Collagen-binding pharmaceutical composition and method of using the same

Abstract

To provide novel collagen-binding therapeutic agents targeting collagen.SOLUTION: A collagen-binding therapeutic agent comprises: two collagen-binding domains linked by a domain linker, and a therapeutic agent linked to at least one of the collagen-binding domains by a therapeutic agent linker, where the collagen-binding agent lacks collagenase activity, and where one of the two collagen-binding domains is selected from a group consisting of polypeptides having specific sequences and the other of the collagen-binding domains is selected from a group consisting of polypeptides having specific sequences.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] (Description of Government Support for Research) This invention was made with government support under National Institutes of Health grant numbers GM103450 and GM103429. The United States government has certain rights in this invention. (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Patent Application 62 / 457,410, filed on February 10, 2017 (the entire contents of which are incorporated herein by reference). (Sequence Listing) This application was filed electronically via EFS - Web and includes an electronic submission sequence listing in text format. The text file includes the sequence listing entitled “2018 - 02 - 09_5965 - 00084_ST25.txt” (created on February 9, 2018) and is 72,312 bytes in size. The sequence listing included in this text file is part of this application and is incorporated herein by reference in its entirety. (Technical Field)

Background Art

[0002] Collagen is the major structural protein found in the extracellular spaces of diverse animal tissues. Collagen can be found in fibrous tissues such as, for example, skin, ligaments, and tendons. Collagen is also abundant in bone, cartilage, the cornea, blood vessels, and muscle tissue. Abnormal or damaged collagen underlies several pathological conditions, including wounds and collagenopathy. Collagenopathy represents many diseases in which collagen structure or collagen formation is not normal. This group of diseases results in a wide range of symptoms, including bone defects, blood vessel defects, and skin defects. Many of these diseases have no available treatment or only ineffective treatments. Agents that target collagen are also promising agents for delivering therapeutic agents to various animal tissues or multiple sub-sites within a specific tissue. Effective delivery of therapeutic agents to multiple sites within the body of a subject in need of a particular therapeutic agent is a growing field. Targeted delivery systems enable the therapeutic agent to be most active at the site where it is needed, while minimizing the off-site effects of the therapeutic agent that can cause unwanted toxicity and side effects. The use of targeted liposomes or polypeptides (such as antibodies) to direct therapeutic agents to specific sites within the body has proven successful, but additional delivery agents are required. Therefore, there is a need in the art for novel agents that target collagen and can be used to treat tissues with damaged or normal collagen, or to deliver appropriate therapeutic agents to such tissues.

Summary of the Invention

[0003] In one aspect, a collagen-binding agent is provided. The collagen-binding agent can include two collagen-binding domains linked by a domain linker. The two collagen-binding domains can be selected from any one of the polypeptides of SEQ ID NOs: 1-47, a variant of one of SEQ ID NOs: 1-47, or a fragment of one of the polypeptides of SEQ ID NOs: 1-47. SEQ ID NOs: 1-39 define the individual collagen-binding domains (i.e., ColG s3a, ColG s3b, ColH s3) of the ColG and ColH proteins of various bacterial species. SEQ ID NOs: 40-47 define the tandem collagen-binding domains found in the ColG proteins of various bacterial species. See also FIG. 5. The collagen-binding agent of the present invention can also further include a therapeutic agent linked to the collagen-binding agent by a therapeutic agent linker. The therapeutic agent can be selected from the following: FGF, parathyroid hormone (PTH), PTH / PTHrP receptor agonist, PTH / PTHrP receptor antagonist, bone morphogenetic protein (BMP), G-CSF, BMP-2, BMP-3, anti-sclerostin antibody, growth hormone, IGF-1, VEGF, TGF-β, KGF, TGF-α, TGF-β1, TGF-β receptor, CT, GH, GM-CSF, EGF, PDGF, seriprorolol, activin, and connective tissue growth factor. In another aspect, a pharmaceutical composition is provided. The pharmaceutical composition can include any of the collagen-binding agents described herein and a pharmaceutical carrier. In yet another aspect, a method of treating a condition is also provided. The method can include administering to a subject, in an amount effective to treat the condition, any of the collagen-binding agents or pharmaceutical compositions described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0004]

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[0005] By way of non-limiting example, the inventors herein report for the first time the high-resolution structure of a tandem collagen-binding agent of bacteria. The pseudo-symmetrical arrangement of the tandem collagen-binding agent resulting from gene duplication and fusion enables the agent to recognize a unique niche of collagen fibrils and promote the degradation of collagen. The structure of the tandem collagen-binding agent also shows that it can be wedged between collagen molecules in the same direction. Together with the previously recognized preference of the collagen-binding domain for the underwound regions of collagen, the structure suggests that the tandem collagen-binding agent targets the damaged regions of collagen fibrils for bacterial collagenase (ColG). Based on this new knowledge of the mode of interaction between the tandem collagen-binding agent and collagen, the inventors disclose a novel collagen-binding agent. The novel binding agent can be used to treat damaged collagen in tissue or to specifically target and deliver therapeutic agents to tissues containing non-damaged or damaged collagen. The tandem collagen-binding agents provided herein can enable a stronger binding to collagen by cross-linking two collagen fibrils. This novel binding interaction of the collagen-binding agents described herein provides for the use of novel and unexpected therapeutic agents. This enhanced binding affinity and cross-linking of collagen molecules can render these tandem collagen-binding agents more effective than their single-binding counterparts for certain applications. Applications can include skin applications.

[0006] In one aspect, a collagen-binding agent is provided. The collagen-binding agent can include two collagen-binding domains linked by a domain linker. As used herein, "collagen-binding domain" refers to a polypeptide that binds to collagen. In some embodiments, the collagen-binding domain can bind to collagen with a Kd of less than 500 μM, 100 μM, 10 μM, 1 μM, 500 nM, 100 nM, 10 nM, 1 nM, or 0.1 nM. The determination of whether a collagen-binding domain binds to collagen can be performed, for example, as described in U.S. Patent Publication 2010 / 0129341, which is hereby incorporated by reference in its entirety. Briefly, the collagen-binding domain is incubated with collagen in a binding buffer, followed by passing the mixture through a filter that allows the collagen-binding domain to pass through but blocks the collagen, thus trapping substances that bind to the collagen. Subsequently, the filtrate is assayed for the presence of the collagen-binding domain. Appropriately, at least 80%, 85%, 90%, 95%, 98% or more appropriately at least 99% of the collagen-binding domain is retained by the filter of this assay compared to when filtration is performed in the absence of collagen.

[0007] The collagen-binding domain can be a bacterial collagen-binding domain. The collagen-binding domain can be a protein derived, inter alia, from the bacterial ColG protein (Matsushita et al., (1999) J. Bacteriol. 181:923-933), or the bacterial ColH protein (Yoshihara et al., (1994) J. Bacteriol. 176: 6489-6496). As shown in Figure 1, ColG is a class I collagenase found in diverse bacterial species (including Clostridium species). ColH is also a class II collagenase found in diverse bacterial species (including Clostridium species). The collagen-binding domain can also be any one of the polypeptides provided by SEQ ID NOs: 1-39 (the said SEQ ID NOs define the collagen-binding domains of diverse bacterial species (such as Clostridium and Bacillus species)). Those skilled in the art will recognize that other members of these collagen-binding protein families (i.e., ColG and ColH) may also be useful in the compositions and methods described herein. Suitably, the collagen-binding agent lacks collagenase activity. "Collagenase activity" refers to the ability of a polypeptide to degrade or break down collagen. For example, as shown in Figure 1A, the ColG and ColH proteins have a collagenase module, and the said module contains several subdomains that enable these proteins to degrade or break down collagen. In some embodiments, the collagen-binding agent lacks these or similar collagenase modules.

[0008] The collagen-binding agent can include two collagen-binding domains, and the domains may be the same type of collagen-binding domain or different types of collagen-binding domains. For example, as illustrated in FIG. 1A, the bacterial ColG protein has two types of collagen-binding domains, an s3a collagen-binding domain and an s3b collagen-binding domain. The bacterial ColH protein has only one collagen-binding domain known as the s3 domain. Thus, the two collagen-binding domains of the collagen-binding agent of the present disclosure may both be ColG s3a domains, both be ColG s3b domains, both be ColH s3 domains, or any combination thereof. Furthermore, the two collagen-binding domains of the collagen-binding agent of the present disclosure may be derived from the same bacterial species or different bacterial species. For example, one of the collagen-binding domains can be ColG s3b of Clostridium histolyticum ( Clostridium histoliticum ), while the other collagen-binding domain can be the ColG 3b domain of Brevibacillus brevis ( Brevibacillus brevis ). The two collagen-binding domains of the collagen-binding agent of the present disclosure can be selected from any one of the polypeptides of SEQ ID NOs: 1-47 or those polypeptides shown in FIGS. 5 and 12. SEQ ID NOs: 1-39 define individual collagen-binding domains (i.e., ColG s3a, ColG s3b, ColH s3) derived from ColG and ColH proteins of various bacterial species. SEQ ID NOs: 40-47 define tandem collagen-binding domains found in ColG proteins of various bacterial species. See also FIG. 5.

[0009] The collagen-binding domain of the present invention may be a variant of the polypeptide of SEQ ID NOs: 1-47. As used herein, the term "wild-type" is a technical term understood by those skilled in the art and means the typical form of a given polypeptide that occurs naturally, as distinguished from a "variant" or "mutant" form. As used herein, a "variant", "mutant" or "derivative" refers to a polypeptide molecule having an amino acid sequence different from the reference protein or polypeptide molecule. A variant can have one or more insertions, deletions or substitutions of amino acid residues as compared to the reference molecule. A variant or mutant can include a fragment of the reference molecule. For example, a collagen-binding domain variant can have one or more insertions, deletions or substitutions of at least one amino acid residue as compared to the "wild-type" collagen-binding domain polypeptide. The polypeptide sequences of the "wild-type" collagen-binding domains of various bacterial species are presented as SEQ ID NOs: 1-47. These sequences can be used as reference sequences. A "deletion" in a polypeptide refers to a change in the amino acid sequence that results in the absence of one or more amino acids. A deletion removes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more amino acid residues. Deletions can include internal deletions and / or terminal deletions (e.g., N-terminal truncation, C-terminal truncation or both) of the reference polypeptide. "Insertion" and "addition" in a polypeptide refer to a change in the amino acid sequence that results in the addition of one or more amino acid residues. An insertion or addition can refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid residues. A variant of the collagen-binding domain can have an N-terminal insertion, a C-terminal insertion, an internal insertion, or any combination of N-terminal insertion, C-terminal insertion and internal insertion.

[0010] With respect to variant collagen binding domains and agents, the terms “% sequence identity,” “percent identity,” or “% identity” refer to the percentage of residues that match between at least two amino acid sequences aligned using a standard algorithm. Methods for aligning amino acid sequences are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions (described in more detail below) generally preserve the charge and hydrophobicity at the substitution site and thus preserve the polypeptide's structure (and thus function). The percent identity of amino acid sequences can be determined as is known in the art (see, e.g., U.S. Patent No. 7,396,664, which is hereby incorporated by reference in its entirety). A suite of well-known free and available sequence comparison algorithms is provided by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information, which can be used at its website from several sources including NCBI (Bethesda, Md.). The BLAST software suite includes a variety of sequence analysis programs including “blastp” (blastp is used to align a known amino acid sequence with other amino acid sequences in various databases). The sequence identity of a polypeptide can be measured over the full length of a fully defined (e.g., defined by a particular SEQ ID NO.) polypeptide sequence or over a shorter length, e.g., over the full length of a fragment obtained from a larger defined polypeptide sequence, where the fragment is, for example, at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. Such lengths are merely exemplary, and it will be understood that any fragment length supported by the sequences shown herein in a table, drawing, or sequence listing may be used for purposes of describing a length over which percent identity can be measured. As described herein, the variant collagen-binding domain and the agent can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1-47.

[0011] The polypeptide sequences of the variant collagen-binding domain and the agent contemplated herein can include conservative amino acid substitutions as compared to the reference amino acid sequence. For example, the variant collagen-binding domain and the agent can include conservative amino acid substitutions as compared to the reference molecule. A "conservative amino acid substitution" is a substitution of one amino acid for a different amino acid that is predicted to have the least interference with the properties of the reference polypeptide. In other words, a conservative amino acid substitution substantially preserves the structure and function of the reference sequence. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the substitution region (e.g., the conformation of beta-sheets or alpha-helices), (b) the charge or hydrophobicity of the molecule at the substitution site, and / or (c) the size of the side chain. The disclosed variant collagen-binding domain and the agent described herein can have one or more functional or biological activities exhibited by the reference polypeptide (e.g., one or more functional or biological activities exhibited by the wild-type collagen-binding domain and the agent (i.e., SEQ ID NOs: 1-47 (see FIGS. 5 and 12)). Suitably, the disclosed variant collagen-binding domain and the agent retain at least 20%, 40%, 60%, 80%, or 100% of the collagen-binding activity of the reference polypeptide (i.e., SEQ ID NOs: 1-47 (see FIGS. 5 and 12)).

[0012] Figure 5 shows the sequence alignment of some collagen-binding domains of bacteria included as SEQ ID NOs: 40-47. As can be seen from this sequence alignment, these proteins have a relatively small amount of sequence identity, but they are all expected to bind to collagen in a similar manner and are thought to have a similar conformation. Accordingly, any of the polypeptides shown in Figure 5 (i.e., SEQ ID NOs: 40-47) or their collagen-binding domains, variants or fragments can be used in the compositions and methods described herein. In Figures 5 and 12, the amino acid residues important for collagen-binding activity are highlighted in blue. Calcium-binding residues are shown in blue and structurally important residues are shown in green. Based on this alignment, it will be immediately apparent to those skilled in the art that various amino acid residue changes (i.e., substitutions, deletions, etc.) are possible without substantially affecting the collagen-binding activity of the polypeptide. For example, those skilled in the art will understand that the amino acid residues shown in black are likely to be modifiable without substantially affecting the collagen-binding activity of the polypeptide. Those skilled in the art will also understand that substitutions in the reference collagen-binding domain or agent can be based on the same position in another collagen-binding domain or agent from another bacterial species or another amino acid residue present. For example, at position 966, the shown collagen-binding domain has a tyrosine (Y) or phenylalanine (F) residue. Accordingly, one exemplary modification apparent from the sequence alignment of Figure 5 is the Y966F substitution of the collagen-binding domain or agent. Similar modifications could be made at each position of the sequence alignment shown in Figure 5. Sequence numbers: 1 - 39 also contain collagen binding domains not shown in Figure 5, some of which are included in Figure 12. However, one of ordinary skill in the art could align these polypeptide sequences with the polypeptide sequences shown in Figures 5 and 12 to determine what additional variants could have arisen with respect to these additional collagen binding domains. Accordingly, the polypeptide has 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the collagen binding polypeptides provided herein. The variant polypeptide still has the ability to bind to collagen.

