RU2790992C2 - Gremlin-1 inhibitor for the treatment of bone fracture or bone defect - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the treatment of a bone fracture or bone defect. The use of a gremlin-1 activity inhibitor for the production of a drug for the treatment of a bone fracture or bone defect and a method for the treatment of a bone fracture or bone defect, including the introduction of a therapeutically effective amount of a gremlin-1 activity inhibitor, are proposed. The inhibitor is an anti-gremlin 1 antibody or its functionally active fragment. The antibody contains the sequence SEQ ID NO: 3 or 4 for HCDR1, the sequence SEQ ID NO: 5 for HCDR2, the sequence SEQ ID NO: 6 for HCDR3, the sequence SEQ ID NO: 7 for LCDR1, the sequence SEQ ID NO: 8 for LCDR2 and the sequence SEQ ID NO: 9 for LCDR3.

EFFECT: inventions accelerate the healing and fusion of bone tissue with segmental gap defects, and can provide improved therapy for the treatment or prevention of non-fusion of a fracture.

16 cl, 8 dwg, 6 tbl, 8 ex

Description

The present invention relates to methods for treating a bone fracture or bone defect. The invention discloses the effective use of anti-gremlin-1-antibody to accelerate the healing and connection of bone tissue in segmental rupture defects; and demonstrates that inhibitors of gremlin-1 activity may provide improved therapy for the treatment or prevention of fracture nonunion.

State of the art

A bone fracture is a tear or crack in bone tissue and can result from traumatic injury such as a fall or blow, but can also result from diseases that adversely affect the integrity of the bone.

Unstabilized bone fractures heal by endochondral ossification, which is initiated by the formation of a blood clot or hematoma. This is due to the inflammatory response that modulates immune cells and surrounding skeletal stem cell populations. Subsequently, the hematoma is replaced by a mineralized cartilaginous callus through the action of various growth factors, including transforming growth factor beta (TGFβ) (Cho et al.; 2002), fibroblast growth factors (FGFs) (Schmid et al.; 2009), and bone morphogenic proteins (BMPs) (Yu et al.; 2010). Under the action of osteoclasts and osteoblasts, the mineralized callus is replaced by bone tissue. The final stage of remodeling involves the replacement of bone tissue with lamellar bone. This process can take many years to complete depending on the patient's age and disease state.

In clinical practice, bone fractures are usually treated with fixation, using a support such as a splint, cast, or brace. In extreme cases involving complex fractures, surgery and the use of internal and external fixators, which are attached directly to the bone, may be required. Even with these measures, in about 10% of patients the tissue repair process is insufficient (Einhorn et al.; 2014), resulting in delayed bone union (failure to achieve union 6 months after fracture) or no union. Nonunion is defined as incomplete healing within 9 months, combined with no radiographic evidence of fracture healing observed for 3 consecutive months (Buza et al.; 2016). Current surgical treatments for nonunion fractures and critical bone defects are often limited in terms of the quantity and quality of materials available. Commonly used therapies include autologous or allogeneic graft, but they carry the additional risk of donor site damage (Goulet et al.; 1997) and infection (Bostrom et al.; 2005), respectively.

A bone defect is the loss of bone tissue due to injury or disease.

There is currently a large unmet medical need for improved treatment of bone fractures and bone defects. Therefore, it is an object of the present invention to provide new methods for the treatment of a bone fracture or bone defect.

The present invention provides inhibitors of gremlin-1 activity for use in the treatment of a bone fracture or bone defect. The invention discloses the effective use of an anti-gremlin-1 antibody to promote healing and bone reconnection in segmental gap defects; and demonstrates that inhibitors of gremlin-1 activity may provide improved therapy for the treatment or prevention of fracture nonunion.

Description of the invention

Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as generally understood by a person skilled in the art. All publications mentioned here are incorporated by reference.

It is understood that any of the embodiments described herein may be combined.

The present invention provides an inhibitor of gremlin-1 activity for use in the treatment of a bone fracture or bone defect. The invention also provides the use of an inhibitor of gremlin-1 activity for the manufacture of a medicament for the treatment of a bone fracture or bone defect. The invention also relates to a method for treating a bone fracture or bone defect comprising administering a therapeutically effective amount of an inhibitor of gremlin-1 activity.

Gremlin-1 (also known as Drm and CKTSF1B1) is a 184 amino acid glycoprotein that is a member of the DAN protein family with a secreted cysteine knot (along with Cerberus and Dan, among others). Gremlin binds to and inhibits the signaling ability of BMP-2, 4 and 7 along with established pro-angiogenic function, possibly through VEGFR2 agonism. The main role of gremlin-1 is manifested during the developmental period, where it is vital for the formation of the kidneys and the formation of the limb rudiment.

It is known that a signaling pathway involving the bone morphogenetic protein (BMP) controls the formation of endochondral bone, where gremlin-1 (GREM1) is one of the natural antagonists of this pathway due to its binding to BMP2, BMP4 and BMP7 (Hsu et al.; 1998) . Conditional deletion of GREM1 in osteoblasts leads to sensitization of BMP signaling/activity and increased bone formation in vivo (Gazzerro et al.; 2007), while conditional overexpression in the same cell type causes osteopenia and spontaneous fractures (Gazzerro et al.; 2005 ). In addition, although total knockout is embryonic lethal in BL6, 49% of fetuses survived beyond 24 h after birth in a mixed C57BL/6/FVB genetic background, and while skeletal developmental defects were present in high numbers, increased rates of bone formation could observed (Canalis et al.; 2012). Despite this developmental function of GREM1, there is no evidence to suggest that inhibition of this protein alone will promote bone fracture repair in postnatal life. Indeed, although endochondral bone formation is the main mechanism of skeletogenesis in the embryonic stages, the mechanisms that regulate cell recruitment are different processes compared to fracture repair in the postnatal period (Ferguson et al.; 1999). The role of inflammation has been cited as a key factor in bone repair in adults, so the developmental factors that control skeletal processes cannot simply be extrapolated to postnatal repair mechanisms.

As used here, the term gremlin-1 usually has the sequence with the identification number UniProt O60565 (SEQ ID NO: 1). The term gremlin-1 may also refer to a gremlin-1 polypeptide that:

(a) contains or consists of the amino acid sequence of SEQ ID NO: 1 with or without an N-terminal signal peptide, i. e. may contain or consist of the mature peptide sequence shown in SEQ ID NO: 21; or

(b) is a derivative having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence of SEQ ID NO: 1 with or without an N-terminal signal peptide (as shown in SEQ ID NO: 21) that retains gremlin- 1, such as the amino acid sequence of SEQ ID NO: 20;

(c) represents variants thereof, where such variants typically share at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, or 95% identity with SEQ ID NO: 1 (or SEQ ID NO: 20 or 21) (or even about 96%, 97%, 98% or 99% identity). In other words, such variants may retain about 60% to about 99% identity with SEQ ID NO: 1, preferably about 80% to about 99% identity with SEQ ID NO: 1, more preferably about 90% to about 99% identity with SEQ ID NO: 1 and most preferably about 95% to about 99% identity with SEQ ID NO: 1. Options are further described below.

