro-domain is cleaved, it becomes activated, leaving behind an active enzyme composed of a catalytic domain that forms a catalytic pocket and a hemopexin-like domain (PDB:1PEX) that is described as functioning as a docking / interaction domain for the substrate collagen II (Col II). It was hypothesized that the catalytic pocket is the optimal region for inhibiting the enzymatic activity of MMP13. However, there are several important issues to consider when trying to enhance the immune response to the catalytic pocket. Firstly, in proMMP13, the prodomain masks the catalytic pocket, preventing access to the pocket to enhance the immune response. Secondly, activated MMP13 has a short half-life, which is mainly due to autoprotein degradation. This short half-life hinders the development of a strong immune response. Thirdly, the catalytic domain sequence is highly conserved across species. Therefore, any expected immune response, even if elevated, will be weak.

[0193] 6.2.1 Immune Strategy To address these issues and increase the likelihood of successfully obtaining MMP13 inhibitors that bind to the catalytic pocket, sophisticated and elaborate immunotherapy strategies involving various formats of MMP13 were devised. Ultimately, the following immunizations were performed: (a) Three llamas were immunized with a cleaved MMP13 variant consisting of a catalytic domain and containing mutant F72D, which protects MMP13 from autoprotein degradation; (b) Three llamas were immunized with the same cleavage-type MMP13 variant as in (a), which included mutation E120A in addition to mutation F72D to inactivate enzyme function; (c) Three llamas were immunized with full-length proMMP13 protein; and (d) Three llamas were immunized with a mixture of plasmids encoding either the secreted proMMP13 variant (V123A) or the GPI-anchored proMMP13 variant (V123A). The V123A mutation has been described as causing a weak interaction between Cys104 and catalytic zinc ions for MMP13, resulting in spontaneous autoactivation.

[0194] 6.2.2 Serum potency Serum titers were determined for proMMP13 and the catalytic domain (F72D). In general, animals immunized with the protein proMMP13(c) or DNA encoding proMMP13 V123A(d) showed a good immune response to proMMP13 but a weak response to the catalytic domain (F72D). Animals immunized with the catalytic domain (F72D)(a) or the inactive catalytic domain (F72D, E120A)(b) showed no immune response to the catalytic domain or only a weak immune response.

[0195] 6.2.3 Building the Library Despite low serum titers for the catalytic domain, we were confident that extensive screening would enable the identification of inhibitory binders to the catalytic pocket. RNA was extracted from PBLs (primary blood lymphocytes) and used as a template for RT-PCR to amplify nanobodies encoding gene fragments. These fragments were cloned into the phagemide vector pAX212, enabling the production of phage particles displaying nanobodies fused with His6- and FLAG3- tags. The phages were then processed according to the standard protocol (Phage Display of Peptides and Proteins: A Laboratory Manual 1). st Prepared and preserved according to Edition, Brian K. Kay, Jill Winter, John McCafferty, Academic Press, 1996. The average size of the 18 immunoassay libraries obtained was approximately 5*108 They were individual clones.

[0196] 6.3 Primary Screening Phage display selection was performed using 18 immunoassay libraries and two synthetic nanobody libraries. The libraries underwent 2–4 rounds of enrichment for different combinations of human proMMP13, activated human, rat, and canine MMP13, and the human MMP13 catalytic domain (F72D) using different antigen presentation formats. Individual clones from the selected outputs were screened using broad-spectrum MMP fluorescence peptide substrates for binding to human and rat MMP13 via ELISA (using periplasmic extracts from E. coli cells expressing nanobody) and inhibitory activity via fluorescence peptide assays measuring the increase in fluorescence during peptide hydrolysis. Nanobodies that showed binding via ELISA but not inhibitory activity via fluorescence peptide assays were further screened using collagen degradation assays. The collagen degradation assays used native substrates instead of peptide substitutes. We hypothesized that nanobodies interfere with collagen degradation but not peptide degradation, since collagen has a much larger interaction surface with MMP13 than fluorescence peptides. However, the collagen degradation assay was incompatible with the periplasm extract, requiring the use of purified nanobodies.

[0197] 6.3.1 Campaign 1 In the first selection campaign, MMP13 was presented as a directly coated antigen. This resulted in high hit rates in ELISA (binding assay) of human and rat MMP13, including diversity of nanobodies that bound to human MMP13, exhibiting broad binding signals in ELISA. The majority of nanobodies were found to cross-react with rat MMP13. However, very low hit rates were obtained in fluorescence-generating peptide assays (inhibition assays). Furthermore, the observed inhibition was incomplete, and some of these nanobodies did not exhibit rat cross-reactivity. Since monovalent, completely inhibitory nanobodies were not obtained, the inventors hypothesized that the correct epitope was not targeted and that the conditions used during selection were not optimal. However, testing of the selection conditions was hampered by the absence of a positive control. Ultimately, it was found that direct coating inhibited enzyme activity.

[0198] 6.3.2 Campaign 2 In Campaign 2, selection was performed in solution using biotinylated MMP13. However, when activated human and rat MMP13 captured via a non-neutralizing antibody was used instead of directly coated proMMP13, the ELISA hit rate was significantly lower than in Campaign 1. The hit rate was also very low in the fluorescence-generating peptide assay. However, one nanobody was found that completely inhibited the collagen degradation assay. Since the pro-form of the enzyme was used to concentrate the binder, we hypothesized that the key epitopes were masked by the propeptide.