[0013] The collagen binding domain of the present invention can be the full - length version of sequence numbers: 1 - 47 or a fragment of sequence numbers: 1 - 47. Said fragment has at least 8, 16, 32, 64, 100 or more than 100 consecutive amino acids of any one of sequence numbers: 1 - 47. As used herein, a "fragment" is a portion of an amino acid sequence that is identical to the reference sequence, but is shorter than the reference sequence. A fragment can include up to, but not including, the full - length of the reference sequence minus at least 1 amino acid. In some embodiments, the fragment can include at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 consecutive amino acid residues of the reference polypeptide (i.e., sequence numbers: 1 - 47). The fragment can preferably be selected from a certain region of the molecule. The term "at least fragment" encompasses the full - length polypeptide. A fragment of a collagen binding domain or agent includes or consists essentially of a continuous portion of the amino acid sequence of the full - length collagen binding domain or agent (i.e., sequence numbers: 1 - 47). A fragment can include N - terminal truncation, C - terminal truncation, or both truncations as compared to the full - length collagen binding domain or agent. N - terminal and / or C - terminal truncation can include the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the reference sequence (i.e., sequence numbers: 1 - 47).

[0014] The collagen-binding domains or agents contemplated herein can also be further modified in vitro or in vivo to include non-amino acid moieties. These modifications can include, but are not limited to: acylation (e.g., O-acylation (ester), N-acylation (amide), S-acylation (thioester)), acetylation (e.g., addition of an acyl group to the N-terminus or lysine residue of a protein), formylation, lipoylation (e.g., attachment of lipoic acid, a C8 functional group), myristoylation (e.g., attachment of myristic acid, a C14 saturated acid), palmitoylation (e.g., attachment of palmitic acid, a C16 saturated acid), alkylation (e.g., addition of an alkyl group, such as methyl, to a lysine or arginine residue), isoprenylation or prenylation (e.g., addition of an isoprenoid group, such as farnesol or geranylgeraniol), C-terminal amidation, glycosylation (e.g., addition of a glycosyl group to asparagine, hydroxylysine, serine or threonine, resulting in a glycoprotein). Unlike glycation (the non-enzymatic binding of sugars), polysialylation (e.g., addition of polysialic acid), glypiation (e.g., formation of a glycosylphosphatidylinositol (GPI) anchor), hydroxylation, iodination (e.g., iodination of thyroid hormones), and phosphorylation (e.g., addition of a phosphate group, usually to serine, tyrosine, threonine or histidine) are representative of other possible modifications.

[0015] The two collagen-binding domains can be the ColG or ColH collagen-binding domain from a bacterial species. In some embodiments, the two collagen-binding domains can be any one of the polypeptides of SEQ ID NOs: 1-39, one polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1-39, or one fragment of at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 contiguous amino acids of any one of SEQ ID NOs: 1-39. The two collagen-binding domains can be the ColG s3b domain of a bacterial species and the ColG s3a domain, s3c domain, or ColH s3 domain of a bacterial species. In some embodiments, one of the collagen-binding domains can be any one of the polypeptides of SEQ ID NOs: 15-30, one polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with any one of SEQ ID NOs: 15-30, or one fragment of at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 consecutive amino acids of any one of SEQ ID NOs: 15-30, where the other collagen-binding domain can be any one of the polypeptides of SEQ ID NOs: 1-14 and 31-39, one polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1-14 and 31-39, or one fragment of at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 consecutive amino acids of any one of SEQ ID NOs: 1-14 and 31-39.

[0016] Both of the two collagen-binding domains can be the ColG s3b domain of a bacterial species. In some embodiments, the two collagen-binding domains can be any one of the polypeptides of SEQ ID NOs: 15-30, one polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with any one of SEQ ID NOs: 15-30, or one fragment of at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 consecutive amino acids of any one of SEQ ID NOs: 15-30. The two collagen-binding domains can be selected from the ColG s3a domain, s3b domain, or ColH s3 domain of a bacterial species. In some embodiments, the two collagen-binding domains are any one of the polypeptides of SEQ ID NOs: 1-14 and 31-39, any one of SEQ ID NOs: 1-14 and 31-39 and a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 consecutive amino acids of any one of SEQ ID NOs: 1-14 and 31-39. The two collagen-binding domains can be tandem collagen-binding domains derived from the bacterial ColG protein. In some embodiments, the collagen-binding agent is any one of the polypeptides of SEQ ID NOs: 40-47, any one of SEQ ID NOs: 40-47 and a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or at least 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 consecutive amino acids of any one of SEQ ID NOs: 40-47.

[0017] The collagen-binding agent can comprise two collagen-binding domains linked by a domain linker. As used herein, a "domain linker" can include a covalent bond and / or a linker or spacer moiety. For example, the two collagen-binding domains can be linked directly via, for example, a peptide bond or a chemical cross-link, or indirectly via, for example, a linker or spacer polypeptide. Useful domain linkers include homofunctional or heterofunctional linkers together with polypeptides, amino acids, nucleic acids. Particularly useful conjugation reagents that can facilitate the formation of a covalent bond between two collagen-binding domains can include N-hydroxysuccinimide (NHS) esters and / or maleimides. The domain linker can also include a spacer polypeptide. The spacer polypeptide can be of any length and can include conventional or non-conventional amino acids. For example, the spacer polypeptide can be 1-100 amino acids in length, and suitably, the spacer polypeptide is at least 2, 3, 5, 10, 15, 20, 25 or more than 25 amino acids in length such that the two collagen-binding domains of the collagen-binding agent can mediate collagen binding. The spacer polypeptide can include any one of the polypeptides of SEQ ID NOs: 48-55, a GST tag, a Ser linker or a Gly linker (but not limited thereto). In some embodiments, the domain linker can include a tag system. The tag system includes any agent group that can bind to each other with high affinity. Several tag systems are well known in the art and include, but are not limited to, biotin / avidin, biotin / streptavidin, or digoxigenin (DIG) systems. In some embodiments, the tag system includes biotin / avidin or biotin / streptavidin. The domain linker can comprise any one of the polypeptides of SEQ ID NO: 48-55, or one polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with any one of the polypeptides of SEQ ID NO: 48-55

[0018] The collagen-binding agent of the present invention can further include a therapeutic agent linked to the collagen-binding agent by a therapeutic agent linker. As used herein, a "therapeutic agent" can include any suitable pharmaceutical or other active agent. The agents can include, but are not limited to, osteogenesis promoters, antibacterial agents, anti-inflammatory agents, polypeptides (e.g., recombinant proteins, cytokines or antibodies), small molecule chemicals, hormones, growth factors, polynucleotides, carbohydrates, lipids, or any combination of the foregoing. Suitably, the therapeutic agent can promote bone growth, reduce inflammation, and promote collagen stabilization. Suitably, the therapeutic agent is one whose therapeutic effect is present in collagen or damaged collagen regions. The therapeutic agents can include, but are not limited to: FGF, parathyroid hormone (PTH), PTH / PTHrP receptor agonists, PTH / PTHrP receptor antagonists, bone morphogenetic proteins (BMPs), G-CSF, BMP-2, BMP-3, anti-sclerostin antibodies, growth hormone, IGF-1, VEGF, TGF-β, KGF, TGF-α, TGF-β1, TGF-β receptors, CT, GH, GM-CSF, EGF, PDGF, seriprorolol, activin, and connective tissue growth factor. Alternatively, the present invention can also assist in cell therapy. Cell therapy focuses on the administration of exogenous stem cells. One approach is to deliver these cells to the lesion, and laminin could be bound to CBD. Cells treated with the immobilized collagen binder seek the lesion and can remain at the damaged site for a long time to assist in repair. PTH / PTHrP receptor agonists and PTH / PTHrP receptor antagonists are described in WO 2013 / 090770 (the entire disclosure of which is incorporated herein by reference). Suitable FGF proteins include, but are not limited to, bFGF (FGF-2), FGF-4, or FGF-10 (see SEQ ID NO: 56).

[0019] The PTH / PTHrP receptor agonist polypeptide segment may be a synthetic polypeptide or a naturally occurring polypeptide. Such a polypeptide may be a portion of a polypeptide or may contain one or more mutations. The mutations may enhance or impair the PTH / PTHrP receptor as compared to wild-type PTH / PTHrP. The agonist activity of the PTH / PTHrP receptor can be assayed as described in WO2013 / 090770 and is known to those skilled in the art by the cAMP stimulation assay. An agonist will stimulate cAMP synthesis in the described assay. Suitably, the agonist can activate receptor activity at at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 110% or 120% of wild-type PTH(1-34). The PTH / PTHrP receptor agonist polypeptide segment is a PTH or PTHrP polypeptide segment. One human isoform of PTH is SEQ ID NO: 62. One human isoform of PTHrP is SEQ ID NO: 57. Although human isoforms are provided, it will be understood by those skilled in the art that other non-human-derived isoforms can be used as well. Such non-human-derived isoforms can interact with the human PTH / PTHrP receptor and vice versa. The PTH / PTHrP receptor agonist polypeptide segment is residues 1-33 of SEQ ID NO: 62 or can include the foregoing (residues 1-33 of PTH (SEQ ID NO: 57)). The PTH / PTHrP receptor agonist polypeptide segment is residues 1-34 of PTH (SEQ ID NO: 62) or can include the foregoing. In other embodiments, the foregoing is a fragment of residues 1-34 of PTH (SEQ ID NO: 62). In other embodiments, the PTH / PTHrP receptor agonist polypeptide segment is residues 1-84 of PTH (SEQ ID NO: 62) or can include the foregoing. In other embodiments, the PTH / PTHrP receptor agonist polypeptide segment is residues 1-14 of PTH (SEQ ID NO: 62) or residues 1-7 of PTH (SEQ ID NO: 62) or can include the foregoing.The amino acids essential for binding to the PTH receptor as an agonist are amino acids 1, 2, and 5 of PTH. In further other embodiments, the PTH / PTHrP receptor agonist is any other species of PTH or PTHrP polypeptide segment.

[0020] The PTH / PTHrP receptor antagonist can, in certain embodiments, comprise PTH(7-34) (i.e., residues 7-34 of PTH (SEQ ID NO: 62)). In another embodiment, the antagonist is or comprises residues 7-33 of PTH (SEQ ID NO: 62). In other embodiments, the antagonist is a fragment of residues 7-34 of SEQ ID NO: 57. In another embodiment, the PTH / PTHrP receptor antagonist comprises PTH(7-14) (i.e., residues 7-14 of PTH (SEQ ID NO: 62)). In another embodiment, the PTH / PTHrP receptor antagonist comprises PTH / PTHrP ((-1)-33). In another embodiment, the PTH / PTHrP receptor antagonist comprises residues 1-14 of PTH having an N-terminal extension. The addition of an N-terminal extension to PTH or the active N-terminal fragment of PTH converts the PTH peptide to an antagonist. The N-terminal extension can be 1, 2, 3, 4, 5 or more than 5 amino acids in length. Typically, what the amino acids of the N-terminal extension are is not important. In one embodiment, the PTH / PTHrP receptor antagonist comprises residues 1-33 of PTH (SEQ ID NO: 58) having a Gly-Ser extension at its N-terminus. In another embodiment, the PTH / PTHrP receptor antagonist comprises PTHrP(7-34) (i.e., residues 7-34 of SEQ ID NO: 57) or a fragment of residues 7-34 of SEQ ID NO: 57. In another embodiment, the PTH / PTHrP receptor antagonist comprises mouse TIP(7-39) (see, e.g., Hoare S R, Usdin T B. 2002. Specificity and stability of a new PTH1 receptor antagonist, mouse TIP(7-39). Peptides 23:989-98). Other PTH / PTHrP receptor antagonists that can be used in fusion proteins are also disclosed in the Hoare et al. paper. The PTH / PTHrP receptor antagonist can be a fragment of at least 8, 10, 12 or more than 12 amino acids derived from residues 1-34 of SEQ ID NO: 62.In other embodiments, the PTH / PTHrP receptor antagonist can be a PTH / PTHrP receptor antagonist polypeptide of another species.

[0021] In certain embodiments, the therapeutic agent or PTH / PTHrP receptor agonist or antagonist polypeptide segment is on the N-terminal side of the collagen-binding polypeptide of the fusion protein. That is, the two polypeptide segments each have an N-terminal and a C-terminal, and the N-terminal of the collagen-binding polypeptide is directly or indirectly, e.g., via a therapeutic agent linker polypeptide segment (e.g., a PKD, glycine or serine linker), linked to the C-terminal of the therapeutic agent or PTH / PTHrP agonist or antagonist polypeptide. A "therapeutic agent linker" can include a covalent bond and / or a linker or spacer moiety. For example, the collagen-binding agent and the therapeutic agent can be linked directly, e.g., via a peptide bond or chemical cross-link, or indirectly, e.g., via a linker or spacer polypeptide. Useful therapeutic agent linkers include homo- or hetero-functional linkers together with polypeptides, amino acids, nucleic acids. Particularly useful conjugation reagents that can facilitate the formation of a covalent bond between two collagen-binding domains can include N-hydroxysuccinimide (NHS) esters and / or maleimides.