As discussed further below, the residue numbers are usually indicated based on the numbering in the sequence of SEQ ID NO: 1. However, one skilled in the art can easily extrapolate the residue numbering to the sequence of the derivative or variant discussed above. Where residue numbers are indicated, the invention also covers those residues in the sequence of the variant or derivative.

The present inventors crystallized human gremlin-1 alone and in complex with an antibody named Ab 7326 (Fab fragments). Crystallization of gremlin-1 allowed the identification of putative residues at the BMP binding site. In addition, crystallization with Ab 7326, which is an allosteric inhibitory antibody, made it possible to identify residues in the epitope of the antibody (WO 2018/115017 A2). Antibodies that bind to this epitope have the potential to be used as therapeutic agents in the treatment of bone fracture or bone defect.

Gremlin-1 Activity Inhibitors

The gremlin-1 activity inhibitor of the present invention is an agent that reduces or blocks the activity of gremlin-1. The inhibitors of the present invention can partially or completely inhibit the activity of gremlin-1. Inhibitors for use in the present invention include, without limitation, inhibitors that are capable of binding to gremlin-1 or a nucleic acid molecule encoding gremlin-1, or capable of inhibiting the expression of gremlin-1. Such inhibitors can be, without limitation, proteins, polypeptides, peptides, peptidomimetics, nucleic acids (eg, DNA, RNA, antisense RNA and siRNA), carbohydrates, lipids, and small molecules.

In one embodiment, the inhibitor of gremlin-1 activity is an anti-gremlin-1 antibody, or a functional fragment, variant, or derivative thereof.

The term "antibody" in the context of the present description includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding site") or their individual chains. An antibody or immunoglobulin generally refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding site thereof. Each heavy chain consists of a heavy chain variable region (abbreviated here as HCVR or V _H ) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated here as LCVR or V _L ) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The V _H and V _L regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs).

Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

An antibody for use in the present invention may be a monoclonal antibody or a polyclonal antibody, and is usually a monoclonal antibody. An antibody for use in the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanized antibody, or an antigen-binding fragment of any of these.

Polyclonal antibodies can be obtained by conventional methods such as immunization of a suitable animal with the antigen of interest. Then you can take the blood from the animal and isolate the immunoglobulin fraction.

Anti-gremlin-1 antibodies can be prepared where immunization of an animal is needed by administering the polypeptides to an animal, e.g., a non-human animal, using well known and routine protocols, see e.g. Handbook of Experimental Immunology, D.M. Weir (ed.), Vol. 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals can be immunized, such as rabbits, mice, rats, sheep, goats, cows, camels, llamas, or pigs. However, rabbits, mice and rats are usually the most suitable.

Monoclonal antibodies can be prepared by any method known in the art, such as the hybridoma method (Kohler & Milstein, 1975, Nature, 256: 495-497), the trioma method, the human B cell hybridoma method (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R Liss, Inc., 1985).

Antibodies for use in the invention can also be prepared using single lymphocyte antibody production methods by cloning and expression of immunoglobulin variable region cDNA derived from single lymphocytes selected for specific antibody production, e.g., the methods described by Babcook, J. et al., 1996 Proc. Natl. Acad. sci. USA 93 (15): 7843-7848l; W092/02551; WO 2004/051268 and WO 2004/106377.

Antibodies for use in the present invention can also be obtained using various phage display methods known in the art, and these include those disclosed by Brinkman et al. (J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184: 177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24: 952-958), Persic et al. (Gene, 1997, 187, 9-18), Burton et al. (Advances in Immunology, 1994, 57: 191-280) and WO 90/02809; WO 91/10737; W092/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Pat. No. 5,698,426; US patent No. 5223409; US patent No. 5403484; US Pat. No. 5,580,717; US patent No. 5427908; US Pat. No. 5,750,753; US patent No. 5821047; US patent No. 5571698; US patent No. 5427908; US patent No. 5516637; US Pat. No. 5,780,225; US patent No. 5658727; U.S. Patent No. 5,733,743 and U.S. Patent 5,969,108.

Fully human antibodies are antibodies in which the variable regions and constant regions (where present) of both the heavy and light chains are human or substantially identical to human sequences, but not necessarily from the same antibody. Examples of fully human antibodies may include antibodies produced, for example, by the phage display methods described above, and antibodies produced in mice, wherein the mouse immunoglobulin variable region genes and optionally the mouse immunoglobulin constant region genes are replaced with their human counterparts, such as, in general, described in EP 0546073, US Pat.

Alternatively, an antibody of the invention may be prepared by a method comprising immunizing a non-human mammal with a gremlin-1 immunogen; obtaining an antibody preparation from said mammal; obtaining from them monoclonal antibodies that recognize gremlin-1.

Antibody molecules for use in the present invention may include a complete antibody molecule having full length heavy and light chains, or a fragment or antigen-binding site thereof. The term "antigen binding site" of an antibody refers to one or more antibody fragments that retain the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Antibodies and their fragments and their antigen-binding sites can be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (for example, VH or VL or VHH), scFv, bi-, tri-, or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23 (9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for creating and obtaining these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International Patent Applications WO 2005/003169, WO 2005/003170 and WO 2005/003171, and the Fab-dAb fragments described in International Patent Application WO 2009 /040562. Polyvalent antibodies may have many specificities or may be monospecific (see, for example, WO 92/22853 and WO 2005/113605). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for suitability in the same manner as intact antibodies.

In one example, a functionally active antibody fragment for use in the present invention is a Fab, Fab', F(ab') ₂ , Fv, or scFv.

The domains of the constant region of the antibody molecule for use in the present invention, if present, may be selected based on effector functions that may be desired. For example, constant region domains may represent human IgA, IgD, IgE, IgG, or IgM domains. In particular, human IgG constant region domains, especially of the IgG1 and IgG3 isotypes, can be used when antibody effector functions are desired. Alternatively, IgG2 and IgG4 isotypes may be used when antibody effector functions are not required. In one example, the isotype is IgG4P as described in Angal S. et al., Mol Immunol, Vol. 30(1), p105-108, 1993.

An antibody for use in the invention may be made, expressed, generated, or isolated by recombinant methods, for example (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for the immunoglobulin genes of interest, or a hybridoma derived therefrom. , (b) antibodies isolated from a host cell transformed to express an antibody of interest, e.g., from a transfectome, (c) antibodies isolated from a combinatorial recombinant antibody library, and (d) antibodies generated, expressed, generated, or isolated by any in another manner which involves splicing immunoglobulin gene sequences with other DNA sequences.

