Novel Type II Collagen-Binding Protein

Abstract

The present invention relates to novel type II collagen αI chain-specific binding proteins. The invention further relates to type II collagen binding proteins fused or conjugated to further therapeutically or diagnostically active moieties. Further aspects of the invention include the use of these type II collagen binding proteins in medicine.

Description

[Technical Field]

[0001] This invention relates to a novel type II collagen αI chain-specific binding protein. The invention further relates to type II collagen-binding proteins fused to or bound to therapeutically or diagnostically effective components. Further aspects of the invention encompass the use of these type II collagen-binding proteins in medicine. [Background technology]

[0002] Collagen is a structural protein that forms a three-dimensional network structure. Type II collagen forms homotrimers, resulting in fibrous structures. Among the many types of collagen, type II collagen forms the basis of articular cartilage and hyaline cartilage. It makes up 85-90% of the collagen in articular cartilage and 65% of the collagen in the vitreous fluid of the eye. Type II collagen is an important clinical target, particularly in eye diseases and joint diseases.

[0003] Short collagen-binding domains derived from bacterial proteins or other naturally occurring collagen-interacting proteins have been proposed for specific medical applications, including collagen diseases, tissue regeneration, heart disease, or cancer.

[0004] However, a drawback of collagen-binding domains derived from bacterial proteins or other naturally occurring collagen-interacting proteins is their broad cross-reactivity with different types of collagen.

[0005] Diseases affecting the structures of the eye and joints present challenges in terms of drug accessibility. Due to low perfusion, parenteral or oral diagnostic and therapeutic agents do not reach sufficient amounts of the articular cartilage or the vitreous humor of the eye. Topical application to these structures is possible, but a drawback of topical application is that therapeutic agents are generally eliminated from the body too quickly over time, perhaps even before they exhibit significant pharmacological effects.

[0006] Therapeutic or diagnostic interventions involving injections into the vitreous humor or joints of the eye are equally inconvenient for both the patient and the physician. Such interventions are high-risk and painful for the patient.

[0007] Needless to say, there is a strong need to relieve patients and clinics, especially in the case of eye and joint diseases, of frequent, burdensome, and unavoidable treatments.

[0008] Therefore, there is a need to find procedures for more effective treatment and diagnosis, particularly for more effective drug delivery to the eyes and joints. One objective of the present invention is to provide molecules that allow therapeutic agents to remain near diseased tissue, thereby prolonging their beneficial effects.

[0009] To overcome the shortcomings of currently known strategies for treating eye and joint diseases, as well as other ailments, novel collagen-specific binding proteins should possess properties such as specific affinity for type II collagen. In particular, with regard to eye and joint diseases, longer retention times are desirable for more effective treatment, especially considering the burdensome and potentially dangerous application. Therefore, longer local retention of the applied substance is highly desirable to reduce the frequency of interventions and alleviate the burden of treatment.

[0010] Hulme et al. (Journal of Orthopaedic Research 2017, Vol.36(4), pp.1238-1247) describes the type II collagen-binding Avimer™ domain. Rothenflu et al. (Nature Materials 2008, Vol.7(3), pp.248-254) describes a type II collagen ligand with the six-amino acid sequence "WYRGRL". Formica et al. (Journal of Controlled Release 2019, Vol.295, pp.118-129) is concerned with the "WYRGRL" ligand for a cartilage-targeting dexamethasone prodrug. Brown et al. (Acta Biomater. 2019, Vol.93, pp.239-257) is a review article on intra-articular targeting of nanomaterials for the treatment of osteoarthritis.

[0011] This invention provides a novel type II collagen-binding molecule for improved new strategies in the treatment and diagnosis of various diseases. Binding proteins with specificity for type II collagen can enable less medical intervention and safer treatment in several severe diseases, thereby improving quality of life.

[0012] The above objectives and benefits are achieved by the subject matter of the enclosed claims. The present invention addresses the needs presented above by providing an example of a type II collagen-binding protein. The above summary does not necessarily describe all the problems that the present invention solves. [Overview of the project]

[0013] This disclosure provides, without limitation, the following items [1] to

[12] : [1] A type II collagen-binding protein comprising an amino acid sequence having at least 80% sequence identity with any one selected from the group of SEQ ID NOs: 1 to 44, including a type II collagen-binding protein comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15, wherein (i) glutamine (Q) is located at the position corresponding to the 25th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) is located at the position corresponding to the 29th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) is located at the position corresponding to the 33rd position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15. [2] Two, three, four, five, or six type II collagen-binding proteins linked together, as described in item [1]. [3] A binding protein to type II collagen as described in item [2], which is either a homopolymer or a heteropolymer. [4] A binding protein to type II collagen described in item [3], which has an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 27-42. [5] Binding affinity to type II collagen less than 10 μM (K D A type II collagen-binding protein described in any one of the items [1] to [4] that have the following characteristics: [6] A dimer or polymer protein containing a type II collagen-binding protein as described in any one of items [1] to [5]. [7] Monoclonal antibodies and fragments thereof, binding proteins, receptors and receptor domains, receptor-binding ligands and antagonists, extracellular domains and fragments thereof of receptors, non-immunoglobulin-binding proteins, cytokines, chemokines, cytotoxic compounds, enzymes and derivatives thereof, and radionuclides, and type II collagen-binding proteins described in any one of items [1] to [5], or dimers or multimers described in item 6, further fused or bound to at least one therapeutically effective portion, selected at will from any combination of the above. [8] Radionuclides, fluorescent compounds, photosensitizers, tags, enzymes, and non-immunoglobulin-binding proteins, and type II collagen-binding proteins described in any one of items [1] to [5] and [7], or dimeric or polymeric proteins described in item [6] or [7], further fused or bound to at least one diagnostic portion selected at will from any combination of the above. [9] A type II collagen-binding protein as described in any one of items [1]-[5], [7], and [8], preferably for use in medicine, for use in the diagnosis or treatment of diseases such as eye diseases or joint diseases.

[10] A composition comprising a type II collagen-binding protein as described in any one of items [1]-[5], [7], and [8], or a dimer or polymer protein as described in any one of items [6] to [8].

[11] A pharmaceutical composition for the treatment of a disease comprising a type II collagen-binding protein, or a dimer or polymer protein defined in any of the above embodiments, and a therapeutically or diagnostically acceptable carrier and / or diluent.

[12] A method for producing a type II collagen-binding protein, or a dimer or polymer protein, defined in any of the above embodiments, comprising culturing host cells under conditions suitable for obtaining a type II collagen-binding protein and optionally isolating the type II collagen-binding protein.

[0014] This summary does not necessarily describe all features of the present invention. Other embodiments will become apparent from the detailed description that follows. [Brief explanation of the drawing]

[0015] [Figure 1]Figure 1A shows the amino acid sequence of type II collagen-binding protein. The numbers in the top row indicate the position of the corresponding amino acid in the Ig-binding protein. Figure 1B shows the reference sequences of SEQ ID NOs. 2, 10, 11, 12, 14, and 15, which contain the motif 25Q-29Y-33W. Figure 1C shows the dimeric type II collagen-binding protein containing the motif 25Q-29Y-33W, where "monomer 1" represents the N-terminal portion and "monomer 2" represents the C-terminal portion. The monomer domains containing the motif 25Q-29Y-33W are shown in grayscale. [Figure 2] Figure 2 shows highly specific binding to type II collagen, as determined by ELISA. Binding to type I, type III, or type V collagen, and to the unrelated fibronectin domain EDB (67B89), was not observed. Figure 2A shows the binding analysis of the dimer of SEQ ID NO: 2 (CID211999) (a dimer of directly linked monomers). Figure 2B shows the binding analysis of the dimer of SEQ ID NO: 2 (CID212000) (a dimer with a linker between monomers). Figure 2C shows the binding analysis of the tetramer of SEQ ID NO: 2 (CID212001). Figure 2D shows the binding analysis of the dimer of SEQ ID NO: 15 (CID212705). Figure 2E shows the binding analysis of the tetramer of SEQ ID NO: 15 (CID212704). [Figure 3] Figure 3 shows that the tetramer of Sequence ID No. 2 (CID212001) binds to type II collagen with high affinity, as demonstrated by SPR binding analysis. [Figure 4] Figure 4 shows the binding analysis of 211519 (dimer of SEQ ID NO: 11) by SPR. Figure 4A shows the concentration series of 211519 (SEQ ID NO: 37). Binding to type II collagen was confirmed by a transient binding signal from multicycle via core measurements. Figure 4B shows the equilibrium fit of amplitude-KD (amplitude-KD) for type II collagen at 780 nM. Figure 4C shows that 211519 (homodimer of SEQ ID NO: 11) is specific to type II collagen. Binding to type I, type III, and type V collagen was not observed (analysis by SPR). [Figure 5] Figure 5 shows immunohistochemical staining of a pig eye section. Type II collagen-binding protein SEQ ID NO: 39 (dimer of SEQ ID NO: 2, intermer linker, CID212000) stains the retina, internal limiting membrane (arrow), and vitreous humor of the pig eye. [Modes for carrying out the invention]