[0022] The therapeutic agent linker can also include a spacer polypeptide. The spacer polypeptide can be of any length and can include traditional or non-traditional amino acids. For example, the spacer polypeptide can be 1-100 amino acids in length, and suitably, the spacer polypeptide is at least 2, 3, 5, 10, 15, 20, 25 or more than 25 amino acids in length such that the two collagen-binding domains of the collagen-binding agent can mediate collagen binding. The spacer polypeptide can include (but is not limited to) a PKD (polycystic kidney disease) domain from collagenase, a GST tag, a His tag, a Ser linker or a Gly linker. In some embodiments, the therapeutic agent linker can include a tag system. The tag system includes any drug group that can bind to each other with high affinity. Some tag systems are well known in the art and include (but are not limited to) biotin / avidin, biotin / streptavidin, or a digoxigenin (DIG) system. In some embodiments, the tag system includes biotin / avidin or biotin / streptavidin. The therapeutic agent can be linked to the N-terminus or C-terminus of the collagen-binding agent by the therapeutic agent linker. In embodiments where the therapeutic agent includes a polypeptide, the C-terminus or N-terminus of the therapeutic agent can be linked to the N-terminus or C-terminus of the collagen-binding agent by the therapeutic agent linker. Furthermore, the same or different drugs can be linked to both the N-terminus and C-terminus of the collagen-binding agent by two or more therapeutic agent linkers.

[0023] There is provided a pharmaceutical composition comprising any of the collagen-binding agents described herein. The pharmaceutical composition can include a pharmaceutical carrier, excipient, or diluent (i.e., a drug), which are non-toxic at the dosages or concentrations used for cells or animals to which they are exposed. Drugs are often present in an aqueous pH-buffered solution. Examples of pharmaceutical carriers include the following: buffers (e.g., phosphoric acid, citric acid, and other organic acids); antioxidants (including ascorbic acid); low molecular weight (less than 10 residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions (e.g., sodium); and / or nonionic surfactants (e.g., TWEEN TM surfactant, a trademark, polyethylene glycol (PEG), and PLURONICS TM surfactant).

[0024] There is also provided a method of treating a condition. The method can include administering to a subject, in an amount effective to treat the condition, any of the collagen-binding agents or pharmaceutical compositions described herein. "Condition" can include wounds (chronic and acute), hyperparathyroidism, hair conditions (excessive hair growth or hair loss), collagenopathies, and bone conditions. Bone conditions include fractures, osteoporosis, periodontal defects, or other bone defects. Collagenopathies include, but are not limited to, osteogenesis imperfecta (OI), Stickler syndrome, Ehlers-Danlos syndrome, Alport syndrome, Caffey disease, and focal collagen or cartilage damage. Many of these diseases are caused by genetic defects that result in underwound or partially unwound collagen in certain tissues. For example, bone loss due to collagen diseases (such as osteogenesis imperfecta, Stickler syndrome), osteoporosis or other diseases that pose a high risk of fractures to an individual due to collagen deficiency may be treatable by administration of a collagen-binding agent conjugated to an osteoanabolic peptide. The collagen-binding agent can direct the osteoanabolic agent to sites where collagen morphogenesis is abnormal and thus prevent fractures. For example, vascular fragility due to defects such as Ehlers-Danlos syndrome type IV, Alport syndrome, or other diseases where vascular rupture is likely due to collagen dysplasia can be treated by administration of a collagen-binding agent that includes a peptide that stimulates vascular growth or repair. The collagen-binding agent will direct the peptide to regions with collagen damage (which are likely to have damaged blood vessels). The therapeutic agent will stimulate growth and repair at the damaged site and prevent vascular rupture. For example, skin fragility due to abnormalities such as Ehlers-Danlos syndrome, Caffey disease, or other diseases where collagen deficiency leads to skin weakening resulting in hyperelasticity makes the skin prone to bruising and reduces wound healing. Skin and epithelial growth factors can be provided as therapeutic agents. The foregoing will stimulate skin growth and repair, preventin striae, and improve healing when conjugated to a collagen-binding agent and delivered to regions of damaged collagen.

[0025] Methods for treating hyperparathyroidism are also provided herein, the methods including, for example, administering to a subject in need of treatment for hyperparathyroidism a collagen-binding agent linked to PTH. In certain embodiments, the PTH administered to the subject can be PTH from a different species. The effects of PTH agonists and antagonists on hair growth have been studied for nearly 15 years. PTH has a common receptor for parathyroid hormone-related peptide (PTHrP), which is normally produced by dermal fibroblasts. PTHrP affects the proliferation / differentiation of keratinocytes and regulates the hair cycle. Most of the tests on hair growth effects have been performed with PTH antagonists because initial tests indicated that these antagonists were the most effective agents. Both injectable and topical formulations have been tested in animal models of chemotherapy-induced alopecia and SKH-1 hairless mice. Part of the effect of PTH antagonists on hair growth is the transition of hair follicles to dystrophic telogen, which protects hair follicles from chemotherapy-induced damage. However, a clinical trial of a topical PTH antagonist for chemotherapy-induced alopecia by IGI Pharmaceuticals was terminated at Phase 2 because the effects were limited. Accordingly, there is a need for new compositions for treating alopecia. In another aspect, methods for treating hair conditions (excessive hair loss or hair growth) are provided. The methods can include administering to a subject in need of treatment to induce hair growth or stop hair loss a collagen-binding agent linked to a PTH / PTHrP receptor agonist. The methods can be applied to individuals with alopecia, including, but not limited to, chemotherapy-induced alopecia, alopecia areata, male pattern baldness, alopecia caused by polycystic ovary syndrome, or other hair loss. The compositions can be administered topically or as a topical treatment to treat hair loss. In another aspect, a method is provided for retarding the growth or regrowth of hair after a hair removal treatment, said method comprising administering to a subject a collagen binding agent conjugated to a PTH / PTHrP receptor antagonist. In certain embodiments, the collagen binding agent + PTH antagonist composition is applied topically, externally. The collagen binding agent + PTH antagonist can be applied after a hair removal treatment to prevent or retard hair regrowth.

[0026] The subject of the present invention can be any mammal, suitably a human, a domesticated animal (e.g., dog, cat, horse, dairy cow, pig) or a mouse or rat. Treating or treating a condition includes alleviating at least one symptom of the condition, reducing or slowing further progression of the condition, or reducing or slowing spread to non-affected areas of the condition. Treating a subject refers to any type of treatment that confers a benefit on a subject that has or is at risk of developing a particular disease, and includes improving the subject's said condition (e.g., one or more symptoms), delaying progression of the disease, delaying onset of symptoms, or slowing progression of symptoms. As used herein, an effective amount or therapeutically effective amount means an amount of a composition that is sufficient to achieve treatment (as defined above) when administered to a subject for treating a condition, disorder or symptom. The therapeutically effective amount will vary according to the compound, formulation or composition, the disease and its severity, and the age, weight, health and responsiveness of the subject to be treated. The compositions described herein (i.e., the collagen-binding agent and the pharmaceutical composition) can be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transdermal, nasopharyngeal, intralesional, intratumoral, intradermal, or transmucosal absorption. Thus, the compositions can be formulated as ingestible, injectable, topical, or suppository formulations. The compositions can also be delivered in liposomes or sustained-release vehicles. Administration of the compositions to a subject according to the present invention can exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, an increase in beneficial biological effects can be expected with a larger dose of the composition compared to a smaller dose. Furthermore, efficacy is also expected at dosages lower than the level at which toxicity is observed.

[0027] The specific dosage administered in any given case will be adjusted according to one or more of the compositions being administered, the disease to be treated or suppressed, the condition of the subject, and other relevant medical factors (which may modify the activity of the composition or the response of the subject), as is well known to those skilled in the art. For example, the specific dosage for a particular subject will depend on age, weight, general health, diet, timing and manner of administration, rate of excretion, medications being concurrently administered, and the severity of the disease for which the treatment is applied. The dosage for a given patient can be determined conventionally by taking into account, for example, the differential activity of the compositions described herein and known agents, e.g., by routinely comparing them by means of appropriate conventional pharmacological protocols. The maximum dosage for a subject is the highest dosage that does not cause undesirable or unacceptable side effects. The number of variables regarding individual treatment regimens is large, and a considerable range of dosages can be expected. The route of administration will also affect dosage requirements. Administration of the composition will improve the symptoms being treated by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to no treatment. The effective dosage described herein refers to the total amount. That is, when two or more compositions are administered, the effective dosage corresponds to the total amount administered. The composition can be administered as a single dose or as divided doses. For example, the composition can be administered more than once, separated by 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, or more than 3 weeks. The collagen-binding agent or pharmaceutical composition described herein can be administered to a subject one or more times to effectively improve the symptoms to be treated. The appropriate dosage range is on the order of several hundred micrograms of the active ingredient and is in the range of about 0.01 to 10 mg / kg / day, preferably about 0.1 to 1 mg / kg / day. The exact amount of the active ingredient required for administration depends on the judgment of the physician and can be specific to each subject. It will be apparent to those skilled in the art that the therapeutically effective amount of the collagen-binding agent or pharmaceutical composition described herein depends, inter alia, on the administration schedule, whether the composition is administered in combination with other therapeutic agents, the condition and health status of the recipient, and the therapeutic activity of the composition.

[0028] The present disclosure is not limited to the specific details of the components, compositions, or method steps shown herein. The compositions and methods disclosed herein can be made, implemented, used, performed, and / or formed in a variety of manners that will be apparent to those skilled in the art in light of the subsequent disclosure. The phrases and terms used herein are for the purpose of description only and should not be regarded as limiting the claims. The recitations (e.g., first, second, and third) used herein to denote the order of various structures or method steps are not intended to be construed as indicating any particular structure or step, or any specific order or configuration with respect to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly inconsistent with the context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein is merely intended to facilitate the disclosure and does not suggest any limitation on the scope of the disclosure whatsoever unless otherwise specified. No language in this specification and no structure shown in the drawings should be construed as indicating that any unclaimed component is essential to the practice of the disclosed subject matter. The use of the terms “including,” “comprising,” or “having” and variations thereof herein means that the components and their equivalents recited thereafter are included together with additional components. Embodiments recited as including, comprising, or having certain components are also intended to consist essentially of and to consist of those certain components.

[0029] The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. For example, if a concentration range is recited as 1% to 50%, values such as, for example, 2% to 40%, 10% to 30%, or 1% to 3% are specifically intended to be recited within this specification. The foregoing are merely examples of specifically intended values, and all possible combinations of numerical values between and including the minimum and maximum values are to be considered specifically recited in this disclosure. The use of the term “about” to specifically enumerate a quantity or range of quantities is intended to indicate that values very near to the recited quantity, for example, values that may arise due to manufacturing tolerances, device and human error in the implementation of measurements, etc., are included within that quantity. All percentages referring to amounts are by weight unless otherwise indicated. None of the references (including non-patent documents or patent documents cited herein) are admitted to constitute prior art. In particular, it will be understood that, absent specific indication to the contrary, any reference herein to any document is not an admission that any of these documents form part of the common general knowledge in the art in the United States or any other country. Any discussion of the references describes what their authors assert, and the Applicant reserves the right to contest the accuracy and appropriateness of any of the documents cited herein. Absent specific and express indication to the contrary, all references cited herein are hereby incorporated by reference in their entirety. In the event of any discrepancy between any definition and / or description found in the cited references, the present disclosure will control. Absent specific definition or indication to the contrary by context, the terms “a,” “an,” and “the” mean “one or more.” For example, “a protein” or “a RNA” should be construed as “one or more proteins” or “one or more RNAs,” respectively. As used herein, "about," "approximately," "substantially," and "significantly" are understood by those skilled in the art and will vary somewhat depending on the context in which they are used. When these terms are used in a context not clear to one of ordinary skill in the art given the context in which they are used, "about" and "approximately" mean plus or minus 10% or less of the applicable term, and further "substantially" and "significantly" will mean plus or minus >10% of the applicable term. The following examples are intended to be illustrative only and are not intended as limitations on the scope of the invention or the appended claims.

Example

[0030] Activity and binding mechanism of a tandem collagen - binding domain with pseudo - two - fold symmetry Clostridium histolyticum secretes virulence factors (including highly active collagenases Col1G (class I) and ColH (class II)) and invades animal tissues. After the multi-domain ColG attaches itself to insoluble collagen using its tandem collagen-binding domains (CBDs), subsequent hierarchical substrate degradation requires progressive cleavage and re-organization of the fibrils. In this experiment, the structure of the calcium-binding CBD is presented at a resolution of 1.9 Å (R work = 15.0%; R free = 19.6%). The pseudo-twofold organization of the CBDs will allow ColG to act as a wedge between collagen molecules 55 Å apart and strongly assist in fibril re-organization and progressive cleavage. Indeed, the above accelerated the formation of collagen fibrils at a molecular ratio of tandem CBD:collagen of 0.1:1 to 0.5:1. At a molecular ratio of 1:1 or greater, the tandem CBDs retarded fibril formation. To toggle a staple between collagen molecules, the more tightly bound C-side CBD can initiate the binding (the above is [(Pro-Hyp-Gly) 10 3 ​as shown by small-angle X-ray scattering (SAXS) of the tandem CBDs that form a 1:1 complex with it). Subsequently, the weaker-binding N-side CBD can firmly grasp the prone collagen molecule and provide the strongest known fibril binding. The conformational change of the tandem CBD is calcium-dependent and cooperative at pCa values in the range of 3 - 6 as measured by size exclusion chromatography and SAXS. At pCa > 5, the tandem CBD adopts an extended structure that is more readily secreted from bacteria. At host pCa greater than 3 2+ the compact structure observed in the crystal structure is adopted. The binding and activation modes described herein will serve as a guide for the development of site-directed drug delivery vehicles. Abbreviations used: CBD, collagen-binding domain; MALDI-TOF-MS, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; DLS, dynamic light scattering; SAXS, small-angle X-ray scattering.