An antibody for use in the invention may be a human antibody or a humanized antibody. The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, then the constant region is also derived from human germline immunoglobulin sequences. Human antibodies for use in the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis, or somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a human monoclonal antibody can be produced by a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse, having a genome containing a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

Human antibodies can be obtained by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.

The term "derivative" refers to any modified form of an antibody, such as a conjugate of an antibody and another agent or effector molecule.

An effector molecule may include one effector molecule, or two or more such molecules linked so as to form a single molecule that can be attached to antibodies for use in the present invention. When it is desired to obtain an antibody fragment associated with an effector molecule, it can be obtained by standard chemical methods or recombinant DNA methods, in which the antibody fragment is linked either directly or through a binding agent to the effector molecule. Methods for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, ^2nd Ed., Robinson et al., Eds., 1987, pp. 623-53; Thorpe et al. . 1982, Immunol Rev., 62: 119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO 2003/031581. Alternatively, when the effector molecule is a protein or polypeptide, then coupling can be achieved using recombinant DNA procedures, for example as described in WO 86/01533 and EP 0392745.

The effector molecule may increase the in vivo half-life of the antibody and/or decrease the immunogenicity of the antibody and/or enhance delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins or albumin-binding compounds such as those described in WO 2005/117984.

The term "humanized antibody" refers to CDR-grafted antibody molecules in which CDR sequences derived from the germline of other mammalian species, such as mice, have been grafted onto human framework sequences. Additional framework modifications can be made in human framework sequences.

As used herein, the term "CDR-grafted antibody molecule" refers to an antibody molecule in which the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g., murine or rat monoclonal antibody) were grafted onto the heavy and/or light chain variable region scaffold of an acceptor antibody (eg, a human antibody). For a review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more of the specificity-determining residues of any of the CDRs described herein above is transferred to a human antibody framework. (See, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only specificity-determining residues from one or more of the CDRs described above are transferred to a human antibody framework. In yet another embodiment, only the specificity-defining residues from each of the CDRs described above are transferred to a human antibody framework.

When CDRs or specificity-defining residues are grafted, any suitable variable region acceptor framework sequence may be used, taking into account the class/type of donor antibody from which the CDRs are derived, including murine, primate, and human frameworks. Therefore, a CDR-grafted antibody for use in the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs or specificity-determining residues described above. Thus, in one embodiment, a neutralizing CDR-grafted antibody is provided wherein the variable domain includes human acceptor framework regions and non-human donor CDRs.

Examples of human scaffolds that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences can be used; they are available, for example, at: http://www.vbase2.org/ (see Retter et al., Nucl. Acids Res. (2005) 33 (supplement 1), D671-D674).

In a CDR-grafted antibody for use in the present invention, the acceptor heavy and light chains need not be derived from the same antibody and may, if desired, include multiple chains having framework regions derived from different chains.

Also, in a CDR-grafted antibody for use in the present invention, the framework regions do not have to have exactly the same sequence as those of the acceptor antibody. For example, uncommon residues can be replaced with more common residues for a given class or type of acceptor chain. Alternatively, selected residues in the framework regions of the acceptor can be changed to match the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in acceptor frameworks that may need to be modified is described in WO 91/09967.

The person skilled in the art should also be clear that antibodies can be subject to various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) by the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705: 129-134, 1995).

In one embodiment, the heavy chain of the antibody contains a CH1 domain, and the light chain of the antibody contains a CL domain, either kappa or lambda.

Biological molecules, such as antibodies or fragments, contain acidic and/or basic functional groups that give the molecule a net positive or negative charge. The amount of total "observable" charge will depend on the absolute amino acid sequence of the molecule, the local environment of the charged groups in the three-dimensional structure, and the environmental conditions of the molecule. The isoelectric point (pI) represents the pH at which a particular molecule or surface carries no net electrical charge. In one embodiment, an antibody or fragment of the present invention has an isoelectric point (pI) of at least 7. In one embodiment, an antibody or fragment has an isoelectric point of at least 8, such as 8.5, 8.6, 8.7, 8.8, or 9. In one embodiment, the pI of the antibody is 8. Programs such as ** ExPASY http://www.expasy.ch/tools/pi_tool.html (see Walker, The Proteomics Protocols Handbook, Humana Press (2005), 571-607) can be used to predict the isoelectric point of an antibody or fragment.

Antibodies for use in the invention may include at least one, at least two, or all three heavy chain CDR sequences of SEQ ID NO: 4-6 (HCDR1/HCDR2/HCDR3, respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the Ab7326 antibody from the examples, as determined using the Kabat methodology.

The Kabat and Chothia numbering systems for determining CDR sequences are well known in the art (as are other systems). CDR sequences can be determined using any suitable method, and in the present invention, while the Kabat numbering system is commonly used, other methods can also be used. In this case, SEQ ID NO: 3 represents the sequence of HCDR1 Ab7326, determined using the combined definition according to the Chothia & Kabat system.

Antibodies for use in the invention may comprise at least one, at least two, or all three light chain CDR sequences of SEQ ID NO: 7-9 (LCDR1/LCDR2/LCDR3, respectively). These are LCDR1/LCDR2/LCDR3 Ab7326 sequences using Kabat methodology.

In one embodiment, the antibody contains at least the HCDR3 sequence of SEQ ID NO: 6.

Typically, an antibody contains at least one heavy chain CDR sequence selected from SEQ ID NOS: 4-6 and at least one light chain CDR sequence selected from SEQ ID NOS 7-9. The antibody may contain at least two heavy chain CDR sequences selected from SEQ ID NOs: 4-6 and at least two light chain CDR sequences selected from SEQ ID NOs: 7-9. The antibody typically comprises all three heavy chain CDR sequences of SEQ ID NO: 4-6 (HCDR1/HCDR2/HCDR3, respectively) and all three light chain CDR sequences of SEQ ID NO: 7-9 (LCDR1/LCDR2/LCDR3, respectively). The antibodies may be chimeric, human or humanized antibodies.

The antibody may contain the heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12 (HCVR variants 1 and 2 of Ab7326). The antibody may contain a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13 (LCVR options 1 and 2 of Ab7326). The antibody preferably contains the heavy chain variable region sequence of SEQ ID NO: 10 or 12 and the light chain variable region sequence of SEQ ID NO: 11 or 13 (particularly the HCVR/LVCR pairs of SEQ ID NO: 10/11 or 12/13).

Ab7326 variants 1 and 2 differ by one amino acid in the heavy chain variable region and one amino acid in the light chain variable region as follows:

- heavy chain variable region variant 1 contains glutamic acid (E) at position 6 (SEQ ID NO: 10)

- heavy chain variable region variant 2 contains glutamine (Q) at position 6 (SEQ ID NO: 12)

- light chain variable region variant 1 contains serine (S) at position 7 (SEQ ID NO: 11)

- light chain variable region variant 2 contains threonine (T) at position 7 (SEQ ID NO: 13).