[0016] The proteins of the present invention are specifically bound to ocular type II collagen by non-immunoglobulin-binding proteins. The inventors of the present invention have created a solution to the ongoing need in the art by providing type II collagen-specific proteins comprising an amino acid sequence having at least 80% sequence identity with any one selected from the group of SEQ ID NOs: 1-26, 43, and 44. Type II collagen-specific proteins as defined herein are functionally characterized by their high affinity for type II collagen. In particular, the present invention provides type II collagen-binding proteins comprising an amino acid sequence having at least 80% sequence identity with any one selected from SEQ ID NOs: 1-44. Novel type II collagen-specific proteins can broaden therapeutic and diagnostic options. In some embodiments, at least two type II collagen-binding proteins link together to form a polymer, for example, shown in SEQ ID NOs: 27-42.

[0017] Before describing the present invention in more detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may change. It should also be understood that the terms used herein are solely for the purpose of describing specific aspects and embodiments and are not intended to limit the scope of the present invention as reflected in the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. This includes those skilled in the fields of protein engineering and purification, as well as those skilled in the fields of development of novel target-specific binding molecules for technical applications and for therapeutic and diagnostic use.

[0018] Preferably, terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommended)," Leuenberger, HGW, Nagel, B., and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010, Basel, Switzerland).

[0019] Throughout this specification and the subsequent claims, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” should be understood to mean including the integer or step, or group of integers or steps, described, but not to mean excluding other integers or steps, or groups of integers or steps. The terms “comprise(s)” or “comprising” may include the limitation “consists of” or “consisting of,” but only if such limitation is necessary to some extent for any reason.

[0020] Several documents (e.g., patents, patent applications, scientific publications, manufacturer specifications, instructions, GenBank accession number registrations, etc.) may be referenced throughout this specification. Nothing in this specification should be construed as acknowledging that the present invention does not have prior rights to such disclosures by prior art. Some of the documents referenced herein may be characterized as “incorporated by reference.” In the event of any conflict between the definitions and teachings in such incorporated documents and the definitions and teachings set forth herein, the text of this specification shall prevail.

[0021] All sequences referenced herein, along with their entire content and disclosure, are disclosed in the accompanying sequence listings, which form part of the disclosures herein.

[0022] General definitions of key terms used in this application As used herein, the terms “Type II collagen αI chain” or “Type II collagen” mean Uniprot accession number P02458.2, amino acids 182-1241 (SEQ ID NO: 51). The term “Type II collagen” includes all polypeptides that exhibit at least 80%, 85%, 90%, 95%, 96%, or 97% or more, or 100% sequence identity with SEQ ID NO: 51 and possess the functionality of Type II collagen. In particular, this term includes Type II collagen from other mammalian species that exhibit at least 80%, 85%, 90%, 95%, 96%, or 97% or more, or 100% amino acid sequence identity with SEQ ID NO: 51. In preferred embodiments, as used herein, the term “Type II collagen” means human Type II collagen, in accordance with the reference to Type II collagen identified under Uniprot accession number P02458.2.

[0023] The terms "Type II collagen-binding protein," "protein that binds to Type II collagen," or "Col2 BP" refer to proteins that bind to Type II collagen with high affinity.

[0024] The terms “protein” and “polypeptide” mean any chain of two or more amino acids linked by peptide bonds, and do not imply a specific length of the product. Therefore, any other term used to refer to “peptide,” “protein,” “amino acid chain,” or any other term used to refer to a chain of two or more amino acids is included within the definition of “protein,” and the term “protein” can be used in place of or interchangeably with any of these terms. Furthermore, the term “protein” is intended to mean the product of post-translational modifications of polypeptides well known in the art.

[0025] The terms "modification" or "amino acid modification" refer to the substitution, deletion, or insertion of a reference amino acid by another amino acid at a specific position in the parent polypeptide sequence. With the known genetic code and recombinant DNA and synthetic DNA technologies, a skilled scientist can easily construct DNA that codes for amino acid variants.

[0026] The terms “binding affinity” and “binding activity” are interchangeable herein and refer to the ability of a polypeptide to bind to another protein, peptide, or fragment or domain thereof. Typically, binding affinity is the equilibrium dissociation constant (K). D ) are measured and reported and used to evaluate and rank the strength of bimolecule interactions.

[0027] The term "fusion protein" refers to a protein that includes at least one first protein that is genetically linked to at least one second protein. Fusion proteins are created by linking two or more genes that originally encoded separate proteins. Fusion proteins may further include additional domains that are not involved in binding to the target, such as, but are not limited to, multimerization sites, polypeptide tags, polypeptide linkers, and half-life extension sites.

[0028] The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or difference) of the amino acid sequences of two or more proteins. "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the sequence that are identical to those of the reference polypeptide sequence, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage of sequence identity. To determine sequence identity, the sequence of the query protein is aligned to the sequence of a reference protein or polypeptide, for example, one of the polypeptides SEQ ID NOs: 1-44. Sequence alignment methods and sequence comparison algorithms are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide to one of the amino acid sequences SEQ ID NOs: 1-44, a SIM Local similarity program known in the art is preferably employed. For multiple alignment analysis, Clustal Omega, known to those skilled in the art, is preferably used.

[0029] Detailed description of embodiments of this invention The type II collagen-binding proteins defined herein contain an amino acid sequence having at least 80% sequence identity with any one selected from the group of SEQ ID NOs: 1 to 44. In particular, provided herein is a type II collagen-binding protein containing an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15, wherein the type II collagen-binding protein has (i) glutamine (Q) at the position corresponding to the 25th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to the 29th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to the 33rd position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and the type II collagen-binding protein has a binding affinity (K) of less than 10 μM to type II collagen. D) has. In various preferred embodiments, the type II collagen-binding protein comprises an amino acid sequence having at least 85% or 89.5% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15, wherein the type II collagen-binding protein has (i) glutamine (Q) at the position corresponding to the 25th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to the 29th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to the 33rd position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and the type II collagen-binding protein has a binding affinity (K) of less than 10 μM to type II collagen. D ) has. In various other preferred embodiments, the type II collagen-binding protein comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15, preferably at least 91%, 92%, 93%, 94%, or 95%, more preferably at least 96%, 97%, 98%, or 99%, wherein the type II collagen-binding protein has (i) glutamine (Q) at the position corresponding to the 25th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to the 29th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to the 33rd position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and the type II collagen-binding protein has a binding affinity (K) of less than 10 μM to type II collagen. D) are present. Such type II collagen-binding proteins exhibit specific binding affinity to type II collagen, i.e., as shown in Figure 2, no nonspecific binding to the domain (67B89) of type I collagen, type III collagen, type V collagen, or fibronectin was detected. In various embodiments, the type II collagen-binding proteins of this disclosure do not have detectable binding affinity to the domain (67B89) of type I collagen, and / or type III collagen, and / or type V collagen, and / or fibronectin, as measured by methods for measuring binding affinity as described elsewhere in this specification. In preferred embodiments, methods for measuring the binding affinity to the domain (67B89) of type I collagen and / or type III collagen and / or type V collagen and / or fibronectin include any of ELISA, SPR, BLI, KinExA assay, flow cytometry, fluorescence spectroscopy, ITC, ultracentrifugation, RIA or IRMA, and ECL, more preferably any of ELISA, SPR, BLI, KinExA assay, and flow cytometry, even more preferably any of ELISA, SPR, and KinExA assay, still more preferably ELISA or SPR, most preferably ELISA. In various embodiments, the type II collagen-binding protein of this disclosure does not have a detectable binding affinity to type I collagen and / or type III collagen. In various other embodiments, the type II collagen-binding protein of this disclosure does not have a detectable binding affinity to type I collagen and type III collagen and / or type V collagen.In various preferred embodiments, the type II collagen-binding protein comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15, and the type II collagen-binding protein has (i) glutamine (Q) at a position corresponding to the 25th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at a position corresponding to the 29th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at a position corresponding to the 33rd position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and has a binding affinity (K D ) for type II collagen of less than 1 μM, more preferably less than 500 nM. In other embodiments, the above type II collagen-binding protein has a binding affinity (K D ) for type II collagen of less than 200 nM, or less than 100 nM, or even less than 50 nM.