[0031] Clostridium histolyticum (recently proposed to be reclassified as Hathewaya histolytica Hathewaya histolyticum ) based on comparison of 16S rRNA genes) produces collagenases that cause extensive tissue damage during myonecrosis (Lawson and Rainey 2015). The most prominent of these enzymes, ColG (class I) and ColH (class II), are multi-domain enzymes containing an N-terminal collagenase module (s1), one or two polycystic kidney (PKD)-like domains, and one to three C-terminal collagen-binding domains (CBDs) (Figure 1A), and a role assignment in collagen degradation has been proposed (Fields 2013). The C-terminal domains of ColG (s3a, s3b) and ColH (s3) are homologs consisting of approximately 120 amino acids. These domains bind to soluble collagenous peptides and insoluble procollagen fibrils. Their role in the binding to procollagen fibrils is essential for their hierarchical structure disruption (Matsushita, Jung et al. 1998, Matsushita, Koide et al. 2001). Truncating the CBD from full-length ColG or ColH abolishes their ability to degrade procollagen fibrils. Such enzymes can only degrade solubilized collagen or denatured collagen (gelatin). Mutagenesis and collagen-binding assays mapped the binding surface of s3b, while NMR and SAXS studies showed that s3b binds to the underwound region of mini-collagen in a single orientation (Philominathan, Koide et al. 2009, Philominathan, Koide et al. 2012). Recently, high-speed atomic force microscopy demonstrated the ability of ColG to disrupt collagen in real time (Watanabe-Nakayama, Itami et al. 2016). During degradation, ColG moves processively along the procollagen fibril from the C-terminus to the N-terminus to disrupt the fibril. ColG also preferentially targeted more disordered procollagen fibril regions. Insights into the structure regarding the processivity and preference of ColG for disordered regions have been described. Bacterial collagenases require calcium for both full catalytic activity and collagen-binding function. Activation of bacterial collagenases requires a domain rearrangement from an α-helix to a β-sheet triggered by the binding of Ca 2+ to a linker (Wilson, Matsushita et al. 2003, Philominathan, Matsushita et al. 2009, Spiriti and van der Vaart 2010, Sides, Liyanage et al. 2012, Bauer, Wilson et al. 2013, Bauer, Janowska et al. 2015). Full-length apo-ColG has Ca2+ At low concentrations (0.2 - 0.3x10 -6 M), it is relatively flexible inside bacteria and is expected to enable easier secretion of enzymes (Wilson, Matsushita et al. 2003). Upon secretion, the linker chelates with Ca 2+ (about 1.2 mM) to form a rigid structure. Although this has not been shown for ColG, as demonstrated using SAXS and limited proteolysis, the chelation of Ca 2+ actually triggers the full-length ColH to adopt a less flexible and more compact structure (Ohbayashi, Matsumoto et al. 2013).

[0032] Clostridium collagenase has been used successfully for many years as a method for removing necrotic tissue in wounds. Recently, a mixture of ColG and ColH has been approved for use in the treatment of excessive connective tissue formation found, for example, in Dupuytren's disease (Gaston, Larsen et al. 2015). Furthermore, the enzyme has shown potential for the treatment of various types of connective tissue formation (Duarte, Correia et al. 2014) and the isolation of pancreatic islets for transplantation (McCarthy, Breite et al. 2011). In addition to the therapeutic use of full-length collagenase for the removal of connective tissue, non-catalytic segments are used for targeted drug delivery to reduce dosage and minimize side effects. First, Nishi et al. developed a fusion protein of a targeted-inducing segment (s2b-s3) and a growth factor. When injected, the fusion protein maintained activity at the injection site for up to 10 days (Nishi, Matsushita et al. 1998). A fusion protein of s3 and parathyroid hormone (PTH-CBD) has been developed for the treatment of osteoporosis (Ponnapakkam, Katikaneni et al. 2011, Ponnapakkam, Katikaneni et al. 2011, Ponnapakkam, Katikaneni et al. 2012), the prevention and treatment of alopecia (Katikaneni, Ponnapakkam et al. 2012, Katikaneni, Ponnapakkam et al. 2014, Katikaneni, Ponnapakkam et al. 2014, Katikaneni, Seymour et al. 2015). Systemic application is based on the clear target-inducing ability of s3 to blood-accessible regenerating collagen (Stratford, Vu et al. 2014).The less affinity s3 is effective as a systemic drug delivery vehicle in vivo, while the more robust collagen binder s2b-s3 is more effective in wound healing applications when applied to the damaged site together with collagen-based bone graft materials (An, Lin et al. 2015, Fujimaki, Inoue et al. 2015, Saito, Uchida et al. 2015, Uchida, Matsushita et al. 2015). Here we report the crystal structure of tandem CBD in the presence of calcium. We also propose its binding and interaction modes with the collagen substrate and the role of calcium based on the results of gel filtration and SAXS.

[0033] Materials and methods Production, purification, crystallization and structure determination Tandem CBD together with the individual s3a, s3b obtained from C. histolyticum was expressed as a glutathione S-transferase (GST)-fusion protein using the previously described method (Matsushita, Jung et al. 1998). Appropriate initial conditions for growing the crystals of tandem CBD were identified by a high-throughput screen (Hampton Research Crystal Screen HT). Using the said initial conditions, subsequent crystallization was carried out by the hanging-drop method. The crystals of tandem CBD (obtained in the presence of 3 mM calcium (21-26% PEG3350, HEPES pH 7.5, 37 °C)) are orthorhombic (space group, P2 1 2 1 2 1 ) and have the following lattice parameters a = 51.5 Å, b = 54.7 Å, c = 92.0 Å. The crystals grew within 24 hours at 37 °C, but did not grow at 4, 16 and 24 °C. The crystals were temperature-sensitive and could not withstand low temperatures. Therefore, the diffraction data were collected at room temperature with a resolution of 1.9 Å at the in-house X-ray facility using a Rigaku 007 generator with Cu Kα irradiation. The dataset was d *It was processed with TREK (Pflugrath 1999) (Table 1). The structure was analyzed with the molecular replacement program MolRep (CCP4 package). s3b (PDB code 2o8o) was used as the search model (Murshudov, Vagin et al. 1997). One tandem CBD was found in the asymmetric unit. Therefore, V M was 2.5 Å 3 / Da, and the solvent content was 50% (Matthews 1968). The refinement of the tandem CBD was carried out using Refmac_6.1.13 (Murshudov, Vagin et al. 1997). TLS restraints were applied to maintain the atomic chains for which each CBD acts as a TLS group. Babinet scaling was used for bulk solvent refinement. 5% of the data was set aside for monitoring R free . The model was manually adjusted between each refinement cycle using MIFit (McRee 1999). Alternative conformations were constructed for Lys818, Glu945, Tyr970, and Arg1005. The Ramachandran plot for the final structure obtained using the Procheck program (Laskowski, Macarthur et al. 1993) showed 91% of the residues in the core region and 9% of the residues in the additionally allowed regions, but showed nothing in the generously allowed or disallowed regions. The final refinement statistics are shown in Table 1.

[0034] Table 1: Data collection statistics TIFF0007682503000001.tif219153 a R meas =Σ hkl {N (hkl) / N (hkl) -1} 1 / 2 Σ i |I i(hkl) -<I (hkl) >| / Σ (hkl )Σ i I i(hkl) b Data for the highest resolution shell are shown in parentheses c5% of the data excluded from the refining

[0035] Small - angle X - ray scattering Appropriate buffer conditions for small-angle X-ray scattering (SAXS) were confirmed using discontinuous native PAGE. For pCa analysis, tandem CBD was equilibrated against 10 mM HEPES-Na (pH 7.5), 100 mM NaCl, and 2% glycerol. pCa was maintained with a total of 0.2 mM EGTA, and CaCl 2 was added to set the pCa values to 3, 4, 5, and 6. The amount of Ca 2+ required to obtain a given pCa was determined using MAXCHELATOR (Bers, Patton et al. 2010). Tandem CBD: [(POG) 10 3 For complex analysis, the complex was equilibrated against 50 mM HEPES-Na (pH 7.5), 100 mM NaCl, and 2 mM CaCl 2 [(POG) 10 3 was dissolved in 5 mM acetic acid at a concentration of 7.5 mg / mL and stored at 4 °C for 24 hours. Subsequently, the peptide was mixed with tandem CBD. The molar ratio of tandem CBD to mini-collagen was 1:2. Measurements were performed in three concentration series for each sample. For the pCa series, the concentrations of tandem CBD used at pCa 4, 5, and 6 were 1, 3, and 5 mg / mL. At pCa 3, the concentration series used was 2, 4, and 6 mg / mL. [(POG) 10 3 ​​​For the complex with [substance], the concentration series used were 1, 3, and 5 mg / mL. All SAXS data were collected at 10 °C at the Advanced Light Source of Berkley National Lab (SIBYLS beamline, 12.3.1). All data processing was achieved using Primusqt of the ATAS 2.6.1 software package. For each pCa's CBD, the exposure data obtained from the concentration gradient not affected by aggregation or detector saturation were extrapolated to infinite dilution. The radius of gyration (R g ), together with the determination of the maximum diameter and the ab initio shape reconstruction of the extrapolated data, were performed using the dammif function of Primusqt. The χ value calculated at the end of each run showed the agreement between the calculated scattering curve and the experimental scattering curve. In the pCa series, the χ value for each shape was in the range between 0.8 and 1.2. ([POG] 10 ) 3 For the complex with [substance], the χ value is 0.8.

[0036] Collagen fibril formation The effect of tandem CBD addition on the self-assembly of collagen molecules was monitored by measuring turbidity as an increase in optical density at 450 nm at 37 °C. On ice, a 2 mg / mL rat collagen solution was diluted to a final concentration of 0.5 mg / mL (2.4 μM) with 40 mM HEPES buffer (pH 7.5) (300 mM NaCl, 2 mM CaCl 2 added). In the next step, the collagen-binding domain was added at the following molar concentration ratios: 0.1:1, 0.2:1, 0.3:1, 0.5:1, 1:1, 2:1, 3:1, 5:1. Turbidity measurements were performed at 1-minute intervals using a spectrophotometer Filter Max F5 (Molecular Devices) in a 96-well plate. The following parameters were estimated from the turbidity curve: t lag -time at the end of the lag phase, maximum turbidity, and maximum fibril growth rate. Size - exclusion chromatography for analysis Size exclusion chromatography was performed at room temperature on an HPLC system. The HPLC system was equipped with a Superdex 75 column (1 x 30 cm (Pharmacia)) at a flow rate of 0.5 mL / min as described (Wilson, Matsushita et al. 2003). The following proteins were used as molecular mass standards: bovine serum albumin (67.0 kDa), chicken ovalbumin (43.0 kDa), and ribonuclease A (13.7 kDa (Pharmacia)). The measurements were performed in triplicate. Quantitative analysis of binding to collagenous peptides The binding of various CBD proteins to collagenous peptides was measured by surface plasmon resonance using a BIACORE instrument (Biacore, Uppsala, Sweden). The instrument had a sensor chip (CM5 (Biacore)) on which the peptide (Gly-(Pro-Hyp-Gly) 12 ) was immobilized as described (Wilson, Matsushita et al. 2003). Collagen fibril binding assay The binding affinities of three CBDs (s3a, s3b, and s3as3b) for collagen fibers were analyzed. First, 10 mg of porcine skin fibers (Nippi, Inc, Japan) were placed in a 0.2 nm spin column, and 200 mM NaCl and 5 mM CaCl 2It was washed with 50 mM Tris-HCl (pH 7.5) supplemented. Subsequently, 20 μL of a protein mixture containing 0.5 mg / mL of BSA (internal control) and various concentrations (2.5 to 20 mg / mL) of tandem CBD (equilibrated with the above buffer) was added and mixed well with the fibers. After incubating at room temperature for 30 minutes, the supernatant containing unbound tandem CBD was collected and quantified using SDS-PAGE. For this step, an equal volume of unused protein mixture was used as a control. After electrophoresis, the proteins were stained with Coomassie Brilliant Blue R-250, and the relative amounts of the proteins were estimated using ImageJ software (version 1.4.2; National Institutes of Health). Based on these estimates, a calibration curve was created for each CBD, and the amount of CBD in each supernatant was quantified using the above. The results obtained by assaying in triplicate were analyzed by a Scatchard plot, and the dissociation constant (K d ) and the number of binding sites (B max ) were obtained for each CBD.

[0037] Results and discussion Explanation of the structure of tandem CBD The crystal structure of the tandem CBD consisting of s3a and s3b was analyzed at a resolution of 1.9 Å. The s3b segment has been described previously, but the s3a segment is described for the first time in this paper. Both s3a and s3b adopt a very similar β-sandwich 'jelly-roll' (containing 10 β-strands). These CBDs are related by a pseudo two-fold rotational symmetry stabilized by salt bridge and hydrogen bond interactions. The pseudo-symmetry axis (perpendicular to the plane of the page in Figure 1B) is positioned 55 Å away from the collagen-binding pocket of the tandem CBD. Each domain chelates two Ca 2+ , i.e., one Ca 2+One is joined in a double pentagonal pyramid structure and the other in an anti-square prism structure (Bauer, Wilson et al. 2013). The electron density enabled the observation of a propyl-cis-peptide bond between Glu792 and Pro793 and a non-propyl-cis-peptide bond between Glu899 and Asn992. The overlay based on 110 equivalent C α atoms shows that s3a and s3b share an r.m.s.d. of 0.9 Å and deviate significantly only in the loops (r.m.s.d. of 1.1 - 2.5 Å). The loops of both CBDs show the highest B-factor values, while the β-sheet residues show low β-factor values. As expected, the B 2+ -factor values of the residues interacting with Ca are particularly the lowest. The average temperature factor of s3a is lower than that of s3b due to crystal packing. In the previously reported structure of holo-s3b (PDB code 4HPK), the protease-sensitive and highly active loop 960 - 968 was not observed (Philominathan, Koide et al. 2009, Sides, Liyanage et al. 2012, Bauer, Wilson et al. 2013). In the structure presented here, the dynamics of this loop are suppressed by crystal packing contacts. In other respects, s3b in isolated s3b and tandem CBD is substantially identical to each other (C α r.m.s.d. 0.6 Å).