Thus, in one embodiment, the antibody comprises the heavy chain variable region (HCVR) sequence of SEQ ID NO: 10, wherein the glutamic acid residue at position 6 is replaced by a glutamine residue (E6Q); where the numbering of the residues corresponds to the numbering in SEQ ID NO: 10.

In one embodiment, the antibody comprises the heavy chain variable region (HCVR) sequence of SEQ ID NO: 12, wherein the glutamine residue at position 6 is replaced by a glutamic acid residue (Q6E); where the numbering of the residues corresponds to the numbering in SEQ ID NO: 12.

In one embodiment, the antibody comprises a light chain variable region (LCVR) sequence of SEQ ID NO: 11, wherein the serine residue at position 7 is replaced by a threonine residue (S7T); where the numbering of the residues corresponds to the numbering in SEQ ID NO: 11.

In one embodiment, the antibody comprises a light chain variable region (LCVR) sequence of SEQ ID NO: 13, wherein the threonine residue at position 7 is replaced by a serine residue (T7S); where the numbering of the residues corresponds to the numbering in SEQ ID NO: 13.

In one embodiment, the antibody comprises SEQ ID NO: 3 or 4 for HCDR1, SEQ ID NO: 5 for HCDR2, SEQ ID NO: 6 for HCDR3, SEQ ID NO: 7 for LCDR1, SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and wherein the heavy chain variable region comprises a sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity) with the sequence of SEQ ID NO: 10, and the light chain variable region contains a sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) with the sequence of SEQ ID NO: 11.

In one embodiment, the antibody comprises SEQ ID NO: 3 or 4 for HCDR1, SEQ ID NO: 5 for HCDR2, SEQ ID NO: 6 for HCDR3, SEQ ID NO: 7 for LCDR1, SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and where the heavy chain variable region contains a sequence having at least 95% identity (for example, at least 95%, 96%, 97%, 98% or 99% identity), with the sequence of SEQ ID NO: 12, and the variable region of the chain contains a sequence having at least 95% identity (for example, at least 95%, 96%, 97%, 98% or 99% identity) with the sequence of SEQ ID NO: 13.

An antibody may contain a heavy chain (H-chain) sequence:

SEQ ID NO: 14 mouse full-length IgG1 heavy chain version 1, or

SEQ ID NO: 28 mouse full-length IgG1 heavy chain version 2, or

SEQ ID NO: 30 human full-length IgG1 heavy chain version 1, or

SEQ ID NO: 16 human full-length IgG1 heavy chain version 2, or

SEQ ID NO: 22 human full-length IgG4P heavy chain version 1 or

SEQ ID NO: 34 human full-length IgG4P heavy chain version 2, or

SEQ ID NO: 18 Fab Heavy Chain Variant 1, or

SEQ ID NO: 32 heavy chain variants 2 Fab.

An antibody may contain a light chain (L-chain) sequence:

SEQ ID NO: 15 mouse full-length IgG1 light chain version 1, or

SEQ ID NO: 29 mouse full-length IgG1 light chain version 2, or

SEQ ID NO: 31 human full-length IgG1 light chain version 1, or

SEQ ID NO: 17 human full-length IgG1 light chain version 2, or

SEQ ID NO: 23 light chain variants 1 of human full-length IgG4P or

SEQ ID NO: 35 human full-length IgG4P light chain version 2, or

SEQ ID NO: 19 Light Chain Variant 1 Fab, or

SEQ ID NO: 33 variants 2 light chain Fab.

In one example, an antibody contains a pair of heavy chain/light chain sequences:

SEQ ID NO: 14/15 variant 1 mouse full-length IgG1, or

SEQ ID NO: 28/29 variant 2 mouse full-length IgG1, or

SEQ ID NO: 30/31 human full-length IgG1 variant 1 or

SEQ ID NO: 16/17 version 2 human full-length IgG1, or

SEQ ID NO: 22/23 human full-length IgG1P variant 1, or

SEQ ID NO: 34/35 human full-length IgG1P variant 2, or

SEQ ID NO: 18/19 light chain variant 1 Fab or

SEQ ID NO: 32/33 light chain variants 2 Fab.

Variant forms of the respective sequences may be interchanged. For example, an antibody may contain a pair of heavy chain/light chain sequences:

SEQ ID NO: 14/29 heavy chain version 1/light chain version 2 mouse full-length IgG1, or

SEQ ID NO: 28/15 heavy chain version 2/light chain version 2 of mouse full-length IgG1, or

SEQ ID NO: 30/17 heavy chain version 1/light chain version 2 of human full-length IgG1, or

SEQ ID NO: 16/31 heavy chain version 2/light chain version 1 of human full-length IgG1, or

SEQ ID NO: 22/35 heavy chain version 1/light chain version 2 of human full-length IgG4P, or

SEQ ID NO: 34/23 heavy chain version 2/light chain version 1 of human full-length IgG4P, or

SEQ ID NO: 18/33 heavy chain version 1/Fab light chain version 2, or

SEQ ID NO: 32/19 heavy chain version 2/light chain version 1 Fab.

The antibodies may be chimeric, human or humanized antibodies.

Alternatively, the antibody may be, or may include, a variant of one of the specific sequences identified above. For example, a variant may represent a substitution, deletion, or addition of any of the above amino acid sequences.

The variant antibody may contain 1, 2, 3, 4, 5, up to 10, up to 20 or more (typically up to a maximum of 50) amino acid substitutions and/or deletions from the specific sequences indicated above. "Deletion" variants may include the deletion of individual amino acids, the deletion of small groups of amino acids, such as 2, 3, 4 or 5 amino acids, or the deletion of larger amino acid regions, such as the deletion of certain amino acid domains, or other properties. Options for "substitution" typically include replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. . Some properties of the 20 basic amino acids that can be used to select suitable substituents are as follows:

Table 1
Properties of amino acids Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asn polar, hydrophilic, neutral asp polar, hydrophilic, charged (-) Pro hydrophobic, neutral Glu polar, hydrophilic, charged (-) Gln polar, hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+) gly aliphatic, neutral Ser polar, hydrophilic, neutral His aromatic, polar, hydrophilic, charged (+) Thr polar, hydrophilic, neutral ile aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral Lys polar, hydrophilic, charged (+) trp aromatic, hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

"Derivatives" or "variants" generally include those in which, instead of a naturally occurring amino acid, there is an amino acid that represents its structural counterpart. The amino acids used in the sequences can also be derivatized or modified, such as labeled, thereby providing antibody function that has not been substantially altered.

The derivatives and variants described above can be made during antibody synthesis or by modification after it has been made, or when the antibody is in recombinant form using known site-directed mutagenesis, random mutagenesis or enzymatic digestion and/or nucleic acid ligation techniques.