[0199] 6.3.3 Campaign 3 Considering the disappointing results of campaigns 1 and 2, the inventors chose to optimize the presentation of key epitopes in the catalytic domain by using activated MMP13 captured by a non-neutralizing antibody. The same capture format was also used for ELISA. This resulted in high hit rates for both human and rat MMP13 in ELISA. However, although the hit rate in the fluorescence-generating peptide assay was slightly higher than in previous campaigns, the nanobodies still showed only incomplete inhibition, and rat cross-reactivity was weak or nonexistent, suggesting that the captured presentation of MMP13 was still not optimal. Three clones, including C0101040E09 ("40E09"), were found to be positive in the collagen degradation assay despite exhibiting incomplete inhibition in the fluorescence-generating peptide assay. Family members of 40E09 were cloned and sequenced: C0101PMP040E08, C0101PMP042A04, C0101PMP040B05, C0101PMP042D12, C0101PMP042A03, C0101PMP024A08, and C0101PMP040D01 (see Tables A-1 and A-2). Sequence diversity of the CDR region is shown in Tables 6.3.3A, 6.3.3B, and 6.3.3C below. The amino acid sequence of the CDR of clone 40E09 was used as a reference for comparing the CDRs of family members (CDR1 starts at Kabat position 26, CDR2 starts at Kabat position 50, and CDR3 starts at Kabat position 95).

[0200] Table 6.3.3A(40E09 CDR1) [Table 3] * A maximum of one CDR1 mutation (SEQ ID NO: 23) per clone. Table 6.3.3B(40E09 CDR2) [Table 4] * Up to 3 CDR2 mutations in a single clone (SEQ ID NO: 37) Table 6.3.3C(40E09 CDR3) [Table 5] * A maximum of one CDR mutation (SEQ ID NO: 52) per clone.

[0201] 6.3.4 Campaign 4 In the fourth campaign, in addition to the immunogenic nanobody phage display libraries derived from immunization strategies (a) and (c), libraries derived from selection strategies (b) and (d) were also investigated (see Example 6.2.1). The selection strategies focused on the MMP13 catalytic domain (F72D) used in solution. The hit rate of the fluorescence-generating peptide assay increased across the libraries, and many nanobodies showed complete inhibition. Inhibitory nanobodies showed only insufficient binding in ELISA, and the capture presentation of MMP13 used in campaign 3 also confirmed that it was not optimal for the accessibility of key epitopes. As a result, rat fluorescence-generating peptide assays were used instead of ELISA to evaluate rat cross-reactivity. All representatives of the nanobody families with confirmed inhibitory activity were rat cross-reactive, suggesting that the epitopes recognized by this particular set of clones reside within the conserved MMP13 catalytic pocket.

[0202] In summary, any routine manipulation of MMP13 was found to interfere with the enzyme's activity and TIMP-2 binding (data not shown). After modifying and evaluating various parameters, including: different antigens (e.g., proMMP13, catalytic domain (F72D), activated human MMP13, activated rat MMP13, activated canine MMP13); different assays (e.g., ELISA, human fluorescence peptide assay, rat fluorescence peptide assay, human collagen degradation assay); and different assay settings (e.g., changes in coating conditions, in solution, and capture of MMP13), the only successful strategy for identifying the completely inhibiting nanobody was found to be selection using the catalytic domain (F72D) or activated MMP13 species (Campaign 4).

[0203] 6.3.5 Lead Panel The sequences of inhibitory nanobodies identified by fluorescence-generating peptide assays or collagen degradation assays were determined. Based on the sequence information, the nanobodies could be classified into various families. Four families derived from screening campaigns 2 and 3 showed complete inhibition in collagen degradation assays but no activity in fluorescence-generating peptide assays; further shown herein as “Profile 1” clones (see 40E09 and family members). Ten families derived from screening campaign 4 showed inhibitory activity in both collagen degradation assays and fluorescence-generating peptide assays; further shown herein as “Profile 2” clones. A representative clone was selected for each nanobody family, resulting in a total of 14 representative clones. The sequences of the representative clones are shown in Table A-1.

[0204] 6.4 In vitro characterization of monovalent lead panels To further characterize the function of representative nanobody clones, they were recloned into pAX129, transformed into E. coli, and expressed and purified according to standard protocols (e.g., Maussang et al. 2013 J Biol Chem 288(41): 29562-72). These clones were then subjected to various functional in vitro assays.

[0205] 6.4.1 Enzyme assay The potency / efficacy of the nanobodies was tested using fluorescence-generating peptide assays and human collagen degradation assays, both of which were also used during screening (see Example 6.3). Furthermore, a second collagen-based assay (fluorescence-generating collagen assay) was established using higher collagen concentrations compared to the collagen degradation assay, simulating the high collagen concentration conditions in cartilage where the MMP13 inhibitor is expected to be active. In this assay, the fluorescence of intact FITC-labeled collagen substrate is low due to the mutual quenching effect of fluorophores. Upon cleavage, quenching is lost and fluorescence increases.

[0206] Table 6.4.1 shows an overview of the efficacy in enzyme assays. Table 6.4.1 Efficacy of the lead panel in the MMP13 enzyme assay. Efficacy is reported only when the efficacy is approximately 100%. [Table 6]

[0207] Figure 1 shows the dose-response curve for the inhibition of activated human MMP13 when high concentrations of bovine collagen I are used (fluorescent collagen assay). [Table 7] Figure 1: Dose-response curves of profile 1 nanobody (left graph) and profile 2 nanobody (right graph) in fluorescence-generating collagen assay.