[0038] Ca of tandem CBD 2+ Induced transformation The difference in Ca 2+ concentrations inside bacteria and the host ECM could be utilized to efficiently secrete bacterial collagenase in the host. The intracellular Ca 2+ concentration in Clostridium is probably similar to that in E. coli ( Escherichia coli ) (0.2 - 0.3 x 10 -6 M) (Holland, Jones et al. 1999), and this concentration is sufficiently lower than the apparent K 2+ for tandem CBD to Ca d (Figure 2A). However, extracellular tissue fluid has a Ca 2+(Maurer and Hohenester, 1997). When monitored by size-exclusion chromatography, the domain re-orientation of s3a-s3b is a cooperative event induced by Ca 2+ and the SAXS data supported the magnitude of this structural change (Figure 2B). At pCa below 5 (10 μM), the envelope of the tandem CBDs deduced by SAXS is rod-shaped (Figure 2B). The bulge in the envelope is similar to the manner in which an α-helical linker appears in SAXS (Sides, Liyanage et al. 2012). At pCa = 6, the β-strand A’ of s3b also unfolds, greatly increasing the activity of the linker (Sides, Liyanage et al. 2012). When increasing the Ca 2+ concentration, the tandem CBDs gradually adopt a more compact shape (Figure 2B). At pCa = 4 (100 μM), the shape is similar to the crystal structure, except for the bulge suggesting that the linker of s3a maintains its activity. At pCa = 3 (1 mM), the shape is in good agreement with the crystal structure of the tandem CBDs. The domain reorganization modeled by the SAXS-deduced envelope is consistent with the observations made on s3b with a 12-residue-long linker. The linker between s3a and s3b has been shown to undergo a secondary structure transformation from an α-helix to a β-sheet, which is Ca 2+Induced by binding, resulting in a more robust contact between domains (Wilson, Matsushita et al. 2003, Sides, Liyanage et al. 2012). The β-strand A' of s3b unfolds to greatly increase the activity of the linker (Sides, Liyanage et al. 2012). Free energy simulations have shown that calcium ions not only thermodynamically stabilize the cis-peptide bond but also catalyze its formation (Spiriti and van der Vaart 2010). Calcium-dependent structural changes were monitored for full-length ColH by size exclusion chromatography and SAXS (Ohbayashi, Matsumoto et al. 2013). Extending the discovery for tandem CBD, full-length ColG and ColH also 2+ It seems likely to undergo induced domain rearrangement. Active ColG and ColH within bacteria enable rapid secretion and induce maximum damage to the host.

[0039] Mini - collagen - tandem CBD interaction Our results suggest a specific interaction between tandem CBD and collagen fibrils or mini-collagen. Collagen fibrils are constructed by the zigzag arrangement of triple-helical tropocollagen, which is insoluble in water. On the other hand, synthetic collagenous peptides or mini-collagen (mimicking the structure of native tropocollagen and being water-soluble) have been used in the study of individual collagen-protein interactions. The use of mini-collagen also enables quantitative analysis of CBD-collagen interactions. K d values were evaluated for various forms of CBD against insoluble collagen fibrils and mini-collagen, and they showed good agreement (Matsushita, Koide et al. 2001). Tandem CBD was the most robust among the tested CBDs (about ~K d ), and much more robust than the sum of s3a or s3b alone (about 100 μM K d) It binds to collagen fibrils. However, tandem CBD binds to mini - collagen only with the same strength as s3b. Tandem CBD shows cooperative binding when the fixed mini - collagen density is high. The SAXS results for tandem CBD:mini - collagen also presented a 1:1 complex. If the binding affinities of s3a and s3b for mini - collagen are given, the s3b segment of tandem CBD binds in a single direction to the C - terminus of [(POG) 10 3 (Philominathan, Koide et al.2009, Philominathan, Koide et al.2012, Bauer, Wilson et al.2013), and thus the former was modeled as an envelope as such (Figure 2B). ​S3a binds to collagen with lower avidity than s3b, probably because s3a has lost one of the conserved tyrosine residues. When the CBD sequences were aligned, three Tyr residues (970, 994, and 996 of s3b) were well conserved, and introduction of mutations in any of these residues decreased binding to mini - collagen (Wilson, Matsushita et al. 2003). Instead, when the tandem CBD sequences were aligned, differences between the N - terminal CBD and the C - terminal CBD emerged. The equivalent of Tyr996 was not well conserved in the N - terminal CBD, but all three Tyr residues were well conserved in the C - terminal CBD (Figure 2B, Figure 5). s3a also lacked the conserved Tyr970 equivalent, and instead Ser851 occupied that position. His848 near s3a occupied nearly the same space near this residue and was able to fulfill its role to a lesser extent. Gene duplication of CBD clearly needed to reduce the functional importance at position 877 in order to prevent this domain type from becoming immobile on the surface of the fibril. The CBD domain is positioned by an extra β - strand (β - strand A’) and inter - domain interactions. β - strand A’ is present in both s3a and s3b but not in s3, and is thus specific to collagenases with multiple CBDs. The extra β - strand is stabilized by non - conserved side - chain interactions, suggesting that gene duplication was a relatively recent event. The observed 1:1 complex of CBD and mini-collagen indicates that stronger s3b binding initiates the binding. This proposed mechanism is supported by the observation that tandem CBD binds to mini-collagen with the same strength as s3b alone. Nevertheless, tandem CBD binds more strongly to collagen fibrils than s3b alone (Toyoshima, Matsushita et al. 2001). Although s3b initiates the binding and plays a central role, s3a is auxiliary but still plays a central role in the insertion into the gap occurring between collagen molecules 55 Å apart. It has been proposed that CBDs can lie side by side and bind to one tropocollagen molecule (Eckhard and Brandstetter 2011), but our structure suggests that this mode is less likely. Unlike ColG, ColH has two PKD-like domains (s2a and s2b) and only one CBD (s3). Although not a collagen-binding substance itself, s2b enhances the affinity of s3 for collagen (Matsushita, Jung et al. 1998). Although s3a is a collagen-binding substance, s3a can act in the same way as s2b by functioning as a source of weak interactions. This interaction allows the segment to contribute to the overall stronger collagen-binding site and to scan a single tropocollagen for opportunistic binding sites. The development of tandem PKD and tandem CBD segments has made it possible for ColG and ColH to potentially explore various niches in collagen fibrils.

[0040] Collagen fibril - tandem CBD interaction Pseudo two-fold symmetry defines the orientation of the collagen-binding surfaces at opposite ends of the molecule, allowing the tandem CBD to bind to two collagen molecules with the same orientation (Figure 1B). Collagen self-assembles in a process that can be monitored by changes in absorption at 450 nm to form collagen fibrils. Collagen-binding molecules (e.g., s3b) bind to collagen and retard fibril formation (Okano-Kosugi, Matsushita et al. 2009). However, since the tandem CBD appears to be able to wedge itself between two collagen molecules, its ability to promote self-assembly of fibrils was tested. To investigate the effect of tandem CBD on collagen fibril formation, the turbidity of a mixture of tandem CBD and collagen was monitored (Figure 3). Notably, the ratio of tandem CBD to collagen has a significant effect on the lag time before fibril formation. In the absence of tandem CBD (control), the lag time for fibril formation is 19 minutes, consistent with previous observations (Okano-Kosugi, Matsushita et al. 2009). At a tandem CBD:collagen ratio of 0.1:1, the lag time decreases to 15 minutes. When the concentration of tandem CBD is relatively high compared to the proportion of collagen, the lag time increases. At a tandem CBD:collagen ratio of 5:1, the lag time is 31 minutes. Fibril growth at low CBD ratios results in an absorption of 0.0217, which is much higher compared to samples with a high proportion of tandem CBD (absorption was 0.005). The effect on the lag time was inversely proportional to the thickness of the collagen fibrils. Mixtures of 0.1:1 and 0.2:1 of tandem CBD:collagen result in fibrils that are approximately 14% thicker than the collagen fibril control. At a 5:1 ratio, the thickness is 2% lower than the control. At low concentrations, tandem CBD aids collagen alignment and thus promotes the formation of nuclei for collagen self-assembly.The apparently opposing effects on the self-assembly of these two ratios of CBD to collagen can provide important clues for understanding how ColG dissociates the assembly and destroys collagen fibrils, as recently revealed (Watanabe-Nakayama, Itami et al. 2016).

[0041] Collagen degradation by ColG Hydrolysis of collagen fibrils by ColG monitored in real time by high-speed atomic force microscopy revealed the following: (1) Interactions between collagen molecules between fibrils prevent the fitting of collagenase molecules; (2) When exposed to ColG, collagen molecules are reorganized on other fibrils; (3) Disordered D-periodicity renders collagen fibrils sensitive to degradation by ColG; (4) ColG moves progressively from the C-terminus to the N-terminus; (5) At each pass, ColG evenly trims the thickness of the collagen fibril. The structure of tandem CBD gives some clue to the action of ColG. If the location of the slot in the face of the domain facing each other is given, we propose the following collagen binding modes: (i) tandem CBDs wedge the gap between the fibers (Figure 4B); (ii) tandem CBDs wedge the slots found in damaged or reformed collagen fibers (Figure 4C). The effort required to attach a staple to the gap between the fibers might seem as if ColG were being held back. The ability of tandem CBDs to promote fibrillogenesis could have explained how ColG reorganized the collagen fibrils. Mechanical disruption of the D-periodicity of the collagen fibrils could introduce pockets for ColG binding. If the fibrils are damaged by removing the outermost tropocollagen, the CBDs could wedge themselves between the exposed tropocollagens. Such an action gives rise to the interesting possibility of targeted delivery of drugs to damaged collagen using tandem CBDs. Alternatively, tandem CBDs might be able to wedge between two fibrils. The median surface-to-surface distance from one fibril to another is about 3.2 nm in the skin (Kuwaba, Kobayashi et al. 2001). Approximately 15% of the interfibrillar gaps in the skin should be of the order of 6 nm. These results suggest that tandem CBDs could be useful for attaching drugs to damaged tissues. Once wedged between the collagen molecules, the progressive movement of ColG from the C-terminus to the N-terminus is probably driven by the collagenase module, and thus evenly trimmed collagen fibrils are generated (Eckhard and Brandstetter 2011, Eckhard, Schonauer et al. 2011). The high-resolution structure of a bacterial tandem collagen-binding domain is reported for the first time. The pseudo-symmetric organization of the CBDs (resulting from gene duplication and fusion) may enable the CBDs to recognize unique niches in collagen fibrils and promote collagenolysis. The structure of the tandem CBDs shows that it can wedge between collagen molecules with a co-directional orientation. Together with the previously identified preference of the CBDs for the underwound regions of collagen, the tandem CBDs are suggested to direct ColG to damaged regions of collagen fibrils. Such target-directed delivery also opens up a new avenue for drug-targeted delivery where the tandem CBDs can tightly attach drugs to the damaged site.