Variant antibodies may have an amino acid sequence that has greater than about 60% or greater than about 70%, such as 75% or 80%, typically greater than about 85%, such as greater than about 90% or 95% amino acid identity with amino acids. the sequences disclosed here (in particular, the HCVR/LCVR sequences and the sequences of the H and L chains). In addition, the antibody may be a variant that has greater than about 60% or greater than about 70%, such as 75% or 80%, typically greater than about 85%, such as greater than about 90% or 95% amino acid identity with the HCVR/LCVR sequences and the H and L chain sequences disclosed herein while maintaining the exact CDRs disclosed for these sequences. Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with HCVR/LCVR sequences and with H- and L-chains disclosed here (in some circumstances while maintaining accurate CDRs).

Variants typically retain about 60%-about 99% identity, about 80%-about 99% identity, about 90%-about 99% identity, or about 95%-about 99% identity. This level of amino acid identity can be observed over the entire length of the corresponding sequence of SEQ ID NO, or over a portion of the sequence, such as about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.

With respect to amino acid sequences, "sequence identity" refers to sequences that have a specified value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

pairwise alignment parameters - method: exact, matrix: PAM, gap opening penalty: 10.00, gap continuation penalty: 0.10;

multiple alignment options - matrix: PAM, gap open penalty: 10.00, % identity latency: 30, end gap penalty: on, gap split distance: 0, negative matrix: none, gap extension penalty: 0.20 , gap penalty for residues: included, hydrophilic gap penalties: included, hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues that have simply been derivatized.

Antibodies having specific sequences and derivatives and variants that retain the function or activity of these chains are therefore intended for use in the present invention.

"Derivatives" as used herein is intended to include reactive derivatives, for example, thiol-selective reactive groups such as maleimides and the like. The reactive group may be linked directly or via a linker segment to the polymer. It should be understood that the remainder of such a group will in some cases form part of the product in the form of a linking group between the antibody fragment and the polymer.

The polymer may be a synthetic or naturally occurring polymer, for example, an optionally substituted straight or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or a straight or branched polysaccharide, for example a homo- or heteropolysaccharide.

Specific optional substituents that may be present on the synthetic polymer include one or more hydroxy, methyl, or methoxy groups. Specific examples of synthetic polymers include optionally substituted straight or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, especially optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

The size of the polymer may vary if desired, but is typically in the average molecular weight range of 500 to 50,000 Da, such as 5,000 to 40,000 Da, such as 20,000 to 40,000 Da. The size of the polymer, in particular, can be selected based on the intended use of the product, for example, the ability to localize in certain tissues or to increase the half-life in the bloodstream (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, when a product is intended to exit the bloodstream and enter tissues, it may be advantageous to use a low molecular weight polymer, eg, a molecular weight of about 5000 Da. For applications where the product remains in the bloodstream, it may be advantageous to use a higher molecular weight polymer, for example having a molecular weight in the range of 20,000 Da to 40,000 Da.

Suitable polymers include a polyalkylene polymer such as poly(ethylene glycol) or especially methoxypoly(ethylene glycol) or a derivative thereof, and especially with a molecular weight in the range of about 15,000 Da to about 40,000 Da.

In one example, antibodies for use in the present invention are attached to poly(ethylene glycol) (PEG) groups. In one specific example, an antibody is an antibody fragment and the PEG molecules can be attached through any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, e.g., any free amino acid, imino group, thiol, hydroxyl, or carboxyl group. Such amino acids may occur naturally in the antibody fragment, or may be inserted into the fragment using recombinant DNA techniques (see, for example, US Patent 5,219,996; US Patent 5,667,425; WO98/25971, WO2008/038024). In one example, an antibody molecule is a modified Fab fragment, where the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Accordingly, additional amino acids form a modified hinge region containing one or more cysteine residues to which an effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

Antibodies can compete for binding to gremlin-1 or bind to the same epitope as defined above with respect to the H chain/L chain, HCVR/LCVR or CDR sequences. In particular, an antibody can compete for binding to gremlin-1 or bind to the same epitope as an antibody that contains the combination of the sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 of SEQ ID NO: 4/5/6/7 /8/9. An antibody can compete for binding to gremlin-1 or bind to the same epitope as an antibody that contains a pair of HCVR and LCVR sequences SEQ ID NO: 10/11 or 12/13 or full-length chains of SEQ ID NO: 14/15 or 16 /17.

An "epitope" is a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes typically represent a subset of structural epitopes and have residues that directly confer interaction affinity. Epitopes can also be conformational, i.e. consist of non-linear amino acids. In some embodiments, epitopes may include determinants that represent reactive surface groups of molecules, such as amino acids, side sugar chains, phosphoryl groups, or sulfonyl groups, and, in some embodiments, may have specific three-dimensional structural and/or specific charge characteristics. .

Whether an antibody binds to the same epitope that binds or competes with a reference antibody can be readily determined using conventional methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference antibody for use in the invention, the reference antibody is allowed to bind to the protein or peptide under saturation conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody binds to a protein or peptide after saturable binding to the reference antibody, then it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody does not bind to the protein or peptide after saturable binding to the reference antibody, then the test antibody may bind to the same epitope as the epitope bound to the reference antibody of the invention.

In order to determine if an antibody competes for binding with a reference antibody, the binding procedure described above is performed in two orientations. In the first orientation, the reference antibody is allowed to bind to the protein/peptide under saturation conditions, followed by evaluation of the binding of the test antibody to the protein/peptide molecule. In the second orientation, the test antibody is allowed to bind to the protein/peptide under saturation conditions, followed by evaluation of the binding of the reference antibody to the protein/peptide. If in both orientations only the first (saturating) antibody binds to the protein/peptide, then it is concluded that the test antibody and the reference antibody compete for binding to the protein/peptide. As will be appreciated by one of skill in the art, an antibody that competes for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or contiguous epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of another antibody by at least 50%, 75%, 90%, or even 99%, as measured in a competitive binding assay ( see, for example, Junghans et al., Cancer Res, 1990: 50: 1495-1502). Alternatively, two antibodies share the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

Additional routine experimentation (e.g., peptide mutations and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for no observable binding. Experiments of this kind can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

из комплекса гремлин-1-Ab7326 Fab.It was found that the anti-gremlin-1 antibody of the examples, i. antibody Ab7326 binds to the following gremlin-1 residues: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175; where Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, which are present on one gremlin-1 monomer, and Ile131, which is present on the second gremlin-1 monomer. The numbering is based on the sequence identification number SEQ ID NO: 1 UniProt O60565. As discussed in the Examples section, these epitope residues were identified using NCONT analysis at 4

from the gremlin-1-Ab7326 Fab complex.