[0030] In various embodiments, the type II collagen-binding protein is IAAKFDEAQX 10 AADX 14 EILHLPNLTEQQRX 28 YFRX 32 WLX 35 DDPSVSX 42 X 43 X 44 LX 46 X 47 AQX 50 LNDX 54 QAPK (SEQ ID NO: 52) (wherein, individually of each other, X 10 is selected from S and Q, X 14 is selected from S and K, X 28 is selected from H and N, X 32 is selected from R and Q, X 35 is selected from S and R, X 42 is selected from T and P, X 43 is selected from H and T, X 44 is selected from I and V, X 46is selected from T and G, X 47 is selected from Q and T, X 50 is selected from H and Q, and X 54 (Selected from S and D) They have at least 95% amino acid sequence identity. In some embodiments, the type II collagen-binding proteins have at least 95% amino acid sequence identity with SEQ ID NO: 52, where the 25th position is Q, the 29th position is Y, and the 33rd position is W.

[0031] In various embodiments, type II collagen-binding protein is IAAKFDEAQX 10 AADX 14 EILHLPNLTEQQRX 28 YFRX 32 WLX 35 DDPSVSX 42 X 43 X 44 LX 46 X 47 AQX 50 LNDX 54 QAPK(Sequence ID 52)(In the formula, X 10 is selected from S and Q, X 14 is selected from S and K, X 28 is selected from H and N, X 32 is selected from R and Q, X 35 is selected from S and R, X 42 is selected from T and P, X 43 is selected from H and T, X 44 The is selected from I and V, X 46 is selected from T and G, X 47 is selected from Q and T, X 50 is selected from H and Q, and X 54 (Selected from S and D) It has at least 95% amino acid sequence identity. In some embodiments, the type II collagen-binding protein has at least 95% amino acid sequence identity with SEQ ID NO: 52, where the 25th position is Q, the 29th position is Y, and the 33rd position is W.

[0032] In various embodiments, type II collagen-binding protein is IAAKFDEAQSAADSEILHLPNLTE Q QRH Y FRR W LSDDPSVSTHILTQAQHLNDDQAPK(Sequence ID 2, CID211261) It has at least 95% amino acid sequence identity.

[0033] The type II collagen-binding proteins of the present invention disclosed herein have at least 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the type II collagen-binding proteins of the present invention disclosed herein have at least 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 2, where the 25th position is Q, the 29th position is Y, and the 33rd position is W.

[0034] In various embodiments, the type II collagen-binding protein has at least 95% amino acid sequence identity with SEQ ID NO: 15, CID211519delFc. The type II collagen-binding protein of the present invention disclosed herein is Sequence ID 15 IAAKFDEAQQAADKEILHLPNLTE Q QRN Y FRQ W LRDDPSVSPTVLGTAQQLNDSQAPK It has at least 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 15, provided that the type II collagen-binding protein of the present invention disclosed herein has at least 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 15, provided that the 25th position is Q, the 29th position is Y, and the 33rd position is W.

[0035] Functional characteristics. One embodiment has a binding affinity (K) of less than 10 μM to type II collagen. DThis refers to type II collagen-binding proteins having a dissociation constant K. Type II collagen-binding proteins bind to type II collagen with measurable binding affinities of less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, or less than 50 nM. Suitable methods are known to those skilled in the art or described in the literature. Methods for measuring binding affinity are known in themselves and can be selected from, for example, the following methods known in the art: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), biolayer interferometry (BLI), kinetic exclusion assay (KinExA assay), flow cytometry, fluorescence spectroscopy, isothermal titration calorimetry (ITC), ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Some of these methods are described in the examples below. Typically, the dissociation constant K is D It is measured at 20°C, 25°C, or 30°C. Unless otherwise specifically indicated, K as described herein D The value is measured at 25°C using SPR. K D The lower the value, the greater the binding affinity to the biomolecule's binding partner. D The higher the value, the weaker the bond between the bonding partners.

[0036] Structural characteristics of type II collagen-binding protein In various embodiments, the type II collagen-binding proteins disclosed herein have at least 80% sequence identity with any one amino acid sequence of SEQ ID NOs: 1 to 44. The type II collagen-binding proteins disclosed herein have at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% sequence identity with any one amino acid sequence of SEQ ID NOs: 1 to 44.

[0037] The type II collagen-binding proteins disclosed herein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 44.

[0038] In various embodiments, the type II collagen-binding protein disclosed herein comprises or consists of a protein having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 (CID211190). In various embodiments, the type II collagen-binding protein disclosed herein comprises or consists of a protein having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2 (CID211261). In various embodiments, the type II collagen-binding protein disclosed herein comprises or consists of a protein having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 20 (CID210970). In various embodiments, the type II collagen-binding protein disclosed herein comprises or consists of a protein having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 15 (CID211519delFc). The type II collagen-binding proteins disclosed herein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 44.

[0039] In some embodiments, the type II collagen-binding protein has the amino acid sequence of SEQ ID NO: IAAKFDEAQQLAFYQILHLENLTEEQRNAFIQSLRDDPSVSX 42 EVLGEAX 49 KLNX 53 SQAPK(wherein X may be any amino acid, preferably X 42 is selected from L, A, and T, X 49 is selected from I, R, and E, and X 53 (Selected from L, K, and Y) It contains an amino acid sequence that is at least 80% identical to the original. Specific examples are shown in the dimers of SEQ ID NOs. 6, 7, 8, or SEQ ID NOs. 32, 33, 36.

[0040] In some embodiments, the type II collagen-binding protein has the amino acid sequence of SEQ ID NO: 9: IAAKFDEAQQAAFYEILHLPNLTEQQRNYFIQWLRDDPSVSX 42 X 43 VLGX 47 AQX 50 LNDSQAPK(wherein X may be any amino acid, preferably X 42 is selected from L and P, X 43 is selected from E and T, X 47 is selected from H and T, X 50 (Selected from Q, L, and R) It contains an amino acid sequence that is at least 80% identical to the original. Specific examples are shown in the dimers of SEQ ID NOs. 10-12, 25, or SEQ ID NOs. 27, 32-37.

[0041] In some embodiments, the type II collagen-binding protein has the amino acid sequence of SEQ ID NO: 13: IAAKFDEAQQAAFYEILHLPNLTEQQRNYFIQWLRDDPSVSX 42 X 43 VLGX 47 AQX 50 LNDSQAPK(wherein X may be any amino acid, preferably X 42 is selected from L and P, X 43 is selected from E and T, X 47 is selected from H and T, X 50 (Selected from Q, L, and R) It contains an amino acid sequence that is at least 80% identical to the original. Specific examples are shown in the dimers of SEQ ID NOs. 14, 15, 23, 28, 29, 30, 31, and 30, and the tetramer of SEQ ID NO. 42.

[0042] In some embodiments, the type II collagen-binding protein has the amino acid sequence of SEQ ID NO: 16: IAAKFDEAQWAAX 13 LEIVHLPNLTEEQRLX 29 FX 31 YSLRDDPSVSLEVLGEAQKLNDSQAPK (wherein X may be any amino acid, preferably X)13 The selection is made from D and F, and X 29 is selected from K and A, X 31 (Selected from R and I) It contains an amino acid sequence that is at least 80% identical to it. Specific examples are shown in the dimers of SEQ ID NOs. 17-21, or SEQ ID NOs. 28, 29, 30, 31, and 35.