[0042] References for Example 1 An, B., Y.S.Lin and B.Brodsky (2015)."Collagen interactions: Drug design and delivery." Adv Drug Deliv Rev. Bauer, R., K.Janowska, K.Taylor, B.Jordan, S.Gann, T.Janowski, E.C.Latimer, O.Matsushita and J.Sakon (2015)."Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH." Acta Crystallogr D Biol Crystallogr 71(Pt 3): 565-577. Bauer, R., J.J. Wilson, S.T.L. Philominathan, D. Davis, O. Matsushita and J. Sakon (2013). "Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium histolyticum." Journal of Bacteriology 195(2): 318 - 327. Bers, D.M., C.W. Patton and R. Nuccitelli (2010). "A Practical Guide to the Preparation of Ca2+ a Buffers." Calcium in Living Cells 99: 1 - 26. Duarte, A.S., A. Correia and A.C. Esteves (2014). "Bacterial collagenases - A review." Crit Rev Microbiol. Eckhard, U. and H. Brandstetter (2011). "Polycystic kidney disease-like domains of clostridial collagenases and their role in collagen recruitment." Biol Chem 392(11): 1039 - 1045. Eckhard, U., E. Schonauer, D. Nuss and H. Brandstetter (2011). "Structure of collagenase G reveals a chew-and-digest mechanism of bacterial collagenolysis." Nat Struct Mol Biol 18(10): 1109 - 1114. Fields, G.B. (2013). "Interstitial collagen catabolism." J Biol Chem 288(13): 8785 - 8793. Fujimaki, H., G. Inoue, K. Uchida, M. Miyagi, W. Saito, A. Sato and M. Takaso (2015). "Elevation of Microglial Basic Fibroblast Growth Factor Contributes to Development of Neuropathic Pain after Spinal Nerve Ligation in Rats." Spine (Phila Pa 1976). Gaston, R.G., S.E. Larsen, G.M. Pess, S. Coleman, B. Dean, B.M. Cohen, G.J. Kaufman, J.P. Tursi and L.C. Hurst (2015). "The Efficacy and Safety of Concurrent Collagenase Clostridium Histolyticum Injections for 2 Dupuytren Contractures in the Same Hand: A Prospective, Multicenter Study." J Hand Surg Am 40(10): 1963 - 1971. Holland, I.B., H.E. Jones, A.K. Campbell and A. Jacq (1999). "An assessment of the role of intracellular free Ca2+ in E.coli." Biochimie 81(8 - 9): 901 - 907. Katikaneni, R., T.Ponnapakkam, O.Matsushita, J.Sakon and R.Gensure (2014)."Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model." Anticancer Drugs 25(1): 30-38. Katikaneni, R., T.Ponnapakkam, A.Seymour, J.Sakon and R.Gensure (2014)."Parathyroid hormone linked to a collagen binding domain promotes hair growth in a mouse model of chemotherapy-induced alopecia in a dose-dependent manner." Anticancer Drugs 25(7): 819-825. Katikaneni, R., T.Ponnapakkam, H.Suda, S.Miyata, J.Sakon, O.Matsushita and R.C.Gensure (2012)."Treatment for chemotherapy-induced alopecia in mice using parathyroid hormone agonists and antagonists linked to a collagen binding domain." Int J Cancer 131(5): E813-821. Katikaneni, R., A.W.Seymour, R.Gulati, T.Ponnapakkam and R.C.Gensure (2015). "Therapy for Alopecia Areata in Mice by Stimulating the Hair Cycle with Parathyroid Hormone Agonists Linked to a Collagen-Binding Domain." J Investig Dermatol Symp Proc 17(2): 13-15. Kuwaba, K., M.Kobayashi, Y.Nomura, S.Irie and Y.Koyama (2001). "Elongated dermatan sulphate in post-inflammatory healing skin distributes among collagen fibrils separated by enlarged interfibrillar gaps." Biochem J 358(Pt 1): 157-163. Laskowski, R.A., M.W.Macarthur, D.S.Moss and J.M.Thornton (1993). "Procheck - a Program to Check the Stereochemical Quality of Protein Structures." Journal of Applied Crystallography 26: 283-291. Lawson, P.A.and F.A.Rainey (2015). "Proposal to restrict the genus Clostridium (Prazmowski) to Clostridium butyricum and related species." Int J Syst Evol Microbiol. Matsushita, O., C.M. Jung, J. Minami, S. Katayama, N. Nishi and A. Okabe (1998). "A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase." J Biol Chem 273(6): 3643-3648. Matsushita, O., T. Koide, R. Kobayashi, K. Nagata and A. Okabe (2001). "Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase." J Biol Chem 276(12): 8761-8770. Matthews, B.W. (1968). "Solvent content of protein crystals." J Mol Biol 33(2): 491-497. McCarthy, R.C., A.G. Breite, M.L. Green and F.E. Dwulet (2011). "Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria." Transplantation 91(2): 137-145. McRee, D.E. (1999). "XtalView / Xfit--A versatile program for manipulating atomic coordinates and electron density." J Struct Biol 125(2-3): 156-165. Murshudov, G.N., A.A. Vagin and E.J. Dodson (1997). "Refinement of macromolecular structures by the maximum-likelihood method." Acta Crystallogr D Biol Crystallogr 53(Pt 3): 240 - 255. Nishi, N., O. Matsushita, K. Yuube, H. Miyanaka, A. Okabe and F. Wada (1998). "Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain." Proc Natl Acad Sci U S A 95(12): 7018 - 7023. Ohbayashi, N., T. Matsumoto, H. Shima, M. Goto, K. Watanabe, A. Yamano, Y. Katoh, K. Igarashi, Y. Yamagata and K. Murayama (2013). "Solution structure of clostridial collagenase h and its calcium-dependent global conformation change." Biophys J 104(7): 1538 - 1545. Okano-Kosugi, H., O. Matsushita, S. Asada, A.B. Herr, K. Kitagawa and T. Koide (2009). "Development of a high-throughput screening system for the compounds that inhibit collagen-protein interactions." Anal Biochem 394(1): 125 - 131. Pflugrath, J.W. (1999). "The finer things in X-ray diffraction data collection." Acta Crystallogr D Biol Crystallogr 55(Pt 10): 1718 - 1725. Philominathan, S.T., O. Matsushita, R. Gensure and J. Sakon (2009). "Ca2+-induced linker transformation leads to a compact and rigid collagen-binding domain of Clostridium histolyticum collagenase." FEBS J 276(13): 3589 - 3601. Philominathan, S.T.L., T. Koide, K. Hamada, H. Yasui, S. Seifert, O. Matsushita and J. Sakon (2009). "Unidirectional Binding of Clostridial Collagenase to Triple Helical Substrates." Journal of Biological Chemistry 284(16): 10868 - 10876. Philominathan, S.T.L., T. Koide, O. Matsushita and J. Sakon (2012). "Bacterial collagen-binding domain targets undertwisted regions of collagen." Protein Science 21(10): 1554 - 1565. Ponnapakkam, T., R.Katikaneni, E.Miller, A.Ponnapakkam, S.Hirofumi, S.Miyata, L.J.Suva, J.Sakon, O.Matsushita and R.C.Gensure (2011)."Monthly administration of a novel PTH-collagen binding domain fusion protein is anabolic in mice." Calcif Tissue Int 88(6): 511-520. Ponnapakkam, T., R.Katikaneni, T.Nichols, G.Tobin, J.Sakon, O.Matsushita and R.C.Gensure (2011)."Prevention of chemotherapy-induced osteoporosis by cyclophosphamide with a long-acting form of parathyroid hormone." J Endocrinol Invest 34(11): e392-397. Ponnapakkam, T., R.Katikaneni, H.Suda, S.Miyata, O.Matsushita, J.Sakon and R.C.Gensure (2012)."A single injection of the anabolic bone agent, parathyroid hormone-collagen binding domain (PTH-CBD), results in sustained increases in bone mineral density for up to 12 months in normal female mice." Calcif Tissue Int 91(3): 196-203. Saito, W., K. Uchida, O. Matsushita, G. Inoue, H. Sekiguchi, J. Aikawa, H. Fujimaki and M. Takaso (2015). "Acceleration of callus formation during fracture healing using basic fibroblast growth factor - kidney disease domain - collagen - binding domain fusion protein combined with allogenic demineralized bone powder." J Orthop Surg Res 10: 59. Sides, C. R., R. Liyanage, J. O. Lay, S. T. L. Philominathan, O. Matsushita and J. Sakon (2012). "Probing the 3 - D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo - versus holo -) by Limited Proteolysis MALDI - TOF MS." Journal of the American Society for Mass Spectrometry 23(3): 505 - 519. Spiriti, J. and A. van der Vaart (2010). "Mechanism of the calcium - induced trans - cis isomerization of a non - prolyl peptide bond in Clostridium histolyticum collagenase." Biochemistry 49(25): 5314 - 5320. Stratford, R., Jr., C.Vu, J.Sakon, R.Katikaneni, R.Gensure and T.Ponnapakkam (2014). "Pharmacokinetics in rats of a long-acting human parathyroid hormone-collagen binding domain peptide construct." J Pharm Sci 103(2): 768-775. Toyoshima, T., O.Matsushita, J.Minami, N.Nishi, A.Okabe and T.Itano (2001). "Collagen-binding domain of a Clostridium histolyticum collagenase exhibits a broad substrate spectrum both in vitro and in vivo." Connect Tissue Res 42(4): 281-290. Uchida, K., O.Matsushita, N.Nishi, G.Inoue, K.Horikawa and M.Takaso (2015). "Enhancement of periosteal bone formation by basic fibroblast-derived growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase." J Tissue Eng Regen Med. Watanabe-Nakayama, T., M. Itami, N. Kodera, T. Ando and H. Konno (2016). "High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils." Sci Rep 6: 28975. Wilson, J. J., O. Matsushita, A. Okabe and J. Sakon (2003). "A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation." Embo Journal 22(8): 1743 - 1752.

Example

[0043] Acceleration of bone formation during fracture healing by basic fibroblast growth factor fused with a tandem collagen - binding domain of Clostridium histolyticum class I Increased retention of recombinant basic fibroblast growth factor (bFGF) at the fracture site accelerates bone formation during fracture healing. We previously constructed a fusion protein consisting of bFGF, a polycystic kidney disease domain (PKD; s2b), and a collagen-binding domain (CBD; s3) derived from Clostridium histolyticum class II collagenase (ColH). We reported that the combination of the fusion protein and a collagen-like peptide, poly(Pro-Hyp-Gly) 10 (SEQ ID NO: 59) induces proliferation and callus formation of mesenchymal cells at the fracture site. C. histolyticum is also known to produce class I collagenase (ColG) (having tandem CBDs (s3a and s3b) at the C-terminus). First, we prepared a collagenase peptide (H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2(Accession number: 60)) The binding affinity of four collagen anchors derived from two cross-linked clostridial collagenases was examined by surface plasmon resonance, revealing that tandem CBD (s3a-s3b) has the highest affinity for collagenous peptides. Subsequently, we constructed fusion proteins consisting of bFGF and single CBD (bFGF-s3b) or tandem CBD (bFGF-s3a-s3b) and compared their biological capabilities with those of a previous fusion construct (bFGF-s2b-s3). Tests using a fracture model showed that bFGF-s3a-s3b exhibited the highest ability to induce mesenchymal cell proliferation and bone formation. Collectively, collagen anchors with higher collagen-binding affinity act to enable bFGF to exhibit higher biological activity. Therefore, poly(Pro-Hyp-Gly) 10 / bFGF-(CBD) 2 The composite appears to have the potential ability to promote bone fracture healing in the clinical setting. Basic fibroblast growth factor (bFGF) is a mitogenic protein with angiogenic properties and is required for bone remodeling during early bone repair (1;2). Recombinant human bFGF has shown efficacy in the regeneration of bone fractures and defects in animal models of osteoporosis (3;4). In two recent clinical trials, treatment with bFGF accelerated bone healing at osteotomy and tibial fracture sites (5;6). The findings of these trials strongly indicate that bFGF promotes bone remodeling and regeneration, but exogenously added bFGF rapidly diffuses from the bone defect site. Clostridium histolyticum (the causative agent of pathological gas gangrene) secretes two classes of collagenase (class I, ColG and class II, ColH). These enzymes generally contain a catalytic domain (s1), a polycystic kidney disease domain (PKD, s2 (SEQ ID NO: 61)), and a collagen-binding domain (CBD, s3). However, the copy numbers of PKD and CBD of the C-terminal collagen anchor differ between ColG and ColH, being s2-s3a-s3b and s2a-s2b-s3, respectively (7;8). We previously showed that a fusion protein consisting of bFGF and the CBD derived from ColH (bFGF-s3) or PKD-CBD (bFGF-s2b-s3) accelerates bone formation in the rat femur compared to native bFGF when loaded on a collagen sheet (9). When combined with a high-density collagen sheet, bFGF-s2b-s3 promoted bone formation more strongly than bFGF-s3 (10). When combined with the collagen-like peptide poly(Pro-Hyp-Gly) 10 (SEQ ID NO: 59), bone formation was also induced more strongly compared to bFGF alone in a mouse bone fracture model (11). In a more recent study, a fusion protein consisting of galectin and tandem CBDs (s3a and s3b) derived from ColG showed higher collagen-binding activity than the corresponding protein fused with PKD and CBD (S2b and s3) derived from ColH (12). Based on these findings, we hypothesized that a bFGF fusion protein containing tandem CBDs of ColG would increase the retention of bFGF at the fracture site by enhancing collagen-binding activity, leading to improved bone formation and fracture repair. Therefore, we evaluated the dissociation constants in vitro between various collagen anchors and mini-collagen. According to those results, we constructed fusion proteins consisting of bFGF and single CBD (bFGF-s3b) or tandem CBDs (bFGF-s3a-s3b) derived from ColG, and examined the bone-forming ability of these fusion proteins together with two previously constructed fusion proteins (consisting of bFGF and a collagen anchor derived from ColH).

[0044] Materials and methods Mini - collagen peptides and collagen - like polypeptides Mini-collagen peptide, H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 (SEQ ID NO: 60) was synthesized by an N-(9-fluorenyl)methoxycarbonyl (Fmoc)-based method on Rink-amide resin (Novabiochem, San Diego, CA). In each cycle, the Fmoc-amino acid (5 equivalents (Novabiochem)) was reacted for 90 minutes in the presence of N,N'-diisopropylcarbodiimide (5 equivalents (Wako Pure Chemical, Osaka, Japan)) and 1-hydroxybenzotriazole (5 equivalents (Wako Pure Chemical)) in N,N-dimethylformamide. Fmoc deprotection was carried out with 20% (v / v) piperidine in DMF for 20 minutes. The peptide cleavage and deprotection steps were carried out at room temperature for 4 hours by treatment with a standard trifluoroacetic acid (TFA) scavenger cocktail (TFA: m-cresol: thioanisole: water: ethanedithiol, 82.5:5:5:5:2.5 (v / v)). The peptide was purified by HPLC, and the HPLC was performed using CH 3 CN water (both containing 0.05% (v / v) TFA). The purity of the product was confirmed by RP-HPLC, and the said HPLC was performed using a Cosmosil 5C18-AR-II column (4.6 x 250 mm (Nacalai Tesque)) with a linear gradient of CH 3 CN water (both containing 0.05% (v / v) TFA). Mass spectrometry analysis was performed using a Bruker Autoflex III MALDI-TOF MS (Bruker Daltonics, Leipzig, Germany). H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 (SEQ ID NO: 60): C 159 H232 N 44 O 52 ([M+H] + ) The calculated MS (MALDI-TOF) m / z value for ([M+H] ) was 3590.7 and the experimental value was 3590.6. H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 (SEQ ID NO: 60) The HPLC profile of (SEQ ID NO: 60) is shown in Figure 11. Collagen-like polypeptide, poly(Pro-Hyp-Gly) 10 (SEQ ID NO: 59) was obtained from a commercial source (PHG Co., Ltd., Hyogo, Japan) (13). Collagen anchors derived from Clostridium collagenase, ColG and ColH CBD (s3) and PKD-CBD (s2b-s3) derived from C. clostridium class II collagenase ColH were purified as previously described (14). CBD (s3b) and CBD-CBD (s3a-s3b) derived from C. clostridium class I collagenase ColG were purified as previously described (17). Quantitative analysis of the binding of collagen anchors to collagenous peptides The binding of the collagen anchor to the mini-collagen peptide was measured by surface plasmon resonance using a BIACORE instrument (Biacore, Uppsala, Sweden) in the same manner as previously reported (15). Briefly, the peptide was dissolved in 10 mM sodium acetate (pH 6.0) at a concentration of 0.1 mg / mL and covalently immobilized on a CM5 sensor chip (Biacore) using the standard amine coupling procedure recommended by the manufacturer. Resonance was measured at 25 °C at a flow rate of 20 μL / min in the following solution: 10 mM HEPES sodium (pH 7.4), 150 mM NaCl, 1 mM CaCl 2 and 0.005% Tween-20. After each binding step, the chip was regenerated with a 180-s pulse of 0.1 M HCl. The value of the apparent dissociation constant K D (app) was calculated from the equilibrium binding data for eight protein concentrations (100 nM - 300 μM) by directly fitting to the following equation by the least squares method: cRU = cRU maxx[Protein] / (K D +[Protein]) (Eq.1) where cRU is the equilibrium response corrected for the bulk refractive index error using a pseudo-conjugate flow cell blocked with ethanolamine, [Protein] is the concentration of the analyte, and K D is the dissociation constant. Collagen - binding bFGF Four collagen-binding bFGF fusion proteins (CB-bFGF) were used in this study (Figure 6). Two fusion proteins, bFGF-s3 and bFGF-s2b-s3, were prepared as previously described (10) (the fusion proteins consist of bFGF and CBD or PKD-CBD derived from ColH). To prepare bFGF-s3b(ColG), the DNA of the expression plasmid pCHG115 was digested with BamHI and EcoRI in the linker region and ligated to the hbFGF-encoding DNA fragment pretreated with BglII and EcoRI (pCHG115 DNA encodes a fusion protein between GST and the C-terminal collagen-binding domain (s3b(ColG))). Escherichia coli DH5α was transformed with the ligation mixture, and the nucleotide sequence of the resulting plasmid (pCHG115-hbFGF) was confirmed by Sanger sequencing. Escherichia coli BL21 CodonPlus RIL (Agilent Technologies, Santa Clara, CA) was transformed with the plasmid to express the GST-bFGF-s3b fusion protein. The fusion protein was purified, and the GST moiety was cleaved as previously described (10). Another fusion protein (bFGF-s3a-s3b) consisting of tandem CBDs derived from bFGF and ColG was generated in the same manner using another expression plasmid pCHG112.