Therefore, antibodies for use in the invention can bind to an epitope that contains at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175 (with residue numbering based on SEQ numbering). ID NO: 1). Antibodies for use in the invention may bind to an epitope that contains 2, 3, 4, 5, 6, 7, 8 or all 9 of these residues (preferably at least 5 residues).

Antibodies for use in the invention may also recognize an epitope where Ile131 is present on a different gremlin-1 monomer with different residues.

While these residues are for a specific human gremlin-1 sequence, one skilled in the art can extrapolate the positions of these residues to other relevant gremlin sequences using conventional techniques. Therefore, antibodies that bind to epitopes containing corresponding residues in these other gremlin sequences are also contemplated for use in the present invention.

To screen for antibodies that bind to a particular epitope, a conventional cross-blocking assay can be performed, such as described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY). Other methods include alanine mutant scanning, peptide blotting (Reineke (2004) Methods Mol Biol 248:443-63), or peptide digestion analysis. In addition, techniques such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science: 9: 487-496). Such methods are well known in the art.

от остатка паратопа антитела.Antibody epitopes can also be determined using x-ray crystallography. Therefore, antibodies for use in the present invention can be evaluated by X-ray crystallography of an antibody associated with gremlin-1. Epitopes, in particular, can be identified by determining residues on gremlin-1 within 4

from the rest of the paratope of the antibody.

Antibodies can be tested for binding to gremlin-1, for example, by standard ELISA or Western blotting. The ELISA assay can also be used to screen for hybridomas that show positive reactivity with the target protein. Binding selectivity of an antibody can also be determined by monitoring antibody binding to cells expressing the target protein, such as by flow cytometry. Thus, the screening method may include the step of identifying an antibody capable of binding to gremlin-1 by performing an ELISA or Western blotting or flow cytometry.

Antibodies can selectively (or specifically) recognize gremlin-1. An antibody or other compound "selectively binds" or "selectively recognizes" a protein when it binds with preferential or high affinity to the protein for which it selectively but does not substantially bind or bind with low affinity to other proteins. The selectivity of an antibody can be further examined by determining whether the antibody binds to other related proteins, as discussed above, or how much it distinguishes them. Antibodies for use in the invention typically recognize human gremlin-1.

The antibodies may also be cross-reactive to related proteins or to human gremlin-1 and other species gremlin-1.

The term "specific (or selective)" antibody should be understood that the antibody binds to the protein of interest without significant cross-reactivity with any other molecule. Cross-reactivity can be assessed by any suitable method described here. The cross-reactivity of an antibody can be considered significant if the antibody binds to another molecule by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to the protein of interest. An antibody that is specific (or selective) can bind less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40% to another molecule 35%, 30%, 25%, or 20% of the strength with which it binds to the protein of interest. An antibody can bind to another molecule with a strength of less than about 20%, less than about 15%, less than about 10%, or less than about 5%, less than about 2%, or less than about 1%, with which it binds to the presenting protein interest.

Thus, antibodies suitable for use in the present invention may have a high binding affinity for (human) gremlin-1. The antibody may have a dissociation constant (K _D ) below <1 nM and preferably <500 pM. In one example, the antibody has a dissociation constant (K _D ) below 200 pM. In one example, the antibody has a dissociation constant (K _D ) below 100 pM. Various methods can be used to determine the binding affinity of an antibody to its target antigen, such as surface plasmon resonance analysis, saturation assay, or immunoassays such as ELISA or RIA, which are well known to those skilled in the art. An exemplary method for determining binding affinity is surface plasmon resonance analysis on a BIAcore™ 2000 instrument (Biacore AB, Freiburg, Germany) using CM5 sensor chips as described by Krinner et al., (2007) Mol. Immunol. Feruary; 44(5): 916-25. (Epub 2006 11 May)).

Anti-gremlin-1 antibody of the examples, i.e. Ab7326 is an allosteric inhibitor of gremlin-1 activity that binds to an epitope remote from the BMP binding site (WO 2018/115017 A2). Ab7326 binds to gremlin-1 with exceptionally high affinity with a Kd value of <100 pM and is expected to be particularly suitable for use in the present invention.

An inhibitor of gremlin-1 activity may interfere with any of the functions of gremlin-1, but generally reduces the binding of gremlin-1 to BMPs (BMP 2, 4 and/or 7). Gremlin-1 is a negative regulator of the BMP and thus reduced binding enhances signaling through the BMP.

BMP binding and signaling can be detected by any method known in the art. The examples of this application describe two functional assays to test how much an agent reduces the binding of gremlin-1 to BMP. Example 3 describes the analysis of the Id1 reporter gene, where the Id1 gene is the target gene of the BMP signaling pathway. The signal enhancement in this assay can be used to determine how much the agent reduces the binding of gremlin-1 to BMP. Example 5 describes the SMAD phosphorylation assay. SMAD 1, 5 and 8 are phosphorylated in the BMP signaling pathway. Therefore, the increase in SMAD phosphorylation can be used to determine how much the agent reduces the binding of gremlin-1 to BMP.

Once a suitable antibody has been identified and selected, the amino acid sequence of the antibody can be identified by methods known in the art. Genes encoding an antibody can be cloned using degenerate primers. The antibody can be obtained recombinantly using conventional methods.

Examples of DNA sequences encoding the full length heavy chains and light chains of Ab7326 are provided in the sequence listing:

SEQ ID NO: 24 (human IgG1 heavy chain version 1)

SEQ ID NO: 25 (human IgG1 light chain DNA variant)

SEQ ID NO: 26 (Human IgG4P DNA Heavy Chain Variant 1)

SEQ ID NO: 27 (human IgG4P light chain version 1).

Pharmaceutical compositions, doses and dosing regimens

The gremlin-1 activity inhibitor for use in the present invention may be in a pharmaceutical composition. The pharmaceutical composition will typically be sterile and will typically include a pharmaceutically acceptable carrier and/or adjuvant. The pharmaceutical composition for use in the invention may further comprise a pharmaceutically acceptable adjuvant and/or carrier.

Pharmaceutical compositions for use in the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent molecule and does not produce any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. The carrier may be suitable for parenteral administration, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, intraspinal, or other parenteral route of administration, such as by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration such as topical, epidermal or mucosal routes of administration. The carrier may be suitable for oral administration. Depending on the route of administration, the inhibitor may be coated with a material that protects it from acids and other natural conditions that can inactivate the inhibitor.

Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous vehicles that can be used in pharmaceutical compositions for use in the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it is desirable to include isotonic agents, eg sugars, polyalcohols such as mannitol, sorbitol or sodium chloride, in the composition.

Pharmaceutical compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for providing a high drug concentration.

Pharmaceutical compositions for use according to the invention may contain additional active ingredients.

Also provided are kits containing an inhibitor of gremlin-1 activity and instructions for use in the treatment method of the invention.