[0208] Profile 1 nanobodies demonstrated complete efficacy in collagen degradation assays under low collagen concentration conditions, but their efficacy decreased in fluorescence-generating collagen assays under high collagen concentration conditions (Figure 1, left panel). Furthermore, the comparator drug MSC2392891A showed even weaker efficacy in the fluorescence-generating collagen assay (Figure 1). Representative nanobodies of Profile 2 were potent and fully effective in both collagen degradation assays and fluorescence-generating collagen assays. In these assays, most nanobodies were more active than the comparator drug. These Profile 2 representatives showed comparable potency (see Table 6.4.1) and efficacy (Figure 1, right panel) in fluorescence-generating peptide assays in humans, cynomolgus monkeys, rats, dogs, and cattle.

[0209] 6.4.2 Binding assay The ELISA configuration was used to evaluate binding affinity. However, this assay proved suitable only for evaluating affinity of profile 1 nanobodies and not for profile 2 nanobodies (see Example 6.3). The results are shown in Table 6.4.2A. Table 6.4.2A: Binding affinity (EC50) of Profile 1 nanobody evaluated by ELISA [Table 8]

[0210] In conclusion, the binding affinity of profile 1 nanobody was comparable across the three species tested (i.e., a difference of less than 10 times). Clone 40E09 had the second-best binding affinity to human MMP13, but showed the best affinity across all species. Since profile 2 nanobodies were found to compete with TIMP-2 for binding to MMP13, a TIMP-2 competitive ELISA was set up and used to evaluate the affinity of profile 2 nanobodies. In particular, profile 1 nanobodies do not compete with TIMP-2. The results are shown in Table 6.4.2B. Table 6.4.2B: Efficacy (IC50) of Profile 2 nanobodies evaluated by TIMP-2 competitive ELISA. [Table 9] In conclusion, the evaluation was similar to the potency obtained by enzyme assays, with nanobodies 516G08, 529C12, and 62C02 showing the highest inhibitory activity, followed by nanobodies 59F06, 525C04, and 63F01.

[0211] 6.4.3 Selective Assay To determine the selectivity of nanobodies for MMP13 compared to MMP1 and MMP14, a fluorescence-generating peptide assay was used. MMP1 and MMP14 are two closely related members of the MMP family. Since profile 1 nanobodies did not show inhibition in the MMP13 fluorescence-generating peptide assay, only profile 2 nanobodies could be tested. TIMP-2, a non-selective MMP inhibitor, was used as a positive control for these assays. The results are shown in Figure 2. [Table 10] Figure 2. Selectivity of MMP13 lead nanobodies All nanobodies were highly selective. They did not exhibit MMP1 inhibition, and for some nanobodies, only very slight inhibition was observed at high concentrations of MMP14.

[0212] 6.4.4 Epitope Binning For epitope binning, panels of monovalent nanobodies derived from profile 1 and profile 2 were tested against purified nanobodies 40E09 (profile 1) in a competitive ELISA. The results of the competitive ELISA are shown in Figure 3. [Table 11] Figure 3. Competitive ELISA of 0.6 nM biotinylated 40E09 against a panel of profile 1 and profile 2 nanobodies of human full-length MMP13 captured via mouse anti-human MMP13 mAb (R&D Systems #MAB511). MMP13 was activated by incubation with APMA at 37°C for 1 hour and 30 minutes. Profile 1 nanobodies 32B08, 43B05, 43E10, and 40E09 (positive control) all compete with 40E09. On the other hand, Profile 2 nanobodies 59F06, 62C02, 63F01, 513C04, 516G08, 517A01, and the negative control cAbLys do not compete with 40E09. Therefore, it is thought that the profile 1 nanobody belongs to a different epitope bin (let's call it "bin 1") than the profile 2 nanobody (let's call it "bin 2").

[0213] 6.5 Divalent structures As demonstrated in Example 6.4.1 above, profile 1 nanobodies did not show inhibition in the fluorescence-generating peptide assay (see Table 6.4.1). We then began investigating the effect of combining profile 1 nanobodies and profile 2 nanobodies. As the best species cross-reactive binder (see Example 6.4.2), nanobody 40E09 was selected as representative of profile 1 nanobodies. For profile 2, three nanobodies were selected: 516G08, 62C02, and 517A01. Nanobodies from profile 1 and profile 2 were linked in a divalent format using a 35GS linker (see Table 6.5; Nb(A)-35GS-Nb(B)). The divalent nanobodies were cloned into pAX205, transformed into P. pastoris, and expressed and purified according to standard procedures. To evaluate the potency of these bivalent constructs, a rat fluorescence-generating peptide assay was used, but the MMP13 concentration was lowered (0.15 nM instead of 1.33 nM) compared to the screening setting to improve the sensitivity of the assay. The bivalent constructs were tested in this adapted fluorescence-generating peptide assay and human fluorescence-generating collagen assay. The obtained data is summarized in Table 6.5. Table 6.5: Efficacy of divalent nanobodies in rat fluorescence-generating peptide assays (adjusted for higher sensitivity) and human fluorescence-generating collagen assays. [Table 12]

[0214] The results show that the bivalent construct, combining profile 1 nanobodies and profile 2 nanobodies, is more potent than their monovalent building blocks in a matched rat fluorescence-generating peptide assay, exhibiting up to a 40-fold improvement in potency. Improved potency was also observed in a human fluorescence-generating collagen assay (up to 4-fold). In both assays, the bivalent construct was equally potent against the positive non-selective control TIMP-2. Therefore, while profile 1 nanobodies are not particularly inhibitory on their own, when combined with divalent constructs, they enhanced the potency of profile 2 nanobodies.

[0215] 6.6 Biophysical Characterization To facilitate patient convenience, a low administration frequency and high retention of therapeutic compounds are preferred. Therefore, it is preferable that the nanobodies have high stability. To test stability, five representative 2-nanometer profiles and one representative 1-nanometer profile were subjected to biophysical characterization.