[0045] Proliferation assay As previously described (9), the periosteum was collected from the distal femur of 10-week-old Wistar rats and subsequently digested with 0.2% type I collagenase (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 37°C for 2 hours. The digested samples were passed through a 40-μm filter to obtain a single cell suspension of nucleated cells. The cell suspension was seeded at 1x10 4 cells / cm 2 in a 6-well culture plate containing: α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The plates were incubated at 37°C for 7 days in a 5% CO 2 atmosphere and subsequently, passage 0 (P0) cells were detached from the plate surface by treatment with 0.25% trypsin and 1 mM EDTA for 5 minutes. The cells were collected and seeded at 1.25x10 3 cells / well in a 96-well plate. Subsequently, α-MEM containing bFGF, bFGF-s3(ColH), bFGF-s2b-s3(ColH), bFGF-s3b(ColG), or bFGF-s3a-s3b(ColG) was added to the culture supernatant at concentrations of 0 (control), 0.1, and 10 pM. Two days after treatment, cell proliferation was evaluated. The water-soluble tetrazolium (WST) assay kit (Cell Count Reagent SF; Nacalai Tesque, Kyoto, Japan) was used for the evaluation according to the manufacturer's protocol and the previously described procedure (9). Fracture model A femoral fracture model was created using 9-week-old C57BL / 6J mice. The mice were given a standard solid diet for rodents (CRF-1 (Oriental Yeast, Tokyo, Japan)) and housed in a semi-barrier system at Nippon Charles River Laboratories (Kanagawa, Japan) under controlled environmental conditions (temperature, 23 ± 2°C; humidity, 55 ± 10%; lighting, 12-hour light / dark cycle). First, a 4-mm incision was made near the patella in the mid-sagittal plane of the left knee under aseptic conditions. After making a 0.5-mm hole with a drill in the intracondylar notch, a 0.2-mm tungsten guide wire was inserted retrogradely into the intramedullary canal, and a femoral segment was removed by a lateral approach using a wire saw (diameter 0.22 mm). To fix the fracture, a stainless steel screw (diameter 0.5 mm) was inserted into the intramedullary canal after removing the guide wire. Immediately after fracture creation, the following were injected into the fracture site: PBS (control), 0.058 nmole of bFGF, bFGF-s3, bFGF-s2b-S3, bFGF-s3b, or poly(Pro-Hyp-Gly) containing bFGF-s3a-s3b 10 (SEQ ID NO: 59) (n = 8 for each treatment). The dose of bFGF was determined based on the results of a previous study (11). The treatment of all animals was in accordance with the guidelines of the Animal Ethics Committee of Kitasato University. Quantification of new bone volume and bone mineral content To quantify the volume of new bone and bone mineral content in the control and treatment regions, femurs were harvested from sacrificed mice 4 weeks after fracture treatment and stored at 4 °C for 48 h in 4% paraformaldehyde. The femurs were transferred to PBS and imaged using an inspeXio SMX-90CT microfocus X-ray CT system (Shimadzu, Tokyo, Japan) with the following settings: acceleration voltage, 90 kV; current, 110 mA; voxel size, 20 lm / pixel; and matrix size, 1024x1024. The volume of new bone and bone mineral content were quantified from the micro-CT images of the whole femur in a 10-mm region (500 slices) of interest in the midfemur using Tri-3D-Bon three-dimensional (3D) image analysis software (Ratoc System Engineering, Tokyo, Japan) as previously described (14;16). Bone mineral content was estimated by comparing the measured density of each femur sample in the micro-CT image with that of a hydroxyapatite (HA) calibration curve. The calibration curve was constructed by plotting data from phantom images prepared using 200, 300, 400, 500, 600, 700 and 800 mg HA / cm 3 New bone was defined as having a threshold density of 300 mg / cm 3 or greater. Histological evaluation Poly(Pro-Hyp-Gly) 10 / bFGF-s3a-s3b(ColG) on bone formation, femurs were excised from control and treated animals 14 days after fracture induction. The femur samples were decalcified in 20% EDTA solution for 28 days and subsequently the remaining tissues were embedded in paraffin. The femurs were sectioned (3 μm) along the long axis in the frontal plane and the resulting sections were stained with hematoxylin and eosin (HE) for morphological analysis. Statistical analysis The differences among PBS (control), bFGF, bFGF-s3, bFGF-s2b-s3, bFGF-s3b, and bFGF-s3a-s3b were examined using one-way analysis of variance with Fisher's least significant difference (LSD) test. The significance level was set at p < 0.05. All statistical analyses were performed using SPSS software (version 19.0 (SPSS Inc., Chicago, IL)).

[0046] Results Binding affinity of collagen anchors The dissociation constants for the four collagen anchor mini-collagen peptides, H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 (SEQ ID NO: 60) were measured by surface plasmon resonance (Table 2). s2b-s3 (PKD-CBD(ColH)) showed a lower Kd value compared to s3 alone (CBD(ColH)), while the values of s3b (CBD(ColG)) and s3a-s3b (CBD-CBD(ColG)) were approximately 10-fold lower than those of the ColH anchor. It can be predicted that the ColG-derived anchor binds more tightly to the collagenous peptide than the ColH-derived anchor.

[0047] Table 2: Binding affinities of various collagen anchors TIFF0007682503000002.tif33156 Regarding Table 2, the anchor proteins were dissolved in HBS-Ca buffer at concentrations ranging from 1x10 -7 M to 3x10 -4 M. The binding of the collagenous peptide, H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 (SEQ ID NO: 60) was measured by surface plasmon resonance. The data were directly fitted to the equation described in the Materials and Methods section by the least squares method, and values for the apparent dissociation constant (K D ) and uncertainty were calculated. In vitro biological activity of the fusion protein The biological activities of four CB-bFGFs were evaluated by measuring the in vitro proliferation of rat periosteal mesenchymal cells (Figure 7). Two days after treatment with 0.1 pM bFGF-s3a-s3b(ColG), the number of cultured periosteal mesenchymal cells increased significantly compared to the control (α-MEM) treatment group. In contrast, no significant increase was detected in cells treated with bFGF, bFGF-s3(ColH), bFGF-s2b-s3(ColH), or bFGF-s3b(ColG). However, when the concentration of bFGF or CB-bFGF was increased to 1 or 10 pM, the cell number increased significantly with all forms of growth factors compared to the α-MEM treatment group. CB - bFGF / poly(Pro - Hyp - Gly) 10 In vitro callus formation induced by the composite Poly(Pro-Hyp-Gly) 10 The gels were mixed with PBS, bFGF (control), or one of the four prepared CB-bFGFs and applied to the fracture sites of the mouse femurs. After 4 weeks of recovery, the callus formation at the fracture sites was evaluated by micro-CT image analysis (Figure 8A - 8F). Compared to the fracture sites injected with PBS, the callus volume and bone mineral content were significantly higher at the sites treated with bFGF or CB-bFGF fusion proteins in combination with Poly(Pro-Hyp-Gly) 10 (Figure 9, P < 0.05). However, it should be noted that bFGF-s2b-s3(ColH), bFGF-s3b(ColG), and bFGF-s3a-s3b(ColG) resulted in higher callus volumes and bone mineral contents compared to bFGF (Figure 9, P < 0.05). Among the three fusion proteins, bFGF-s3a-s3b(ColG) showed the highest bone repair efficacy, and its callus volume and bone mineral content were significantly higher than those of all other groups (Figure 9, P < 0.05). Histomorphometric findings Poly(Pro-Hyp-Gly) 10To elucidate the mechanism by which / bFGF-s3a-s3b(ColG) accelerates new bone formation, histological evaluation of the treated fracture sites was performed 2 weeks after the first detection of callus formation in a mouse femoral fracture model. Larger calluses were observed at the fracture sites treated with either bFGF-s2b-s3(ColH), bFGF-s3b(ColG) or bFGF-s3a-s3b compared to the control group (Figs. 10A - 10F). Notably, the callus formed by bFGF-s3a-s3b treatment was significantly larger than that observed in the other treatment groups (Figs. 10A - 10F). This finding is for poly(Pro-Hyp-Gly) 10 / bFGF-s3a-s3b(ColG) was shown to accelerate the proliferation of periosteal cells at the early stage of fracture healing.

[0048] Discussion Clostridial collagenase has a collagen anchor at its C-terminus. The anchor binds to collagenous peptides having a collagen fibril and triple helix structure, but does not bind to denatured collagen (gelatin) (17). The anchor is formed by two types of domains, PKD and CBD (the former strengthens the binding of the latter). The enzyme has a collagen anchor formed by various copy numbers of PKD and CBD. Previously, we showed the callus-inducing ability of a composite material formed by a collagen carrier (high-density collagen sheet / powder or demineralized bone matrix) and anchor-fused bFGF (bFGF-PKD-CBD) (the anchor is formed by a single copy of each of PKD and CBD derived from Clostridium histolyticum class II collagenase (ColH)) (14; 16). Recently, we attempted to use a collagenous peptide gel instead of the collagen carrier. This is because collagenous peptides can be more easily applied by injection. In a fracture model of aged mice, poly(Pro-Hyp-Gly) 10 and the novel composite material formed with bFGF-PKD-CBD is for poly(Pro-Hyp-Gly) 10and induced stronger bone formation than those formed with bFGF (11). Therefore, we were able to speculate that we could optimize the efficacy of this composite material by switching the anchors with diverse binding affinities to collagenous peptides. To estimate the binding affinity of diverse anchors to the peptide carrier (poly(Pro-Hyp-Gly) 10 ), we synthesized a longer collagenous peptide, H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 as a ligand for the surface plasmon resonance assay. The first triplet, Pro-Arg-Gly in the peptide was introduced to maintain the water solubility and triple helix structure of the peptide. Since the peptide is immobilized at the N-terminus on the sensor chip, it would be acceptable to predict that the changes near the N-terminus do not significantly affect the binding. For this peptide, a single CBD (Colg s3b) presented a K 8 value (5.72 ± 0.473x10 D M) similar to the previously reported K -5 value (4.54 ± 0.15x10 D M) for a shorter collagenous peptide (G(POG) -5 ), indicating that the quantitative analysis performed here is reproducible. The collagen anchors (s3a-s3b and s3b) derived from class I enzyme (ColG) showed significantly higher affinity to this peptide than those derived from class II enzyme (ColH) (s2b-s3 and s3), suggesting that the former is a more appropriate anchor for the peptide carrier than the latter. The presence of the additional CBD (s3a) did not significantly enhance the binding of ColG s3b to this synthetic peptide, suggesting that the peptide used in this assay is still too short to allow simultaneous binding of two CBDs. Another possibility is that the binding between S3a and the collagen peptide is too weak to be reflected by the apparent K D value. Binding assays using longer collagenous peptides and / or small-angle X-ray diffraction tests may be necessary to solve this problem. If one assumes that efficacy correlates with binding affinity, greater heterotopic bone formation might be expected when using ColG anchor-fused bFGF in combination with collagenous peptides. Therefore, we prepared CB-bFGF having one of the anchors described above. To confirm that the bFGF moiety of each CB-bFGF construct was intact, a cell proliferation assay was performed in vitro. Four CB-bFGFs and bFGF promoted cell proliferation in a dose-dependent manner. This indicates that bFGF has activity despite the diverse anchor moieties. The specific activity of bFGF-CBD-CBD(s3a-s3b) seems to be slightly higher than that of other CB-bFGFs at low concentrations (0.1 - 1.0 pM), which might be due to the binding of this CB-bFGF to collagen produced by mesenchymal cells. Subsequently, poly(Pro-Hyp-Gly) 10 and the osteogenic potential of the complex formed with any of the four CB-bFGFs were compared using a mouse femoral fracture model. Poly(Pro-Hyp-Gly) 10 When combined with the gel-like carrier formed, bFGF-CBD(ColG) and bFGF-CBD-CBD(ColG) induced significantly greater heterotopic bone formation and significantly larger callus at fracture healing at 2 weeks compared to bFGF, bFGF-PKD-CBD(ColH), or bFGF-CBD(ColH). Among the test substances, bFGF-CBD-CBD(ColG) resulted in the highest callus volume and bone mineral content. The binding affinity of the collagen anchor can affect the osteogenic activity of the corresponding fusion protein. This is because rapid diffusion from the defect site of the target molecule would limit their osteogenic potential (10). Previously, we showed that bFGF-PKD-CBD(ColH) has a higher binding affinity for the collagen carrier and induces stronger bone formation compared to bFGF-CBD(ColH) (10). Among the collagen anchors used in this study, ColG CBD showed approximately 10-fold higher affinity for the collagenous peptide than the ColH anchor. This finding is consistent with the in vivo results (in vivo, bFGF fused with the ColG anchor accelerated osteogenesis more effectively when combined with the collagenous peptide compared to ColH anchor-fused bFGF). Overall, the osteogenic potential appears to correlate with the binding affinity of the collagen anchor, at least within the range we examined. Furthermore, when introduced together with the collagen-like polypeptide, poly(Pro-Hyp-Gly) 10 it is also likely that tandem CBD(ColG) increases the retention time of bFGF at the fracture site and thus accelerates bone formation. These results suggest that bFGF-CBD-CBD(ColG) combined with poly(Pro-Hyp-Gly) 10 is a promising therapeutic agent for the stimulation of bone repair in a clinical setting. Previous studies have shown that fusion proteins of growth factors and CBD have superior biological activities compared to natural growth factors (18; 19). For example, Han et al. (18) reported that bone morphogenetic proteins containing CBD derived from von Willebrand factor enhanced the in vitro alkaline phosphatase activity of mouse osteoblast cell line MC3T3-E1. Since bFGF stimulates periosteal mesenchymal cells (9; 10; 16), we examined the mitogenic ability of bFGF and several CB-bFGF fusion proteins using rat periosteal mesenchymal cells and showed that bFGF-CBD-CBD stimulated the highest cell mitogenic activity among the proteins examined at a low concentration of 1 pM. Collectively, the findings of this study showed that bFGF-CBD-CBD has both high collagen-binding affinity and biological activities, including the ability to stimulate callus formation during fracture healing. A recombinant collagen-binding bFGF fusion protein containing tandem CBDs of C. clostridium class I collagenase ColG induced strong bone formation when injected together with a collagen-like peptide, poly(Pro-Hyp-Gly) 10 into the fracture site of mouse femurs. The high osteogenic properties of bFGF-CBD-CBD / poly(Pro-Hyp-Gly) 10 suggest that this composite has the potential ability to promote fracture healing in a clinical setting.