Agents for use in the invention, or formulations or compositions thereof, may be administered for therapeutic and/or prophylactic treatment.

In therapeutic applications, the agents are administered to a subject already suffering from a disorder or condition in an amount sufficient to cure, alleviate, or partially alleviate the condition or one or more of its symptoms. Such therapeutic treatment may lead to a reduction in the severity of symptoms or an increase in the frequency or duration of asymptomatic periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount".

In prophylactic applications, the agents are administered to a subject at risk of developing a disorder or condition in an amount sufficient to prevent or lessen the subsequent adverse effects of the condition or one or more of its symptoms. An amount sufficient to achieve this is defined as a "prophylactically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury, as well as the body weight and general condition of the subject.

The subject to whom the administration is carried out may be a human or non-human animal. The term "non-human animal" includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, dogs, cats, horses, sheep, cows, chickens, amphibians, reptiles, and the like. people.

An agent or pharmaceutical composition for use in the invention may be administered by one or more routes of administration using one or more of a variety of routes known in the art. As will be appreciated by one of skill in the art, the route and/or mode of administration will vary depending on the desired results. Examples of routes of administration of agents or pharmaceutical compositions for use in the invention include parenteral routes such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous or intraspinal routes of administration, for example by injection or infusion. Alternatively, the agent or pharmaceutical composition may be administered by a non-parenteral route, such as a topical, epidermal, or mucosal route. The agent or pharmaceutical composition may be formulated for oral administration.

A suitable dose of inhibitory agent or pharmaceutical composition for use in the invention can be determined by a qualified medical practitioner. Actual dosage levels of active ingredients in pharmaceutical compositions for use in the present invention may vary so as to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and route of administration, without being toxic to the patient. The selected dosage level will depend on many pharmacokinetic factors, including the potency of the particular compositions used, route of administration, time of administration, rate of elimination of the particular compound used, duration of treatment, age, sex, body weight, condition, general health, and prior medical history of the patient being treated. treatment, and similar factors well known in medicine.

A suitable dosage may, for example, be in the range of about 0.01 µg/kg to about 1000 mg/kg body weight, typically about 0.1 µg/kg to about 100 mg/kg body weight of the patient being treated. For example, a suitable dosage may be from about 1 μg/kg to about 10 mg/kg of body weight per day, or from about 10 μg/kg to about 5 mg/kg of body weight per day.

Dosing regimens can be adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single dose may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as dictated by the therapeutic situation. As used here, a single dosage form refers to physically discrete units suitable as unit doses for subjects to be treated; where each unit contains a predetermined amount of the active agent, calculated to obtain the desired therapeutic effect in combination with the required pharmaceutical carrier.

Administration may be in single or multiple doses. Multiple doses can be administered by the same or different routes and at the same or different injection sites. Alternatively, dosages may be provided with a sustained release formulation, in which case less frequent administration is required. The dosage and frequency of administration may vary depending on the half-life of the inhibitory agent in the patient and the desired duration of treatment.

Agents, formulations, or pharmaceutical compositions for use in the invention may be administered in conjunction with one or more other therapeutic agents. The combined administration of two or more agents can be achieved in various ways. Both may be administered together in a single composition, or they may be administered in separate compositions as part of a combination therapy. For example, one may be administered before, after, or at the same time as the other.

Therapeutic indications

The gremlin-1 activity inhibitors of the present invention are provided for the treatment of a bone fracture or bone defect. A bone fracture is a tear or crack in bone tissue and can result from traumatic injury such as a fall or blow, but can also result from diseases that adversely affect the integrity of the bone. A bone defect is the loss of bone tissue due to injury or disease.

A fracture can represent a break in any bone in the body.

A bone defect can be a bone defect in any bone in the body.

In one embodiment, the bone fracture is a delayed or nonunion fracture. A delayed union fracture is defined as a fracture that does not achieve union within 6 months of the fracture. A non-union fracture is defined as incomplete healing within 9 months, combined with no radiographic evidence of fracture healing observed for 3 consecutive months. (Einhorn et al.; 2014; Buza et al.; 2016). Examples of fractures that tend to develop with a delayed or nonunion include those of the tibia, distal radius, femoral neck, and navicular.

In one embodiment, a bone fracture or bone defect occurs as a result of a disease that adversely affects the integrity of the bone. Examples of diseases that adversely affect bone integrity include, but are not limited to, osteoporosis, osteogenesis imperfecta, diabetes, Paget's disease, rheumatoid arthritis, ankylosing spondylitis, multiple myeloma, primary bone cancer (eg, osteosarcoma, Ewing's sarcoma, and chondrosarcoma), cancer with bone metastases (eg, breast cancer, prostate cancer, and lung cancer), diffuse idiopathic skeletal hyperostosis, osteomyelitis, renal failure, Duchenne muscular dystrophy, and thalassemia.

Brief description of the figures

Figure 1 shows the percentage recovery of the signal for antibodies obtained by immunization in the analysis of the reporter gene HEK-ID1.

Figure 2 shows the percentage recovery of the signal for antibodies obtained from the library, in the analysis of the reporter gene HEK-ID1.

Figure 3 shows the results of HEK-ID1 reporter gene analysis with titration of human gremlin (Figure 3A) and mouse gremlin (Figure 3B), and the effect of antibody 7326 (shown as PB376 antibody) in reconstitution of BMP signaling.

Figure 4 shows a structural model of the gremlin-Fab complex highlighting possible BMP and Fab epitope binding regions.

In FIG. 5 shows an examination of the area devoid of callus/bone tissue during fracture repair in the acquired x-rays. The area within the defect that was devoid of tissue was quantified using image analysis and then by comparing the control group and the group treated with anti-gremlin 1 antibody. Results are presented as mean ± SD for 10 rats/group. * P<0.05; ** P<0.01; *** P<0.001 as measured by Mann-Whitney U-test.

In FIG. 6 shows LMB (low mineral bone; newly formed bone) and HMB (high mineral bone; mature bone) examination within a 3 mm femoral defect. Panel A: 3D micro-CT analysis of the defect area of the femur to identify newly formed or mature bone. Measured the percentage of bone volume/tissue volume and compared all subjects (as a whole) in the control group and the group treated with anti-gremlin 1 antibody. A control group was also compared with an experimental group treated with anti-gremlin 1 antibody in animals divided into low response responders (LR incomplete connection) and high response responders (HR complete connection). Results are presented as mean ± standard deviation. * P<0.05; ** P<0.01; *** P<0.001 as measured by Mann-Whitney U-test. Panel B: Representative micro-CT showing 3D images of bone volume in the LR and HR groups in control and after treatment with anti-gremlin 1 antibody.