[0216] 6.6.1 Thermal Shift Assay The thermal stability of wild-type anti-MMP13 nanobodies was investigated using a thermal shift assay (TSA). The results are shown in Table 6.6.1. Table 6.6.1: Tm of monovalent nanobody at pH 7 [Table 13] The Tm value at pH 7 is in the range of 65°C to 83°C, indicating good to very good stability characteristics.

[0217] 6.6.2 Analytical SEC The polymerization and aggregation tendencies of a selected panel of five representative anti-MMP13 nanobodies were investigated by analytical size exclusion chromatography (aSEC). A summary of the results is shown in Table 6.6.2. Table 6.6.2: Analytical SEC parameters of MMP13 lead nanobodies [Table 14] The retention times for the five representative nanobodies were within the expected range for monovalent nanobodies (7.6–8.2 minutes), and the relative area of ​​the major peak and the overall recovery rate exceeded 90% for all nanobodies. The biophysical properties based on TSA and aSEC were considered suitable for further development of all nanobodies tested.

[0218] 6.7 Selection of clones for further development and sequence optimization Based on the functional and biophysical properties of representative nanobodies and divalent constructs, four exemplary lead nanobodies: 62C02, 529C12, 80A01, and the divalent construct C01010080 ("0080", consisting of profile 2 nanobodies 517A01 and profile 1 nanobodies 40E09) were selected for further development. The inventors have undertaken the optimization of the amino acid sequences of the lead panel ("sequence optimization" or "SO"). In the sequence optimization process, the following are attempted: (1) knocking out sites for post-translational modification (PTM); (2) humanizing parent nanobodies; and (3) knocking out potential existing antibody epitopes. At the same time, it is desirable to maintain or further improve the functional and biophysical properties of the nanobodies.

[0219] 6.7.1 Post-translational modification (PTM) The post-translational modifications (PTMs) evaluated were Met oxidation, Asn deamidation, Asp isomerization, Asn glycosylation, and pyroglutamate formation. The E1D mutation (typically incorporated to prevent pyroglutamate formation) was not analyzed during sequence optimization but was present in the formatted nanobodies. The mutation was accepted in all MMP13 read nanobodies except for 62C02, where a 12-fold decrease in potency was observed. Therefore, it was decided not to incorporate the E1D mutation into the 62C02 building block. To evaluate potential PTM, forced oxidation and thermal stress were applied to the lead panels. No modifications were observed in the lead panels except for C0101517A01 ("517A01") and C0101080A01 ("80A01").

[0220] Under forced oxidation and temperature stress conditions, C0101080A01 became more susceptible to Asp isomerization and Met oxidation. However, the degree of isomerization and oxidation varied under evaluation conditions, slightly below or above the thresholds applied in each case. Ultimately, amino acid residues 54-55, 100d-100e, M100j, and 101-102 were identified as residues responsible for PTM. In particular, all of these residues are located in either the CDR2 or CDR3 region and are therefore potentially involved in target binding. To address different requirements, NKK libraries with mutations in these residues were constructed and then screened by fluorescence-generating peptide assays to assess potential loss of potency and screened for biophysical properties (Tm). Surprisingly, mutations at various positions in these CDRs were found to be possible without significant loss of potency, i.e., maintaining over 80% inhibition. In the D100dX library, nine amino acid ("AA") substitutions showed over 80% inhibition (E, G, A, P, T, R, M, W, and Y). In the case of the M100jX library, 16 AA substitutions showed inhibition exceeding 85% (all except T, C, and H). In the case of the D101X library, 16 AA substitutions showed inhibition exceeding 90% (all except D, F, and P). In the case of the Y102X library, most clones showed inhibition of over 90%. Therefore, it was considered that amino acid position 102 could be mutated to any residue. The following conserved mutations are particularly preferred: D100dE and M100jL.

[0221] A summary of preferred mutations in the CDR region is provided in Tables 6.7.1A, 6.7.1B, and 6.7.1C below. The amino acid sequence of the CDR of clone 80A01 was used as a reference for comparing the CDRs of family members. According to Kabat numbering, CDR1 begins at amino acid residue 26, CDR2 begins at amino acid residue 50, and CDR3 begins at amino acid residue 95. Table 6.7.1A(80A01SO CDR1) [Table 15] * A maximum of 0 CDR1 mutations in a single clone (SEQ ID NO: 28) Table 6.7.1B(80A01SO CDR2) [Table 16] * A maximum of 0 CDR2 mutations in a single clone (SEQ ID NO: 43) Table 6.7.1C(80A01SO CDR3) [Table 17] * Up to four CDR mutations (SEQ ID NO: 57) in a single clone X1 = E, G, A, P, T, R, M, W, Y X2=A,R,N,D,E,Q,Z,G,I,L,K,F,P,S,W,Y and V X3 = A, R, N, C, E, Q, Z, G, H, I, L, K, M, S, T, W, Y and V X4 = A, R, N, D, C, E, Q, Z, G, H, I, L, K, M, F, P, S, T, W and V

[0222] Under forced oxidation and temperature stress conditions, C0101517A01 tended to be deamidated. Ultimately, amino acid residues N100b and N101 were identified as deamidation-sensitive residues by temperature stress experiments. However, these residues are located in the CDR3 region and are potentially involved in target binding. Therefore, knocking out the deamidation site may affect binding. In this case, an NKK library with mutations in the deamidation-sensitive residues was constructed and then screened using fluorescence-generating peptide assays to assess potential loss of potency and screen for biophysical properties (Tm). Unexpectedly, mutations of N100b to Q or S, or N101 to Q or V, prevented deamidation, while simultaneously increasing Tm by 1–3°C compared to the parent nanobody C0101517A01, although the potency remained equivalent. Four preferred variants are shown in Table 6.7.1D; this also summarizes the results of TSA and fluorescence-generating peptide assays. A summary of preferred mutations in CDR is shown in the table below. Table 6.7.1D. Data summary of the C0101517A01 sequence-optimized variant in the second round: [Table 18]