[0043] In some embodiments, the type II collagen-binding protein has the amino acid sequence of SEQ ID NO: 43: NAAXXDXAQXSAXXEIXXLPNLTTRQKQKFIYSLQRDPSVSKEVLGEAQKLNDSQAPK It contains an amino acid sequence that is at least 80% identical to it. Specific examples are shown in SEQ ID NOs: 1 and 26.

[0044] In some embodiments, the type II collagen-binding protein has the amino acid sequence of SEQ ID NO: 44: IAAKFDEAQSAADSEILHLPNLTEQQRHYFRRWLSDDPSVSXXXLXXAQXLNDXQAPK It contains an amino acid sequence that is at least 80% identical to it. Specific examples are shown in the dimers of SEQ ID NOs. 2, 24, 38, and 39, and the tetramer of SEQ ID NO. 41.

[0045] In various embodiments of the present invention, the type II collagen-binding proteins disclosed herein may have one or more, preferably two, amino acid residue deletions at the N-terminus of any of SEQ ID NOs: 1-44. In various embodiments of the present invention, the type II collagen-binding proteins disclosed herein may have one or more, preferably two, amino acid residue deletions at the N-terminus of any of the monomer sequences of SEQ ID NOs: 1-26, 43, and 44, as exemplified by SEQ ID NOs: 22-26. In preferred embodiments of the dimeric and polymeric type II collagen-binding proteins disclosed herein, only the monomer located at the N-terminus of the dimer or polymer may have one or more, preferably two, amino acid residue deletions at the N-terminus of that monomer. In various embodiments of the present invention, the monomeric type II collagen-binding proteins disclosed herein contain at least 47 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44. In various other embodiments of the present invention, the monomeric type II collagen-binding proteins disclosed herein contain at least 48 or 49 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44. In yet another embodiment of the present invention, the monomeric type II collagen-binding protein disclosed herein comprises at least 50 or 51 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44. In various preferred embodiments of the present invention, the monomeric type II collagen-binding protein disclosed herein comprises at least 52 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44. In various other preferred embodiments of the present invention, the monomeric type II collagen-binding protein disclosed herein comprises at least 53 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44. More preferably, the monomeric type II collagen-binding protein disclosed herein comprises at least 54 or 55 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44. In a particularly preferred embodiment of the present invention, the monomeric type II collagen-binding protein disclosed herein comprises at least 56 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44.In various preferred embodiments of the present invention, the monomeric type II collagen-binding protein disclosed herein comprises at least 57 or even 58 amino acid residues from any of SEQ ID NOs: 1-26, 43, and 44.

[0046] In various embodiments of the present invention, the type II collagen-binding proteins disclosed herein may have one or more, preferably two, amino acid residue deletions at the N-terminus of any of the monomer sequences of SEQ ID NOs: 1-26, 43, and 44, as exemplified by SEQ ID NOs: 22-26. In preferred embodiments of the dimeric and polymeric type II collagen-binding proteins disclosed herein, only the monomer located at the N-terminus of the dimer or polymer may have one or more, preferably two, amino acid residue deletions.

[0047] Figure 1C illustrates a dimeric type II collagen-binding protein of the present invention, wherein at least one of the two monomeric domains is characterized by the motif 25Q-29Y-33W. Thus, in various embodiments, a dimeric or multimeric type II collagen-binding protein of the present invention comprises at least one monomer that is a type II collagen-binding protein having an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15, wherein the monomeric type II collagen-binding protein has (i) glutamine (Q) at the position corresponding to the 25th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to the 29th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to the 33rd position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15. In various preferred embodiments, the monomeric type II collagen-binding protein contained in the dimeric or polymeric type II collagen-binding protein of the present invention has a binding affinity (K) of less than 10 μM to type II collagen, as described elsewhere in this specification. D) It has. The monomeric type II collagen-binding protein contained by the dimeric or multimeric type II collagen-binding protein of the present invention may be located at the N-terminus or the C-terminus of the dimeric or multimeric type II collagen-binding protein.

[0048] The multimeric type II collagen-binding protein of the present invention can contain one or more monomeric type II collagen-binding proteins having at least 80% sequence identity with any of SEQ ID NOs: 1-26, 43 and 44, and can contain one or more monomeric type II collagen-binding proteins having at least 80% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15. The monomeric type II collagen-binding protein has (i) glutamine (Q) at the position corresponding to the 25th position of any of SEQ ID NOs: 2, 10, 11, 12, 14 and 15; (ii) tyrosine (Y) at the position corresponding to the 29th position of any of SEQ ID NOs: 2, 10, 11, 12, 14 and 15; and (iii) tryptophan (W) at the position corresponding to the 33rd position of any of SEQ ID NOs: 2, 10, 11, 12, 14 and 15. The monomeric type II collagen-binding protein has a binding affinity (K D ) It has.

[0049] The monomeric type II collagen-binding protein contained by the multimeric type II collagen-binding protein of the present invention may or may not be located at the N-terminus or C-terminus of the multimeric type II collagen-binding protein.

[0050] In various embodiments, the binding protein to type II collagen provided by the present invention comprises an amino acid sequence having at least 80% sequence identity with any one of the dimers of SEQ ID NOs. 27-40, wherein at least one of the two monomers has (i) glutamine (Q) at the position corresponding to position 25 of any of SEQ ID NOs. 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to position 29 of any of SEQ ID NOs. 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to position 33 of any of SEQ ID NOs. 2, 10, 11, 12, 14, and 15; and a binding affinity (K) of less than 10 μM to type II collagen, as described elsewhere in this specification. D The dimerized type II collagen-binding protein has a binding affinity (K) of less than 10 μM to type II collagen. At least one monomer of the dimerized type II collagen-binding protein may be located at either the N-terminus or the C-terminus of a dimerized type II collagen-binding protein having at least 80% sequence identity with any one of SEQ ID NOs: 27-40. In various preferred embodiments, the dimerized type II collagen-binding protein comprises an amino acid sequence having at least 85% or 89.5% sequence identity with any one of SEQ ID NOs: 27-40, and at least one of its two monomers has (i) glutamine (Q) at the position corresponding to position 25 of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to position 29 of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to position 33 of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and a binding affinity (K) of less than 10 μM to type II collagen, as described elsewhere in this specification. DIn another preferred embodiment, the dimeric type II collagen-binding protein has a binding affinity (K) of less than 10 μM to type II collagen. D It is a type II collagen-binding protein that possesses )

[0051] In various embodiments, the binding protein to type II collagen provided by the present invention comprises an amino acid sequence having at least 80% sequence identity with the tetramer of SEQ ID NO: 41 or 42, wherein one, two, three, or all of the four monomers have (i) glutamine (Q) at the position corresponding to position 25 of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to position 29 of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to position 33 of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and a binding affinity (K) of less than 10 μM to type II collagen, as described elsewhere in this specification. DThis is a type II collagen-binding protein having a binding affinity (K) of less than 10 μM to type II collagen. One of up to four monomers may be located at either the N-terminus or C-terminus of the tetrameric type II collagen-binding protein having at least 80% sequence identity with SEQ ID NO: 41 or 42. In various preferred embodiments, the tetrameric type II collagen-binding protein comprises an amino acid sequence having at least 85% or 89.5% sequence identity with SEQ ID NO: 41 or 42, and one, two, three, or all of its four monomers have (i) glutamine (Q) at the position corresponding to the 25th position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to the 29th position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to the 33rd position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and a binding affinity (K) of less than 10 μM to type II collagen, as described elsewhere in this specification. D It is a type II collagen-binding protein that possesses ) In yet another preferred embodiment, the tetrameric type II collagen-binding protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 41 or 42, preferably at least 91%, 92%, 93%, 94%, or 95%, more preferably at least 96%, 97%, 98%, or 99%, wherein one, two, three, or all of its four monomers have (i) glutamine (Q) at the position corresponding to the 25th position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at the position corresponding to the 29th position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at the position corresponding to the 33rd position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and a binding affinity (K) of less than 10 μM to type II collagen, as described elsewhere in this specification. D It is a type II collagen-binding protein that possesses )

[0052] As further disclosed herein, in various embodiments, the dimeric or multimeric type II collagen-binding proteins of the invention can include one or more linker sequences that link the monomeric type II collagen-binding proteins included by the dimeric or multimeric type II collagen-binding proteins. In various embodiments, the linker sequence includes the amino acid sequence GGGS (SEQ ID NO: 45). In various other embodiments, the linker sequence includes the amino acid sequence GGGGS (SEQ ID NO: 46). In still other embodiments, the linker sequence includes, or includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 47) or the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 48).