[0049] References for Example 2 [1] Khan SN, Bostrom MP, Lane JM: Bone growth factors. Orthop Clin North Am 2000;31:375-388. [2] Ueno M, Urabe K, Naruse K, Uchida K, Minehara H, Yamamoto T, Steck R, Gregory L, Wullschleger ME, Schuetz MA, Itoman M: Influence of internal fixator stiffness on murine fracture healing: two types of fracture healing lead to two distinct cellular events and FGF-2 expressions.Exp Anim 2011;60:79-87. [3] Kato T, Kawaguchi H, Hanada K, Aoyama I, Hiyama Y, Nakamura T, Kuzutani K, Tamura M, Kurokawa T, Nakamura K: Single local injection of recombinant fibroblast growth factor-2 stimulates healing of segmental bone defects in rabbits.J Orthop Res 1998;16:654-659. [4] Kawaguchi H, Kurokawa T, Hanada K, Hiyama Y, Tamura M, Ogata E, Matsumoto T: Stimulation of fracture repair by recombinant human basic fibroblast growth factor in normal and streptozotocin-diabetic rats.Endocrinology 1994;135:774-781. [5] Kawaguchi H, Jingushi S, Izumi T, Fukunaga M, Matsushita T, Nakamura T, Mizuno K, Nakamura T, Nakamura K: Local application of recombinant human fibroblast growth factor-2 on bone repair: a dose-escalation prospective trial on patients with osteotomy.J Orthop Res 2007;25:480-487. [6] Kawaguchi H, Oka H, Jingushi S, Izumi T, Fukunaga M, Sato K, Matsushita T, Nakamura K: A local application of recombinant human fibroblast growth factor 2 for tibial shaft fractures: A randomized, placebo-controlled trial.J Bone Miner Res 2010;25:2735-2743. [7] Bauer R, Wilson JJ, Philominathan ST, Davis D, Matsushita O, Sakon J: Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum.J Bacteriol 2013;195:318-327. [8] Bauer R, Janowska K, Taylor K, Jordan B, Gann S, Janowski T, Latimer EC, Matsushita O, Sakon J: Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH.Acta Crystallogr D Biol Crystallogr 2015;71:565-577. [9] Uchida K, Matsushita O, Naruse K, Mima T, Nishi N, Hattori S, Ogura T, Inoue G, Tanaka K, Takaso M: Acceleration of periosteal bone formation by human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase.J Biomed Mater Res A 2014;102:1737-1743.

[10] Uchida K, Matsushita O, Nishi N, Inoue G, Horikawa K, Takaso M: Enhancement of periosteal bone formation by basic fibroblast-derived growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase.J Tissue Eng Regen Med 3-18-2015.

[11] Sekiguchi H, Uchida K, Inoue G, Matsushita O, Saito W, Aikawa J, Tanaka K, Fujimaki H, Miyagi M, Takaso M: Acceleration of bone formation during fracture healing by poly(pro-hyp-gly)10 and basic fibroblast growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase.J Biomed Mater Res A 2016;104:1372-1378.

[12] Fukata Y, Itoh A, Nonaka Y, Ogawa T, Nakamura T, Matsushita O, Nishi N: Direct cytocidal effect of galectin-9 localized on collagen matrices on human immune cell lines.Biochim Biophys Acta 2014;1840:1892-1901.

[13] Kishimoto T, Morihara Y, Osanai M, Ogata S, Kamitakahara M, Ohtsuki C, Tanihara M: Synthesis of poly(Pro-Hyp-Gly)(n) by direct poly-condensation of (Pro-Hyp-Gly)(n), where n=1, 5, and 10, and stability of the triple-helical structure.Biopolymers 10-15-2005;79:163-172.

[14] Saito W, Uchida K, Matsushita O, Inoue G, Sekiguchi H, Aikawa J, Fujimaki H, Takaso M: Acceleration of callus formation during fracture healing using basic fibroblast growth factor-kidney disease domain-collagen-binding domain fusion protein combined with allogenic demineralized bone powder.J Orthop Surg Res 2015;10:59.

[15] Wilson JJ, Matsushita O, Okabe A, Sakon J: A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation.EMBO J 4-15-2003;22:1743-1752.

[16] Saito W, Uchida K, Ueno M, Matsushita O, Inoue G, Nishi N, Ogura T, Hattori S, Fujimaki H, Tanaka K, Takaso M: Acceleration of bone formation during fracture healing by injectable collagen powder and human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase.J Biomed Mater Res A 2014;102:3049-3055.

[17] Matsushita O, Koide T, Kobayashi R, Nagata K, Okabe A: Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase. J Biol Chem 3-23-2001;276:8761-8770.

[18] Han X, Zhang W, Gu J, Zhao H, Ni L, Han J, Zhou Y, Gu Y, Zhu X, Sun J, Hou X, Yang H, Dai J, Shi Q: Accelerated postero-lateral spinal fusion by collagen scaffolds modified with engineered collagen-binding human bone morphogenetic protein-2 in rats. PLoS One 2014;9:e98480.

[19] Shiozaki Y, Kitajima T, Mazaki T, Yoshida A, Tanaka M, Umezawa A, Nakamura M, Yoshida Y, Ito Y, Ozaki T, Matsukawa A: Enhanced in vivo osteogenesis by nanocarrier-fused bone morphogenetic protein-4. Int J Nanomedicine 2013;8:1349-1360. Preferred embodiments of the present invention are as follows. 〔1〕A collagen-binding therapeutic agent comprising two collagen-binding domains linked by a domain linker and a therapeutic agent linked by a therapeutic agent linker to at least one of the collagen-binding domains, wherein the collagen-binding agent lacks collagenase activity, and each of the two collagen-binding domains is selected from the group consisting of any one of the polypeptides of SEQ ID NO: 1-39, a polypeptide having at least 90% sequence identity with any one of SEQ ID NO: 1-39, and a fragment of at least 8 consecutive amino acids of any one of SEQ ID NO: 1-39, the collagen-binding therapeutic agent. 〔2〕The collagen-binding agent according to 〔1〕 above, wherein the therapeutic agent is selected from the group consisting of FGF, parathyroid hormone (PTH), PTH / PTHrP receptor agonist, PTH / PTHrP receptor antagonist, bone morphogenetic protein (BMP), G-CSF, BMP-2, BMP-3, anti-sclerostin antibody, growth hormone, IGF-1, VEGF, TGF-β, KGF, TGF-α, TGF-β1, TGF-β receptor, CT, GH, GM-CSF, EGF, PDGF, seriprorolol, activin, and connective tissue growth factor. 〔3〕The collagen-binding agent according to 〔1〕 or 〔2〕 above, wherein one of the collagen-binding domains is selected from the group consisting of any one of the polypeptides of SEQ ID NO: 15-30, a polypeptide having at least 90% sequence identity with any one of SEQ ID NO: 15-30, and a fragment of at least 8 consecutive amino acids of any one of SEQ ID NO: 15-30, and the other collagen-binding domain is selected from the group consisting of any one of the polypeptides of SEQ ID NO: 1-14 and 31-39, a polypeptide having at least 90% sequence identity with any one of SEQ ID NO: 1-14 and 31-39, or a fragment of at least 8 consecutive amino acids of any one of SEQ ID NO: 1-14 and 31-39. 〔4〕The collagen-binding agent according to any one of 〔1〕 to 〔3〕 above, wherein the two collagen-binding domains are selected from the group consisting of any one of SEQ ID NOs: 15-30, a polypeptide having at least 90% sequence identity with any one of SEQ ID NOs: 15-30, and a fragment of at least 8 consecutive amino acids of any one of SEQ ID NOs: 15-30. 〔5〕The collagen-binding agent according to any one of 〔1〕 to 〔4〕 above, wherein the two collagen-binding domains are selected from the group consisting of a polypeptide of any one of SEQ ID NOs: 1-14 and 31-39, a polypeptide having at least 90% sequence identity with any one of SEQ ID NOs: 1-14 and 31-39, or a fragment of at least 8 consecutive amino acids of any one of SEQ ID NOs: 1-14 and 31-39. 〔6〕The collagen-binding agent according to any one of 〔1〕 to 〔5〕 above, wherein the collagen-binding agent comprises a polypeptide selected from the group consisting of any one of the polypeptides of SEQ ID NOs: 40-47, a polypeptide having at least 90% sequence identity with any one of SEQ ID NOs: 40-47, and a fragment of at least 8 consecutive amino acids of any one of SEQ ID NOs: 40-47. 〔7〕The collagen-binding agent according to any one of 〔1〕 to 〔6〕 above, wherein the domain linker comprises a polypeptide. 〔8〕The collagen-binding agent according to any one of 〔1〕 to 〔7〕 above, wherein the domain linker comprises a polypeptide selected from the group consisting of any one of the polypeptides of SEQ ID NOs: 48-55, or a polypeptide having at least 80% sequence identity with any one of the polypeptides of SEQ ID NOs: 48-55. 〔9〕The collagen-binding agent according to any one of 〔1〕 to 〔8〕 above, wherein the therapeutic agent is selected from the group consisting of polypeptides, hormones, growth factors, cytokines, small molecules, polynucleotides, carbohydrates, and lipids. 〔10〕The collagen-binding agent according to any one of 〔1〕 to 〔9〕 above, wherein the therapeutic agent comprises a polypeptide. [

[11] ] The collagen-binding agent according to any one of [[1]] to [

[10] ] above, wherein the therapeutic agent is selected from the group consisting of FGF, parathyroid hormone (PTH), a PTH / PTHrP receptor agonist, and a PTH / PTHrP receptor antagonist. [

[12] ] The collagen-binding agent according to any one of [[1]] to [

[11] ] above, wherein the therapeutic agent linker contains a polypeptide. [

[13] ] The collagen-binding agent according to any one of [

[10] ] to [

[12] ] above, wherein the C-terminus of the therapeutic agent is linked to the N-terminus of the collagen-binding agent by the therapeutic agent linker. [

[14] ] A pharmaceutical composition comprising any one of the collagen-binding agents according to [[1]] to [

[13] ] above and a pharmaceutical carrier. [

[15] ] A method for treating a condition, comprising the step of administering to a subject any one of the collagen-binding agents according to [[1]] to [

[13] ] above or the pharmaceutical composition according to [

[14] ] above in an amount effective for treating the condition. [

[16] ] The method according to [

[15] ] above, wherein the subject is a mammal. [

[17] ] The method according to [

[16] ] above, wherein the mammal is a human. [

[18] ] The method according to any one of [

[15] ] to [

[17] ] above, wherein the condition is selected from the group consisting of hyperparathyroidism, hair conditions (excessive hair growth or hair loss), collagenosis, wounds, bone conditions, spinal fixation, ischemic heart disease, peripheral neuropathy, and spinal cord injury. [

[19] ] The method according to any one of [

[15] ] to [

[17] ] above, wherein the condition includes a collagenosis selected from the group consisting of osteogenesis imperfecta, Stickler syndrome, Ehlers-Danlos syndrome, Alport syndrome, cafe-au-lait spots, and focal collagen or cartilage damage. [

[20] ] Use of the composition according to any one of [[1]] to [

[14] ] above in the manufacture of a medicament for treating a condition.

Claims

1. A collagen-binding therapeutic agent comprising two collagen-binding domains linked by a domain linker and an FGF polypeptide linked by a therapeutic agent linker to at least one of the collagen-binding domains, wherein the collagen-binding therapeutic agent lacks collagenase activity, one of the two collagen-binding domains is selected from the group consisting of the polypeptides of SEQ ID NO: 1-14, and the other collagen-binding domain is selected from the group consisting of the polypeptides of SEQ ID NO: 15-30.

2. The collagen-binding therapeutic agent according to claim 1, wherein the two collagen-binding domains together comprise a polypeptide selected from the group consisting of any one of the polypeptides of SEQ ID NO: 40-47.

3. The collagen-binding therapeutic agent according to claim 1 or 2, wherein the domain linker comprises a polypeptide.

4. The collagen-binding therapeutic agent according to any one of claims 1 to 3, wherein the domain linker comprises a polypeptide selected from the group consisting of any one of the polypeptides of SEQ ID NO: 48-55.

5. The collagen-binding therapeutic agent according to any one of claims 1 to 4, wherein the therapeutic agent linker comprises a polypeptide.

6. The collagen-binding therapeutic agent according to any one of claims 1 to 5, wherein the C-terminus of the FGF polypeptide is linked to the N-terminus of one of the collagen-binding domains by the therapeutic agent linker.

7. A pharmaceutical composition comprising any one of the collagen-binding therapeutic agents according to claims 1 to 6 and a pharmaceutical carrier.

8. Use of any one of the collagen-binding therapeutic agents according to claims 1 to 6 or the pharmaceutical composition according to claim 7 in the manufacture of a medicament for treating a condition.

9. The use according to claim 8, wherein the condition is selected from the group consisting of hyperparathyroidism, hair conditions (excessive hair growth or hair loss), collagenosis, wounds, spinal fixation, ischemic heart disease, obliterative neuropathy, and spinal cord injury.

10. The use according to any one of claims 8 to 9, wherein the condition comprises a collagenosis selected from the group consisting of osteogenesis imperfecta, Stickler syndrome, Ehlers-Danlos syndrome, Alport syndrome, café au lait spots, and focal collagen or cartilage damage.

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