[0223] A summary of preferred mutations in the CDR region is provided in Tables 6.7.1E, 6.7.1F, and 6.7.1G below. The amino acid sequence of the CDR of clone 517A01 was used as a reference for comparison with the CDRs of other clones. According to Kabat numbering, CDR1 begins at amino acid residue 26, CDR2 begins at amino acid residue 50, and CDR3 begins at amino acid residue 95. Table 6.7.1E(517A01SO CDR1) [Table 19] * A maximum of 0 CDR1 mutations in a single clone (SEQ ID NO: 26) Table 6.7.1F(517A01SO CDR2) [Table 20] * A maximum of 0 CDR2 mutations in a single clone (SEQ ID NO: 41) Table 6.7.1G(517A01SO CDR3) [Table 21] * Up to two CDR mutations (SEQ ID NO: 55) in a single clone.

[0224] 6.7.2 Humanization For humanization, the nanobody sequence was made more homologous to the human IGHV3-IGHJ germline consensus sequence. Specific amino acids in the framework regions that differ between the nanobody and the human IGHV3-IGHJ germline consensus sequence (excluding the nanobody "Hallmark" residue) were replaced with their human counterparts, while maintaining the original protein structure, activity, and stability.

[0225] 6.7.3 Existing antibodies and anti-drug antibodies The inventors conducted an early risk assessment of immunogenicity-related clinical outcomes to guide their sequence optimization strategy. The assessment included both the potential immunogenicity of the drug candidate and the potential impact of anti-drug antibodies based on the mechanism of action and the nature of the final biotherapeutic molecule. The nanobody sequences were evaluated to (i) minimize binding of any naturally occurring existing antibodies and (ii) reduce the likelihood of inducing an immunogenic response that may arise from treatment. Mutations L11V and V89L were introduced into all MMP13 nanobodies.

[0226] 6.7.4 Preferred SO clones In Tables A-1 and A-2, the sequences were plotted based on sequence optimization of the read panel, where the sequences were detailed considering PTM, humanization, and potential existing antibody and anti-drug antibody epitopes.

[0227] 6.8 Bispecific constructs Anti-MMP13 nanobodies need to be preferentially active in target sites such as joints in order to inhibit the cartilage-degrading function of MMP13. Aggrecan is abundant in joints, and the main proteoglycans of the extracellular matrix (ECM) account for approximately 50% of the total protein content. Therefore, at least theoretically, immobilizing anti-MMP13 nanobodies on an aggrecan binder can induce the anti-MMP13 nanobodies to the relevant tissues and improve their retention. The objective of this study was to test the efficacy of the resulting bispecific constructs, obtained by combining the anti-MMP13 conjugate of the present invention with these aggrecan conjugates, using human fluorescence-generating peptide assays and competitive ELISA assays. Various bispecific constructs containing aggrecan conjugates and MMP13 inhibitors were generated and tested as shown in the table. Typical result formats and validity are shown in Tables 6.8A and 6.8B.

[0228] Table 6.8A: Bispecific MMP13-CAP nanobodies were tested using human fluorescence-generating peptide assays. Efficacy was compared to their respective single-specific counterparts. TIMP-2 and MSC2392891A were used as internal references in each assay. SO: Optimized sequence, excluding pre-Ab mutations. SOvar: Non-final SO variant. [Table 22]

[0229] Table 6.8B: Bispecific MMP13-CAP nanobodies were tested using competitive ELISA assays. Efficacy was compared to their respective single-specific counterparts (ALB26 fusions). SO: Optimized sequence (excluding pre-Ab mutations). SOvar: Non-final SO variant. [Table 23]

[0230] The results shown in Tables 6.8A and 6.8B indicate that combining aggrecan conjugates with MMP13 inhibitors does not adversely affect the efficacy of the MMP13 inhibitors. In particular, in most cases, the lead panel MMP13 inhibitors have the same efficacy as the non-selective MMP inhibitor TIMP-2.

[0231] Example 6.9 DMOAD study in an in vivo rat MMT model To further demonstrate the in vivo efficacy of the MMP13 inhibitor fused to the CAP binder of the present invention, a surgically induced medial meniscal tear (MMT) model was used in rats. Briefly, an anti-MMP13 nanobody was conjugated to the CAP binder ("754" or C010100754). One knee of rats was surgically treated to induce OA-like symptoms. Treatment was initiated with an IA injection on postoperative day 3. Histopathology was performed on postoperative day 42. Intermediate and final serum samples were collected for exploratory biomarker analysis. The width of medial and overall parenchymal cartilage degeneration, as well as the rate of reduction in cartilage degeneration, were determined. Twenty animals were used per group. Figure 4 shows the suppression of cartilage breakdown in the medial tibia. The results show that cartilage width was significantly reduced after 42 days with the MMP13-CAP construct compared to the vehicle. These results suggest the following: (a) The CAP portion does not adversely affect the activity of the anti-MMP13 nanobody (754), consistent with the results of Example 6.8; (b) The CAP portion enables the retention of anti-MMP13 nanobodies; and (c) Anti-MMP13 nanobodies have a positive effect on cartilage width.

[0232] [Table 24] Figure 4. Inhibition of cartilage degradation by nanobodies in a rat MMT model. The entire contents of all references cited throughout this application (including references to documents, issued patents, published patent applications, and concurrently pending patent applications) are expressly incorporated herein by reference, in particular with respect to the teachings referenced above.