[0053] The invention further provides a binding protein for type II collagen that does not have a detectable binding affinity for the Fc domain of an immunoglobulin (lacking binding affinity for the Fc domain of an immunoglobulin), and the type II collagen-binding protein includes an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 1 to 44. In various embodiments, the invention provides a binding protein for type II collagen that does not have a detectable binding affinity for the Fc domain of an immunoglobulin, and the type II collagen-binding protein includes an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15, and the type II collagen-binding protein has (i) glutamine (Q) at a position corresponding to the 25th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; (ii) tyrosine (Y) at a position corresponding to the 29th position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and (iii) tryptophan (W) at a position corresponding to the 33rd position of any one of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; as described elsewhere herein, having a binding affinity (K for type II collagen of less than 10 μM DThe present invention provides a type II collagen-binding protein that does not have detectable binding affinity to the Fc domain of immunoglobulins, comprising one or more, preferably two or three, of the following amino acid residues from any of SEQ ID NOs: 1 to 21: aspartic acid (D) (13D) at the position corresponding to the 13th position of any of SEQ ID NOs: 1 to 21; lysine (K) (14K) at the position corresponding to the 14th position of any of SEQ ID NOs: 1 to 21; and / or arginine (R) (31R) at the position corresponding to the 31st position of any of SEQ ID NOs: 1 to 21. In a preferred embodiment, the type II collagen-binding protein of the present invention, which does not have a detectable binding affinity to the Fc domain of immunoglobulin, comprises one or more, preferably two or three, of the following amino acid residues of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15: aspartic acid (D) (13D) at the position corresponding to the 13th position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; lysine (K) (14K) at the position corresponding to the 14th position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15; and / or arginine (R) (31R) at the position corresponding to the 31st position of any of SEQ ID NOs: 2, 10, 11, 12, 14, and 15. Particularly preferred are type II collagen-binding proteins that do not have detectable binding affinity to the Fc domain of immunoglobulins, comprising one or more, preferably two or three, of the following amino acid residues of SEQ ID NO: aspartic acid (D) (13D) at the position corresponding to the 13th position of any of SEQ ID NO: SEQ ID NO: K (14K) at the position corresponding to the 14th position of SEQ ID NO: Accordingly, the present invention provides a binding protein to type II collagen that does not have a detectable binding affinity to the Fc domain of immunoglobulin, wherein the type II collagen binding protein comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 15, and comprises one or more, preferably two or three, of the following amino acid residues of SEQ ID NO: 15: aspartic acid (D) (13D) at the position corresponding to the 13th position of any of SEQ ID NO: 15; lysine (K) (14K) at the position corresponding to the 14th position of SEQ ID NO: 15; and / or arginine (R) (31R) at the position corresponding to the 31st position of SEQ ID NO: 15.In various preferred embodiments, the type II collagen-binding protein lacking a detectable binding affinity to the Fc domain of immunoglobulin further comprises (i) glutamine (Q) at the position corresponding to position 25 of SEQ ID NO: 15; (ii) tyrosine (Y) at the position corresponding to position 29 of SEQ ID NO: 15; and (iii) tryptophan (W) at the position corresponding to position 33 of SEQ ID NO: 15; and a binding affinity (K) of less than 10 μM to type II collagen, as described elsewhere herein. D ) has.

[0054] In various embodiments of the present invention, a binding protein to type II collagen that does not have a detectable binding affinity to the Fc domain of an immunoglobulin (or lacks binding affinity to the Fc domain of an immunoglobulin) is determined to have no detectable binding affinity to the Fc domain of an immunoglobulin (or lacks binding affinity to the Fc domain of an immunoglobulin) by a method for measuring binding affinity as described elsewhere in this specification. In preferred embodiments, the method for measuring the binding affinity to the Fc domain of an immunoglobulin is any of ELISA, SPR, BLI, KinExA assay, flow cytometry, fluorescence spectroscopy, ITC, ultracentrifugation analysis, RIA or IRMA, and ECL, more preferably any of ELISA, SPR, BLI, KinExA assay, and flow cytometry, even more preferably any of ELISA, SPR, and KinExA assay, still more preferably ELISA or SPR, most preferably SPR.

[0055] The terms “Fc region” and “Fc domain” can be used interchangeably herein. The Fc region is the tail region of an immunoglobulin, particularly an antibody, that interacts with a cell surface receptor called, for example, an Fc receptor. Thus, Fc region or Fc domain means the Fc region or Fc domain of an immunoglobulin, particularly an antibody. In various embodiments, the Fc region is derived from mammalian IgG (antibodies), including human IgG, mouse IgG, rat IgG, goat IgG, bovine IgG, guinea pig IgG, and rabbit IgG. The Fc region can also be derived from human IgM or human IgA. In various embodiments, the Fc region is derived from human IgG (antibodies), such as human IgG1 (antibody), human IgG2 (antibody), or human IgG4 (antibody), and more preferably from human IgG1 (antibody).

[0056] The Avimer™ domain is an artificial protein of 30 to 35 amino acids having a rigid structure stabilized particularly by disulfide bonds. In various embodiments of the present invention, the type II collagen-binding protein disclosed herein may or may not have an amino acid sequence that generates disulfide bonds in the type II collagen-binding protein disclosed herein. Accordingly, in various embodiments of the present invention, the type II collagen-binding protein disclosed herein may or may not have an amino acid sequence that includes cysteine ​​(Cys) residues that generate disulfide bonds in the type II collagen-binding protein disclosed herein. More specifically, in various embodiments of the present invention, the type II collagen-binding protein disclosed herein may or may not have an amino acid sequence that includes two or more cysteine ​​(Cys) residues that generate one or more disulfide bonds in the type II collagen-binding protein disclosed herein. In various embodiments, the type II collagen-binding protein disclosed herein may or may not have an amino acid sequence that does not include cysteine ​​(Cys) residues (one or more).

[0057] Multimer. In a preferred embodiment, the type II collagen-binding protein is a multimer comprising several of the type II collagen-binding proteins defined herein. The multimer may contain two, three, four, or more type II collagen-binding proteins. In one embodiment, the type II collagen-binding protein comprises two, three, four, or more type II collagen-binding proteins linked together; that is, the type II collagen-binding protein may be a dimer, trimer, or tetramer, etc. In some embodiments, the multimer is a dimer of the type II collagen-binding proteins defined above.

[0058] In various embodiments, type II collagen-binding proteins are homodimers. A homodimer type II collagen-binding protein is a protein in which two type II collagen-binding proteins having identical amino acid sequences are linked together. In some embodiments, a homomultimer type II collagen-binding protein may contain at least two modules of SEQ ID NO: 11 and have at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 37 (211519). In some embodiments, a homomultimer type II collagen-binding protein may contain at least two modules of SEQ ID NO: 15 and have at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 40 (212705). For example, a homodimer type II collagen-binding protein may contain two monomers of SEQ ID NO: 15 linked together in a head-to-tail orientation. In some embodiments, a homomultimer type II collagen-binding protein may contain at least two modules of SEQ ID NO: 2 and have at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 38 (211999). For example, a homodimer type II collagen-binding protein may contain two monomers of SEQ ID NO: 2 linked to each other in a head-to-tail direction. In some embodiments, a homomultimer type II collagen-binding protein may contain at least two modules of SEQ ID NO: 1. For example, a homodimer type II collagen-binding protein may contain two monomers of SEQ ID NO: 1 (211190) linked to each other in a head-to-tail direction.

[0059] In various embodiments, type II collagen-binding proteins are homotetramers. A tetrameric type II collagen-binding protein is a protein in which four type II collagen-binding proteins with identical amino acid sequences are linked together (see, for example, SEQ ID NOs. 41 and 42, but not limited to these).

[0060] In other embodiments, the polymer is a heterodimer, for example, the two amino acid sequences of a type II collagen-specific protein have different amino acid sequences.

[0061] In some embodiments, the heterodimer type II collagen-binding protein may include SEQ ID NO: 10 and have at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 10 (e.g., the N-terminal monomer of the heterodimers of SEQ ID NO: 36 (210987), SEQ ID NO: 32 (210992), or SEQ ID NO: 33 (211027)).