[0233] Table A-1: ​​Amino acid sequences of anti-MMP13 inhibitors ("ID" refers to the sequence number used herein) [Table 25-1] [Table 25-2] [Table 25-3]

[0234] Table A-2: Sequences of CDRs and frameworks, and preferred combinations provided by Formula I, namely FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (hereafter, "ID" refers to a given sequence number; the first column refers to the ID of the entire ISVD). [Table 26-1] [Table 26-2]

[0235] Table A-3: Amino acid sequences of anti-MMP13 constructs ("ID" refers to the sequence number used herein). [Table 27]

[0236] Table B [Table 28-1] [Table 28-2]

[0237] Table C: Various linker sequences (where "ID" refers to the sequence number used in this specification) [Table 29]

[0238] Table D: Serum albumin-bound ISVD sequences ("ID" refers to the sequence number used herein), including CDR sequences. [Table 30-1] [Table 30-2]

[0239] Table E: Aggrecan-binding ISVD sequences (where "ID" refers to the sequence number used herein), including CDR sequences. [Table 31]

[0240] Table F: Multivalent constructs (where "ID" refers to the sequence number used herein), including CDR sequences. [Table 32-1] [Table 32-2] [Table 32-3]

Claims

1. A polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that binds to a matrix metalloproteinase (MMP).

2. The polypeptide according to claim 1, wherein the MMP is selected from the group consisting of MMP13 (collagenase), MMP8 (collagenase), MMP1 (collagenase), MMP19, and MMP20 (enamelicin), and preferably the MMP is MMP13.

3. The polypeptide according to claim 2, wherein the ISVD that binds to MMP13 does not bind to MMP1 or MMP14 (membrane type).

4. The polypeptide according to claim 3, wherein the ISVD bound to MMP13 includes three complementarity-determining regions (CDR1 to CDR3, respectively), where (i) CDR1 is sequence numbers 27, 28, 25, 26, 23, 29, 30, 31, 32, 33, 34, 35, 36 and 24; and Selected from the group consisting of amino acid sequences having one, two, or three amino acid differences from sequence numbers 27, 28, 25, 26, 23, 29, 30, 31, 32, 33, 34, 35, 36, and 24; (ii) CDR2 is sequence numbers 42, 43, 40, 41, 37, 44, 45, 46, 47, 48, 49, 50, 51, 38 and 39; and Selected from the group consisting of amino acid sequences having one, two, or three amino acid differences from sequence numbers 42, 43, 40, 41, 37, 44, 45, 46, 47, 48, 49, 50, 51, 38 and 39; and (iii) CDR3 is sequence numbers 56, 107, 57, 54, 106, 55, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 53; and Selected from the group consisting of amino acid sequences having 1, 2, 3 or 4 amino acid differences from sequence numbers 56, 107, 57, 54, 106, 55, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 53, The aforementioned polypeptide.

5. The polypeptide according to claim 4, wherein (i) CDR1 is selected from the following group: (a) Sequence ID 23; and (b) An amino acid sequence having one amino acid difference from sequence number 23, where, -At position 7, Y has been changed to R; (ii) CDR2 is selected from the following group: (c) Sequence ID 37; and (d) an amino acid sequence having one, two, or three amino acid differences from sequence number 37, where the amino acid differences are defined as follows: - At position 4, V is changed to T; and / or - At position 5, G is changed to A; and / or -At position 9, N has been changed to H; and (iii) CDR3 is selected from the following group: (e) Sequence ID 52; and (f) An amino acid sequence having one amino acid difference from sequence number 52, i.e., in which Y is changed to S at position 6. The aforementioned polypeptide.

6. The polypeptide according to claim 4, wherein (i) CDR1 is sequence number 26; (ii) CDR2 is sequence number 41; and (iii) CDR3 is selected from the following group: (e) Sequence ID 55; and (f) An amino acid sequence having one or two amino acid differences from sequence number 55, where the amino acid difference is defined as follows: - At position 8, N is changed to Q or S; and / or - At position 19, N is changed to V or Q. The aforementioned polypeptide.

7. The polypeptide according to claim 4, wherein (i) CDR1 is sequence number 28; (ii) CDR2 is sequence number 43; and (iii) CDR3 is selected from the following group: (e) Sequence ID 57; and (f) An amino acid sequence having 1, 2, 3, or 4 amino acid differences from sequence number 57, where amino acid differences are defined as follows: - At position 10, D is changed to E, G, A, P, T, R, M, W or Y; and / or - At position 16, M is changed to A, R, N, D, E, Q, Z, G, I, L, K, F, P, S, W, Y, or V; and / or - At position 17, D is changed to A, R, N, C, E, Q, Z, G, H, I, L, K, M, S, T, W, Y, or V; and / or - At position 18, Y is changed to A, R, N, D, C, E, Q, Z, G, H, I, L, K, M, F, P, S, T, W, or V. The aforementioned polypeptide.

8. A polypeptide according to any one of claims 1 to 16, wherein: -CDR1 is selected from the group consisting of sequence numbers 27, 28, 25, 26, 23, 29, 30, 31, 32, 33, 34, 35, 36 and 24; -CDR2 is selected from the group consisting of sequence numbers 42, 43, 40, 41, 37, 44, 45, 46, 47, 48, 49, 50, 51, 38 and 39; and -CDR3 is selected from the group consisting of sequence numbers 56, 107, 57, 54, 106, 55, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 53. The aforementioned polypeptide.

9. The polypeptide according to claim 17, wherein the ISVD comprises a combination of CDR1, CDR2, and CDR3 selected from the group consisting of: - CDR1 is sequence number 27, CDR2 is sequence number 42, and CDR3 is sequence number 56; - CDR1 is sequence number 28, CDR2 is sequence number 43, and CDR3 is sequence number 107; - CDR1 is sequence number 28, CDR2 is sequence number 43, and CDR3 is sequence number 57; - CDR1 is sequence number 25, CDR2 is sequence number 40, and CDR3 is sequence number 54; -CDR1 is sequence number 26, CDR2 is sequence number 41, and CDR3 is sequence number 106; - CDR1 is sequence number 26, CDR2 is sequence number 41, and CDR3 is sequence number 55; -CDR1 is sequence number 23, CDR2 is sequence number 37, and CDR3 is sequence number 52; - CDR1 is sequence number 26, CDR2 is sequence number 48, and CDR3 is sequence number 62; -CDR1 is sequence number 26, CDR2 is sequence number 41, and CDR3 is sequence number 63; - CDR1 is sequence number 29, CDR2 is sequence number 44, and CDR3 is sequence number 58; - CDR1 is sequence number 30, CDR2 is sequence number 45, and CDR3 is sequence number 58; -CDR1 is sequence number 31, CDR2 is sequence number 46, and CDR3 is sequence number 59; - CDR1 is sequence number 32, CDR2 is sequence number 47, and CDR3 is sequence number 60; -CDR1 is sequence number 33, CDR2 is sequence number 41, and CDR3 is sequence number 61; - CDR1 is sequence number 34, CDR2 is sequence number 49, and CDR3 is sequence number 64; - CDR1 is sequence number 35, CDR2 is sequence number 50, and CDR3 is sequence number 65; -CDR1 is sequence number 36, CDR2 is sequence number 51, and CDR3 is sequence number 66; -CDR1 is sequence number 23, CDR2 is sequence number 39, CDR3 is sequence number 53; and - CDR1 is sequence number 24, CDR2 is sequence number 38, and CDR3 is sequence number 52. The aforementioned polypeptide.

10. The polypeptide according to any one of claims 1 to 9, wherein the ISVD comprises four framework regions (FR1 to FR4, respectively) and three complementarity determination regions CDR1, CDR2, and CDR3, or is essentially composed of these.

11. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide is SEQ ID NO: 1 or a polypeptide having at least 95% sequence identity with SEQ ID NO:

1.

12. The polypeptide according to any one of claims 4, 8, 9, and 10, wherein CDR1 is sequence number 27, CDR2 is sequence number 42, and CDR3 is sequence number 56.

13. The polypeptide according to any one of claims 4 to 10 and 12, wherein ISVD is selected from the group consisting of SEQ ID NOs: 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, and 8.

14. When the polypeptide is determined, for example, by KinExA, K Ｄ is 1E －０７ M to 1E －１３ M, for example, 1E －０８ M to 1E －１２ M, preferably at most 1E －０７ M, preferably 1E －０８ M or 1E －０９ M less than, or 1E －１０ M less than, for example, 5E －１１ M, 4E －１１ M, 3E －１１ M, 2E －１１ M, 1.7E －１１ M, 1E －１１ M, etc., or 5E －１２ M, 4E －１２ M, 3E －１２ M, 1E －１２ M, and binds to MMP13, the polypeptide according to any one of claims 1 to 13.

15. When polypeptides are determined by, for example, competitive ELISA, TIMP-2 competitive ELISA, fluorescence-generating peptide assay, fluorescence-generating collagen assay, or collagen degradation assay, IC ５０ ga 1E －０７ M-1E －１２ During M, for example, 1E －０８ M-1E －１１ A polypeptide according to any one of claims 1 to 14, which inhibits the activity of MMP13 between M.

16. Polypeptides, IC ５０ up to 1E －０７ M, preferably 1E －０８ M, 5E －０９ M, or 4E －９ M, 3E －９ M, 2E －９ M, for example, 1E －９ The polypeptide according to claim 15, which inhibits the activity of MMP13 with M, etc.

17. If the polypeptide is determined by, for example, ELISA, then EC ５０ ga 1E －０７ M-1E －１２ During M, for example, 1E －０８ M-1E －１１ A polypeptide according to any one of claims 1 to 16, which binds to MMP13 between M.

18. If the polypeptide is determined, for example, by SPR, the off-rate is 5E －０４ s －１ A polypeptide according to any one of claims 1 to 14, which binds to MMP13 at a level less than [value missing].

19. The polypeptide according to any one of claims 1 to 18, wherein MMP13 is human MMP13, rat MMP13, canine MMP13, bovine MMP13, cynomolgus monkey MMP13, preferably human MMP13, preferably SEQ ID NO:

115.

20. The polypeptide according to any one of claims 1 to 19, wherein the polypeptide antagonizes the activity of MMP13, for example (i) antagonizes protease activity, preferably aggrecan and / or collagen cleavage, where the collagen is preferably collagen II; and (ii) antagonizes the binding of collagen to the hemopexin-like domain.

21. The polypeptide according to claim 20, wherein the polypeptide blocks the binding of MMP13 to collagen and / or aggrecan by at least 20%, for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

22. The polypeptide according to any one of claims 1 to 20, wherein the polypeptide inhibits the protease activity of MMP13, inhibiting the proteolysis of substrates such as aggrecan, collagen II, collagen I, collagen III, collagen IV, collagen IX, collagen X, collagen XIV and / or gelatin, wherein the collagen is preferably type II collagen.

23. The polypeptide according to any one of claims 1 to 22, comprising at least two ISVDs, wherein at least one ISVD specifically binds to an MMP, preferably an MMP13.