[0062] In some embodiments, the hetero-dimer-binding protein may include SEQ ID NO: 14 and have at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 14 (for example, the N-terminal monomer of the heterodimers of SEQ ID NO: 28 (210970) and SEQ ID NO: 29 (210971), or the C-terminal monomer of the heterodimers of SEQ ID NO: 30 (210972) or SEQ ID NO: 31 (210973)).

[0063] For example, the hetero-dimer binding protein of SEQ ID NO: 27 (209130) contains SEQ ID NOs. 4 and 11 linked to each other in a head-to-tail direction (N-terminus to C-terminus). For example, the hetero-dimer binding protein of SEQ ID NO: 35 (208585) contains SEQ ID NOs. 17 and 12 linked to each other in a head-to-tail direction (N-terminus to C-terminus). For example, the hetero-dimer binding protein of SEQ ID NO: 36 (210987) contains SEQ ID NOs. 10 and 6 linked to each other in a head-to-tail direction (N-terminus to C-terminus). An example of a dimer type II collagen binding protein is shown in Figure 1C.

[0064] In some embodiments, two or more type II collagen-binding proteins are directly linked. In some embodiments, two or more type II collagen-binding proteins are linked via peptide linkers. In various embodiments, two or more type II collagen-binding proteins are linked via peptide linkers of up to 30 amino acids. In other embodiments, two or more type II collagen-binding proteins are linked via peptide linkers of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Examples of linkers are shown in SEQ ID NOs. 45-48.

[0065] The binding protein multimers are generally artificially produced by recombinant DNA technology, which is well known to those skilled in the art.

[0066] Type II collagen-binding proteins are fused to further portions. In various embodiments, type II collagen-binding proteins are fused to or bound to therapeutically effective portions. In other embodiments, type II collagen-binding proteins are fused to or bound to one or more therapeutically effective portions. In some embodiments, such therapeutically effective portions can be selected from monoclonal antibodies and their fragments, extracellular domains of receptors and their fragments, non-immunoglobulin scaffold proteins, cytokines, chemokines, cytotoxic compounds, enzymes and their derivatives, radionuclides, and any combination thereof.

[0067] In various embodiments, type II collagen-binding proteins are fused to or bound to the diagnostic moiety. In other embodiments, type II collagen-binding proteins are fused to or bound to one or more diagnostic moieties. In some embodiments, such diagnostic moieties can be selected from radionuclides, fluorescent compounds, photosensitizers, tags, enzymes, and non-immunoglobulin scaffold proteins, as well as any combination thereof.

[0068] Various embodiments relate to type II collagen-binding proteins fused to therapeutically effective proteins or clinically effective proteins.

[0069] Medical Use. Various embodiments relate to type II collagen-binding proteins disclosed herein for medical use. Specific embodiments relate to the type II collagen-binding proteins for use in the diagnosis or treatment of eye and joint diseases. In some embodiments, the eye disease may be a neovascular eye disease. Neovascular eye diseases may be selected from, but are not limited to, age-related macular degeneration with angiogenesis, myopic choroidal neovascularization, idiopathic choroidal neovascularization, choroidal neovascularization, branch retinal vein occlusion, central retinal vein occlusion, diabetic retinopathy, retinopathy of prematurity, and diabetic macular edema. In some embodiments, the eye disease may be an inflammatory eye disease. Inflammatory eye diseases may include, but are not limited to, a group of conditions such as non-infectious posterior uveitis, non-infectious intermediate uveitis, non-infectious anterior uveitis, idiopathic uveitis, non-infectious panuveitis, uveitis, scleritis, and orbital inflammatory diseases associated with primary inflammatory systemic diseases such as Behçet's disease, sarcoid disease, lupus, juvenile idiopathic arthritis, rheumatoid arthritis, and Wegener's granulomatosis. Examples of joint-related diseases include rheumatoid arthritis, arthropathy, and other joint-related disorders.

[0070] Compositions. Various embodiments relate to compositions comprising type II collagen-binding proteins disclosed herein.

[0071] Various embodiments relate to pharmaceutical compositions for the treatment of diseases, comprising type II collagen-binding proteins disclosed herein, and pharmaceutically acceptable carriers and / or diluents. The pharmaceutical compositions may optionally contain further known auxiliaries and excipients, which include, but are not limited to, stabilizers, surfactants, salts, buffers, colorants, and the like. The compositions may be in the form of liquid formulations, lyophilized products, creams, topical lotions, aerosols, powders, granules, emulsions, or liposomal formulations.

[0072] Pharmaceutical compositions comprising type II collagen-binding proteins as defined herein can be used for the treatment of eye diseases and joint diseases as described above.

[0073] Various embodiments relate to diagnostic compositions for diagnosing diseases comprising type II collagen-binding proteins as defined herein and diagnostically acceptable carriers and / or diluents. These include, but are not limited to, stabilizers, surfactants, salts, buffers, colorants, and the like. The compositions may be in the form of liquid formulations, lyophilized products, granules, emulsions, or liposomal formulations.

[0074] The diagnostic compositions comprising type II collagen-binding protein described herein can be used for the diagnosis of eye diseases and joint diseases as described above.

[0075] The compositions contain a therapeutically or diagnostically effective amount of type II collagen-binding protein as defined herein. The amount of protein administered depends on the organism being treated, the type of disease, the patient's age and weight, and other factors that are known in themselves. Depending on the Galenos formulation, these compositions may be administered by parenteral administration by injection or infusion, systemic administration, intravitreous administration, intra-articular administration, or other conventionally employed methods of application. The type of pharmaceutical formulation depends on the type of disease to be treated, the route of administration, the severity of the disease, the patient being treated, and other factors known to those skilled in the art of medicine.

[0076] Various embodiments relate to methods for treating or diagnosing subjects with ocular diseases, comprising administering a therapeutically effective amount of the type II collagen-binding protein disclosed herein to the eye of a subject. Preferred is a method for treating subjects with ocular diseases selected from age-related macular degeneration with neovascularization (AMD), diabetic macular edema, retinal vein occlusion, and other neovascular retinal diseases.

[0077] Preparation of type II collagen-binding proteins. The type II collagen-binding proteins described herein can be prepared by any of many conventionally known techniques, such as simple organic synthesis strategies, solid-phase assisted synthesis techniques, fragment ligation techniques, or commercially available automated synthesis equipment. Alternatively, these proteins can be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques. Furthermore, they may be prepared by cell-free in vitro transcription / translation, or in combination with conventional synthetic techniques.

[0078] Various embodiments relate to polynucleotides encoding type II collagen-binding proteins disclosed herein. The present invention further provides an expression vector comprising the polynucleotide, and a host cell comprising the isolated polynucleotide or expression vector.

[0079] Various embodiments relate to methods for producing type II collagen-binding proteins as disclosed herein, comprising culturing host cells under suitable conditions that enable the expression of the type II collagen-binding protein, and optionally isolating the type II collagen-binding protein.

[0080] For example, one or more polynucleotides encoding type II collagen-binding protein can be expressed in a suitable host, and the resulting type II collagen-binding protein can be isolated. Vectors containing such polynucleotides are incorporated herein. Further embodiments relate to vectors containing such nucleic acid molecules. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to transfer protein-coding information to a host cell. Furthermore, isolated cells containing such nucleic acid molecules or such vectors are disclosed. Suitable host cells include prokaryotes or eukaryotes. Various mammalian or insect cell culture systems can also be employed for recombinant protein expression.

[0081] One embodiment also relates to a host cell or non-human host possessing the vector. The host cell is a cell that is transformed with a nucleic acid sequence and thereby expresses the gene of interest.

[0082] Suitable conditions for culturing prokaryotic or eukaryotic host cells are well known to those skilled in the art. Cell culture and protein expression for protein production can be carried out on any scale, from small shaking flasks to large fermenters, by applying techniques well known to those skilled in the art.

[0083] One embodiment relates to the method for preparing the binding protein detailed above, the method comprising the following steps: (a) preparing a nucleic acid encoding the type II collagen-binding protein defined herein; (b) introducing the nucleic acid into an expression vector; (c) introducing the expression vector into host cells; (d) culturing the host cells; (e) subjecting the host cells to culture conditions that express the type II collagen-binding protein, thereby producing the type II collagen-binding protein defined herein; (f) optionally isolating the type II collagen-binding protein produced in step (e); and (g) optionally conjugating the type II collagen-binding protein with a further functional moiety defined herein.

[0084] Generally, the isolation of purified type II collagen-binding proteins from culture mixtures can be performed by applying conventional methods and techniques well known in the art, such as centrifugation, precipitation, aggregation, different embodiments of chromatography, filtration, dialysis, concentration, or combinations thereof. Chromatographic methods are well known in the art and are not limited to, but include ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), hydrophobic interaction chromatography, or affinity chromatography.

[0085] To simplify purification, type II collagen-binding proteins can be fused with other peptide sequences that have increased affinity to the separation material. Preferably, such fusions are selected that do not adversely affect the functionality of the type II collagen-binding protein, or that can be separated after purification by introducing specific protease cleavage sites. Such methods are also known to those skilled in the art. [Examples]

[0086] The following examples are provided to further illustrate the present invention. The present invention is particularly exemplified by type II collagen-binding proteins as described above. However, the present invention is not limited thereto, and the following examples merely demonstrate the feasibility of the present invention based on the above description. For a complete disclosure of the present invention, references are also made to the documents cited herein, which are incorporated fully by reference hereto.

[0087] Example 1: Selection and screening of type II collagen α1 chain-binding proteins Library. A proprietary cDNA library was synthesized in-house using randomized oligonucleotides produced with synthetic trinucleotide phosphoramidites (ELLA Biotech) to achieve a well-balanced amino acid distribution by simultaneously excluding cysteine ​​and other amino acid residues at random positions. The corresponding cDNA library was amplified by PCR and ligated to pCD33-OmpA phagemide. The phage library was prepared and purified using aliquots of the ligation mixture in electroporation of E. coli SS320 (Lucigen), and frozen stocks were stored. Unless otherwise indicated, established recombinant genetic engineering methods were used.

[0088] Phage display selection. A phage display was used as the selection system to enrich native libraries against each on-target (immunograde human type II collagen, purchased from Chondrex). In each round, a pre-selection step was performed using empty beads blocked with a Sigmablocker as the off-target. The AIT method was applied, meaning that the target protein was immobilized on magnetic Epoxy M-270 Dynabeads in each round. E. coli ER2738 (Lucigene) was used to infect the cryophage library and to re-amplify the phage pool after each round. Phage amplification and purification were performed using standard methods known to those skilled in the art. Four selection rounds were performed using an automated KingFisher System (Thermo Fisher) to isolate and capture the desired phage-target complexes. The target amount was reduced in each round by decreasing the amount of target-coupled beads. The bound phages were eluted with trypsin and re-amplified. Selection success was analyzed by phage pool ELISA on immuno-grade human type II collagen (Chondrex, 5 μg / ml), porcine type II collagen (prepared in-house from pig eyes, 5 μg / ml), BSA (5 μg / ml), or Sigmablocker-coated medium-binding microtiter plates (Greiner Bio-One). Binding phages were detected using α-M13 HRP-binding antibody (GE Healthcare).

[0089] Cloning of target-binding phage pools into expression vectors: A selection pool showing specific binding to human and / or porcine collagen type II in a phage pool ELISA was amplified by PCR according to methods known in the art, cleaved with an appropriate restriction nuclease, and ligated into a derivative of the expression vector pET-28a (Merck, Germany) containing a C-terminal strep-tag.

[0090] Results: Various phage display selection pools yielded specific signals for their respective on-targets. Controls using BSA and Sigmablocker did not show binding to most pools. The selected pools were sequenced, subcloned, and proceeded to high-throughput screening.

[0091] Primary screening: A primary screening of single variants was performed against directly immobilized immunograde human type II collagen (Chondrex) [c=1 μg / ml]. Binding variants were detected using Strep-Tactin® HRP conjugate. Hits were selected as follows: the signal from the sample was greater than the signal from the negative control.

[0092] Secondary screening. Secondary screening was performed for immuno-grade human type II collagen (Chondrex; on-target) [c=1 μg / ml] and BSA (off-target) [c=1 μg / ml]. Hits were identified as follows: the signal for the variant against the on-target was 10 times greater than the signal against the off-target.

[0093] PhyNexus / SPR. Hits were prepared and purified on a microscale (PhyNexus) and analyzed by SPR measurement. Immunograde human type II collagen (Chondrex) was immobilized on a CM5 chip (GE Healthcare) by amine coupling. Upon binding, the analyte (potential hits) accumulated on the surface, increasing the refractive index. The change in refractive index was measured in real time and plotted as a response unit against time. Hit variants were selected for sequence analysis and laboratory-scale production.

[0094] Example 2: Expression of type 2 collagen-binding protein Affinity-tagged variants were expressed in E. coli BL21(DE3) under the control of the T7 promoter using a low-copy plasmid system. The protein was induced with lactose contained in the culture medium (autoinduction medium) and produced in a soluble form. BL21(DE3) competent cells were transformed with the expression plasmid, spread on selective agar plates (kanamycin), and incubated overnight at 37°C. Single colonies were seeded into 3 ml of 2×YT medium supplemented with 50 μg / ml of kanamycin to prepare a pre-culture, and incubated in a culture tube at 37°C and 200 rpm using a standard orbital shaker for 6 hours. In a 1L Erlenmeyer flask, if the variant had an affinity tag, a 0.3 / OD preculture was seeded into 350ml of ZYM-5052 (0.5% glycerol, 0.2% lactose, 0.05% glucose, 0.5% yeast extract, 1.0% casamino acid, 25mM Na2HPO4, 25mM KH2PO4, 5mM Na2SO4, 2mM MgSO4, and trace elements), or if the variant did not have an affinity tag, a 0.3 / OD preculture was seeded into 350ml of H15 medium (0.89% glycerol, 0.76% lactose, 2.2% glucose, 5% yeast extract, 250mM MOPS, 200mM Tris, 10mM MgCl2, and trace elements) supplemented with 50μg / ml kanamycin, and this was used as the main culture. The culture was transferred to an orbital shaker and incubated at 30°C and 200 rpm. Recombinant protein expression was induced by metabolizing glucose and then introducing lactose into the cells. The cells were cultured overnight for approximately 17 hours to reach a final OD600 of approximately 2-4. The OD600 was measured before collection, and samples adjusted to 0.6 / OD600 were taken out, pelletized, and frozen at -20°C. To collect biomass, the cells were centrifuged at 12000 × g at 22°C for 15 minutes. The weight (wet weight) of the pellet was measured. After storing the cells at -20°C, they were processed.

[0095] Example 3 Purification of type II collagen-binding protein Affinity-tagged proteins were purified by affinity chromatography and size exclusion. Affinity chromatography purification was performed using a Strep-Tactin Superflow high-volume cartridge (IBA Lifesciences), followed by size exclusion chromatography (SEC) using an Akta system and a Superdex® 200 Hiload 16 / 600 column (Cytiva). Fusion variants with Aflibercept were purified by MabSelect SuRe (Cytiva) affinity chromatography, followed by size exclusion chromatography (SEC) using an Akta system and a Superdex® 200 Hiload 16 / 600 column. All chromatographic steps were performed using an AKTA system with a Cytiva matrix. Homogenous species were obtained for all purified ligands, and no aggregation was detected. After SDS-PAGE analysis, the positive fraction was pooled and its protein concentration was measured. Further analysis included SDS-PAGE, SE-HPLC, and RP-HPLC. The protein concentration and molar extinction coefficient were determined by absorbance measurement at 280 nm. Reverse-phase chromatography (RP-HPLC) was performed using a Dionex HPLC system and a PLRP-S (5 μm, 300 Å) column (Agilent). Analytical size exclusion chromatography (SE-HPLC) was performed using a Dionex HPLC system and a Superdex200 increase 5 / 150 GL (Cytiva). Collagen II binding was evaluated by SPR measurement using a ViaCore 3000 system. Purified collagen II was obtained from Chondrex and VEGF-165 from Millipore. The target was immobilized on a CM5-Sensor by NHS / EDC chemistry (immobilization level ~1000 RU). The binding molecule was injected at different concentrations, and affinity was calculated using the Langmuir 1:1 binding model. Table 2 shows the expression and purification results of the selected type II collagen-binding protein (or polymers containing such protein). TIFF0007854204000001.tif163170

[0096] Example 4: Protein binding analysis using SPR The purified protein was immobilized onto a CM-5 sensor chip (GE Healthcare) using NHS / EDC after PDEA activation, and 110-140 RU was obtained using a ViaCore 3000 system (GE Healthcare). The chip was equilibrated with SPR running buffer (PBS 0.05% Tween pH 7.3).