24. The polypeptide according to claim 23, wherein at least two ISVDs specifically bind to an MMP, preferably an MMP13.

25. A polypeptide comprising two or more ISVDs, each individually specifically bound to MMP13, wherein a) At least the "first" ISVD specifically binds to the first antigenic determinant, epitope, portion, domain, subunit, or conformation of MMP13; and here, b) At least the “second” ISVD specifically binds to a second antigenic determinant, epitope, partial, domain, subunit, or conformation of MMP13, which is distinct from the first antigenic determinant, epitope, partial, domain, subunit, or conformation; The aforementioned polypeptide.

26. The polypeptide according to claim 25, wherein the "first" ISVD that specifically binds to MMP13 is selected from the group consisting of SEQ ID NOs: 111, 11, 110, 10, 112, 12, 109, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.

27. The polypeptide according to claim 25 or 26, wherein the "second" ISVD that specifically binds to MMP13 is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8.

28. The polypeptide according to any one of claims 1 to 27, further comprising, preferably, at least one ISVD specifically bound to aggrecan, selected from the group consisting of SEQ ID NOs: 166, 167, and 168.

29. The polypeptide according to any one of claims 1 to 28, comprising at least two ISVDs that specifically bind to aggrecan.

30. The polypeptide according to claim 29, wherein at least two ISVDs that specifically bind to aggrecan may be the same or different.

31. The polypeptide according to claim 29 or 30, wherein at least two ISVDs that specifically bind to aggrecan are independently selected from the group consisting of SEQ ID NOs: 166 to 168.

32. The polypeptide according to any one of claims 28 to 31, wherein the ISVD that specifically binds to aggrecan specifically binds to human aggrecan [SEQ ID NO: 105].

33. The polypeptide according to any one of claims 28 to 32, wherein the ISVD that specifically binds to aggrecan specifically binds to canine aggrecan, bovine aggrecan, rat aggrecan; porcine aggrecan; mouse aggrecan, rabbit aggrecan; cynomolgus monkey aggrecan and / or rhesus monkey aggrecan.

34. The polypeptide according to any one of claims 28 to 33, wherein ISVD, which specifically binds to aggrecan, preferably binds to cartilage and / or cartilaginous tissue such as the meniscus.

35. The polypeptide according to any one of claims 1 to 34, wherein the polypeptide is stable in synovial fluid (SF) at 37°C for at least 7 days, for example, 14 days, 21 days, 1 month, 2 months, or 3 months.

36. The polypeptide according to any one of claims 1 to 35, further comprising ISVD that binds to serum albumin.

37. The polypeptide according to claim 36, wherein the ISVD that binds to serum albumin essentially consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), where CDR1 is SEQ ID NO: 157, CDR2 is SEQ ID NO: 158, and CDR3 is SEQ ID NO:

159.

38. The polypeptide according to claim 37, wherein the ISVD that binds to serum albumin is selected from the group consisting of ALB135 (SEQ ID NO: 193), ALB129 (SEQ ID NO: 144), ALB8 (SEQ ID NO: 142), ALB23 (SEQ ID NO: 143), and ALB132 (SEQ ID NO: 145).

39. The polypeptide according to any one of claims 23 to 38, wherein at least two ISVDs are linked to each other directly or via a linker.

40. The polypeptide according to claim 39, wherein the linker is selected from the group consisting of SEQ ID NOs: 125 to 141, preferably SEQ ID NO:

129.

41. A polypeptide according to any one of claims 1 to 40, further comprising C-terminal elongation.

42. The polypeptide according to claim 41, wherein the C-terminal extension is C-terminal extension (X)n, where n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4 or 5 (and preferably 1 or 2, for example 1); and each X is an independently selected (preferably naturally occurring) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

43. The polypeptide according to any one of claims 1 to 42, wherein the polypeptide has at least 80%, 90%, 95%, or 100% sequence identity with any one of sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 109, 110, 111, 112, 160, 161, 162, 163, 164, 165, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, or 192.

44. For example, a method for treating or preventing a disease or disorder of an individual in which MMP13 activity is involved, the method comprising administering to the individual a polypeptide according to any one of claims 1 to 43 in an amount effective for treating or preventing the symptoms of the disease or disorder.

45. The method according to claim 44, wherein the disease or disorder is selected from the group consisting of arthropathy and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and agricanopathies.

46. A polypeptide according to any one of claims 1 to 43, for use as a pharmaceutical.

47. A polypeptide according to any one of claims 1 to 43 for use in treating or preventing conditions comprising arthropathy and chondrodysplasia, arthritis, such as osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumatic laceration or detachment, chondrodysplasia, costochondritis, spondyloepiphyseal dysplasia, herniated disc, lumbar disc degenerative disease, degenerative joint disease, and relapsing polychondritis, osteochondritis dissecans and agricanopathies.

48. A polypeptide according to any one of claims 1 to 43, wherein the polypeptide cross-blocks the binding of at least one polypeptide represented by any one of sequence numbers 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, and 8 to MMP13, and / or the binding of at least one polypeptide represented by any one of sequence numbers 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, and 8 to MMP13.

49. The binding of a polypeptide to MMP13 by any one of the polypeptides represented by sequence numbers 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, and 8 is cross-blocked and / or at least by sequence numbers 111, 11, 112, 12, 109, 9, 110, 10, 1, 13, 14, 15, 1 The binding to MMP13 is cross-blocked by a polypeptide represented by any one of 6, 17, 18, 19, 20, 21, 22, 2, 3, 4, 5, 6, 7, and 8, wherein the polypeptide comprises at least one VH, VL, dAb, immunoglobulin single variable domain (ISVD) that specifically binds to MMP13, and the binding to MMP13 modulates the activity of MMP13.

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