[0097] Upon binding, the target analyte accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as a response to time or as resonance units. The analyte was serially diluted and applied to the tip at a flow rate of 30 μl / min. Binding was performed for 120 seconds, and dissociation for 120 seconds. After each run, the tip surface was regenerated with 30 μl of regeneration buffer (10 mM glycine, pH 2.0) and equilibrated with running buffer.

[0098] Coupling tests were conducted using ViaCore 3000 (GE Healthcare). Data evaluation was performed using the manufacturer's BIAevaluation 3.0 software with a Langmuir 1:1 model (RI=0). The evaluated dissociation constant (K) was... D The values ​​shown are standardized for immobilized proteins. Measurements were taken in real time, and the change in refractive index is plotted as the response to time [seconds] or as resonance units [RU]. The results are shown in Table 3 and Figures 3 and 4. 211190 (SEQ ID NO: 1) showed additional binding to type I and type III collagen (additional binding to type II collagen), but not to type V collagen (it also did not show additional binding to the extradomain B of control, BSA, or fibronectin). TIFF0007854204000002.tif72170

[0099] Example 5. K of the binding protein D Measurement (ELISA) High-binding plates (Greiner, 781061) were immobilized overnight at 4°C with 2.5 μg / ml recombinant human collagen type II (h Col II, Chondrex, CHX-20051), rabbit collagen type II (rb Col II prepared from rabbit eyes), porcine collagen type II (p Col II, prepared from pig eyes), human collagen type I (h Col I, Sigma, C5483-1MG), human collagen type V (h Col V, Abcam, ab7537), human collagen type III (h Col III, Abcam, ab7535), and 67B89 (fibronectin domain produced by E. coli). The ELISA plates were washed three times with PBST (PBS + 0.1% Tween) and blocked at room temperature for 2 hours with 3% BSA / 0.5% Tween / PBS. After washing with PBST, 1.1 μM ~ 7 × 10⁶ was added. -4 Dilution series of nM dimers (CID211999, CID212000), 100 nM ~ 7 × 10⁻⁶ -6 Dilution series of nM tetramers (CID212001 and CID212704) and dilution series of 10 μM to 2 nM dimers (CID212705) were incubated on ELISA plates at room temperature for 1 hour. The plates were washed with PBST and incubated with biotin-labeled anti-StrepTag antibody (Antikorper-online, ABIN1573895) at a 1:10.000 dilution at room temperature for 1 hour. Binding was visualized using 1:10.000 dilution of α-rabbit IgG-HRP (Sigma, A9169) and TMB Plus von KEM EN TEC Diagnostic, Kat-Nr.4395 as the substrate.

[0100] Results. ELISA analysis clearly confirmed specific binding to type II collagen. The homodimers of SEQ ID NO: 2 (CID211999 and CID212000) and SEQ ID NO: 15 (CID212705) showed high binding affinity (1-2 orders of magnitude nM K). DThe tetramer of SEQ ID NO: 15 (CID212704) and the tetramer of SEQ ID NO: 2 (CID212001) exhibit high binding affinity, for example, 3-digit picomolar binding affinity to different types of type II collagen. No nonspecific binding was detected to the domains (67B89) of type I collagen, type III collagen, type V collagen, or fibronectin (Figure 2).

[0101] Furthermore, the binding of type II collagen-binding proteins was analyzed by ELISA. For example, 209130 (SEQ ID NO: 27) showed specific binding of 0.22 μM to human type II collagen and 0.11 μM to porcine type II collagen. 211138 (SEQ ID NO: 34) showed specific binding of 0.45 μM to human type II collagen and 0.81 μM to porcine type II collagen. 210987 (SEQ ID NO: 36) showed specific binding of 0.46 μM to human type II collagen. 211519 (SEQ ID NO: 37) showed specific binding of 0.7 μM to both human type II collagen and porcine type II collagen. 211190 (SEQ ID NO: 1) showed specific binding to human type II collagen, as well as human type I and human type III collagen, but not to human type V collagen.

[0102] Example 6: Analysis of specific protein binding by IHC Sections of formalin-fixed, paraffin-embedded pig eyes were deparaffinized with xylol and rehydrated with ethanol in a decreasing concentration series. For antigen retrieval, the slices were incubated in TE buffer, pH 9.0, at 110°C for 5 minutes. Peroxidase blocking solution (Novolink Polymer) TM After blocking endogenous peroxidase with a Detection System (Leica, RE7140-K), slices were incubated with 5 μM or 500 nM CID211999 (dimer), CID212000 (dimer), and CID212001 (tetramer) at room temperature for 45 minutes. Next, rabbit anti-StrepTag antibody (Genscript, A00026) at a 1:500 dilution and Novolink Polymer were added.TM Protein binding was detected using a detection system. A 1:10 dilution of anti-collagen type II antibody (Abcam, #ab34712) was used as a positive control. Tissue slices were incubated with the antibody for 30 minutes. The washing step was performed with TBS pH 7.6. For visualization, AEC staining (Dako, K3461) was used, and after washing with water, the nuclei were stained with Meyer hematoxylin solution (Merck, 1.09249.0500). See Figure 5 for the results. Similar results to those shown in Figure 5 were obtained for SEQ ID NO: 38 (dimer SEQ ID NO: 2, CID211999) and SEQ ID NO: 41 (tetramer of SEQ ID NO: 2, CID212001).

Claims

1. A type II collagen-binding protein comprising an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of Sequence IDs 2, 11, and 15, (i) Place glutamine (Q) at the position corresponding to the 25th position of the selected sequence; (ii) tyrosine (Y) at the position corresponding to the 29th position of the selected sequence; and (iii) having tryptophan (W) at the position corresponding to the 33rd position of the selected sequence; Binding affinity (K) to type II collagen is less than 10 μM. Ｄ A type II collagen-binding protein characterized by having ) and not having detectable binding affinity to type I collagen, type III collagen, and type V collagen.

2. The type II collagen-binding protein according to claim 1, wherein two, three, four, five, or six type II collagen-binding proteins are linked to one another.

3. The type II collagen-binding protein according to claim 2, which is a homopolymer or a heteropolymer.

4. The type II collagen-binding protein according to claim 1 or 2, comprising an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 27 to 42.

5. A dimer or polymer protein comprising a type II collagen-binding protein as described in any one of claims 1 to 4.

6. A type II collagen-binding protein according to any one of claims 1 to 4, or a dimer or multimer protein according to claim 5, further fused or bound to at least one therapeutically effective portion, which is optionally selected from a monoclonal antibody or fragment thereof, a binding protein, a receptor or receptor domain, a receptor-binding ligand or antagonist, an extracellular domain or fragment thereof of a receptor, a non-immunoglobulin-binding protein, a cytokine, a chemokine, a cytotoxic compound, an enzyme, and derivatives thereof, as well as a radionuclide, and any combination thereof.

7. A radionuclide, a fluorescent compound, a photosensitizer, a tag, an enzyme, and a non-immunoglobulin-binding protein, and a type II collagen-binding protein according to any one of claims 1 to 4 and 6, further fused to or bound to at least one diagnostic portion, optionally selected from any combination of the above, or a dimer or polymer protein according to claim 5 or 6.

8. A composition comprising a type II collagen-binding protein according to any one of claims 1 to 4, 6, and 7, or a dimer or polymer protein according to any one of claims 5 to 7.

9. A pharmaceutical composition for the treatment of a disease comprising a type II collagen-binding protein defined in any one of claims 1 to 4, 6, and 7, or a dimer or polymer protein according to any one of claims 5 to 7, and a therapeutically or diagnostically acceptable carrier and / or diluent.

10. A method for producing a type II collagen-binding protein defined in any one of claims 1 to 4, 6, and 7, or a dimer or polymer protein according to any one of claims 5 to 7, comprising culturing host cells under conditions suitable for obtaining a type II collagen-binding protein and optionally isolating the type II collagen-binding protein.

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