Figure 7 shows the results of histomorphometric analysis of the defect of the femur. Percentage of bone volume/tissue volume (BV/TV (%)), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were compared between the control group and the anti-gremlin 1 antibody treated group. Results are presented as mean ± SD for 10 rats/group. * P<0.05; ** P<0.01; *** P<0.001 as measured by Mann-Whitney U-test.

In FIG. 8 shows the correlation of 3D micro-CT analysis and 2D histomorphometric analysis of total BV/TV%. Correlations were made in both groups on LMB (mineral-poor bone; newly formed bone) and HMB (mineral-rich bone; mature bone) within 3 mm of the femoral defect and compared with 2D histomorphometry evaluation data (n=20). Evaluation by Pearson's analysis indicates a significant correlation of BV/TV% between 3D micro-CT analysis and 2D histomorphometric analysis.

The following examples illustrate the invention.

Examples

Example 1 Expression, Purification, Refolding and Structuring of a Protein.

Protein expression and production of inclusion bodies

A truncated human gremlin-1 coding sequence (SEQ ID NO: 20) optimized for expression in E. coli was cloned into a modified pET32a vector (Merck Millipore) using BamHI/XhoI to obtain a vector encoding an N-terminal tagged gremlin sequence. 6His-TEV (pET-hGremlin1).

Expressed sequence:

MGSSHHHHHHSSGENLYFQGS AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD; SEQ ID NO: 2 (with non-gremlin residues labeled 6His-TEV in italics). Sequence numbering is based on UniProt O60565 and SEQ ID NO: 1.

The pET-hGremlin1 DNA plasmid was used to transform BL21 (DE3) cells. A single ampicillin resistant colony was harvested from the LB/Amp agar plate and used to inoculate 100 ml of LB/Amp starter culture. After shaking (200 rpm) for 16 hours at 37° C., 25 ml of starter culture was used to inoculate 500 ml of 2xTY/Amp medium. The culture was shaken (250 rpm) at 37° C. until an OD 600 of 3 was reached. Then 20 ml of MOPS + glycerol nutrient mixture (1M MOPS pH 7.4, 40% glycerol, 0.5% MgSO ₄ ) was added to the culture. 0.42% MgCl ₂ ), was induced with 300 μM IPTG and then incubated at 17°C, 180 rpm for 16 h. Cells were harvested by centrifugation (4000 g for 20 min at 4°C).

Cell pellets from centrifugation were resuspended in lysis buffer (PBS pH 7.4, 0.35 mg/ml lysozyme, 10 μg/ml DNase and 3 mM MgCl ₂ ) at 4°C, and the insoluble fraction was collected by centrifugation at 3500 g for 30 min at 4°C. Centrifuged inclusion bodies were washed three times by resuspension in wash buffer (50 mM Tris, 500 mM NaCl, 0.5% Triton X-100, pH 8.0) followed by centrifugation at 21,000 g for 15 min. Two additional washes were performed using wash buffer without Triton X-100.

Solubilization

Inclusion bodies were resuspended in denaturing buffer (8 M urea, 100 mM Tris, 1 mM EDTA, 10 mM Na ₂ S ₄ O ₆ and 100 mM Na ₂ SO ₃ , pH 8.5), stirred for 16 h at room temperature and clarified by centrifugation at 21000g for 15 min.

Cleaning up before refolding

The solubilized inclusion bodies were loaded onto a Sephacryl S-200 26/60 (120 ml) column equilibrated with 8 M urea, 50 mM MES, 200 mM NaCl, 1 mM EDTA, pH 6.0 buffer. Fractions containing gremlin-1 protein were diluted with 6 M urea, 20 mM MES, pH 6.0 and loaded onto HiTrap SP HP cation exchange columns and eluted in a 1 M NaCl gradient with 10 column volumes (10 CV). Fractions containing purified denatured hGremlin-1 protein were pooled.

Refolding

Denatured purified Gremlin-1 protein was added dropwise to refolding buffer (50 mM Tris, pH 8.5, 150 mM NaCl, 5 mM GSH and 5 mM GSSG, 0.5 mM cysteine, 5 mM EDTA, 0.5 M arginine ) to a final concentration of 0.1 mg/ml and incubated at 4°C with constant stirring for 5 days. After 5 days, the gremlin-1 protein was dialyzed against 20 mM HEPES, 100 mM NaCl, pH 7.5.

After dialysis, the protein was applied to a HiTrap heparin column and eluted using a gradient of 0-100% heparin elution buffer (20 mM HEPES, 1 M NaCl, pH 7.5) using 20 CV. A correctly folded protein is eluted at 1 M NaCl, while any misfolded protein is eluted at lower salt concentrations.

The protein eluted at 1 M NaCl was concentrated and further purified on an S75 26/60 column equilibrated with 20 mM Hepes, pH 7.5, 1 M NaCl.

The protein was characterized by SDS-PAGE (shift in gel) and was shown to have the expected molecular weight and proper disulfide bonding using liquid chromatography, mass spectrometry (LC-MS) and was active in cellular assay (ID1 reporter assay).

Determination of the structure of gremlin-1

Gremlin-1 protein crystals were grown using the hanging drop method by mixing a solution of gremlin-1 at a concentration of 6.6 mg/ml and 0.1 M citric acid at pH 4, 1 M lithium chloride and 27% polyethylene glycol (PEG) 6000 in a ratio of 1:1. Before data collection, the crystals were cryoprotected by adding 20% glycerol to the crystallization buffer. Diffraction data were collected on a Diamond Light Source and processed using XDS (Kabsch, Wolfgang (2010) Acta Crystallographica Section D 66, 125-132). Diffraction statistics data are summarized in the table below:

table 2
Diffraction statistics data Diffraction statistics

)Wavelength (

) 0.97949 Spacer group C2 Cell dimensions a=84.55

, b=107.22

, c=77.09

; α=90.00°, β=120.43°, γ=90.00° Resolution range* (

) 26.19-2.72 ( 2.79-2.72) Completeness (%) 98.5 (99.0) Plurality 3.4 (3.4) I/sigma 9.6 (2.0) merge 0.095 (0.622) Refinement statistics Resolution range (

) 26.19-2.72 R _cryst 0.24 R _free 0.29 Rmsd communication (

)** 0.013 R.m.s.d. angles (°) 1.782 * values in brackets correspond to the shell with the highest resolution;
**rmsd standard deviation.

The structure of gremlin-1 was resolved by molecular substitution using Phaser (McCoy et al., J Appl Cryst (2007), 40, 658-674) and a gremlin-1 model accessible from specific coordinates of the gremlin-1/Fab complex. The resulting gremlin-1 model contained four copies of the gremlin-1 monomer organized as two dimers. Model adjustments were made using Coot (Emsley et al. Acta Crystallographica Section D: Biological Crystallography 66(4), 486-501) and coordinates were refined using Refmac (Murshudov et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallographica Section D: Biological Crystallography 2011;67(Pt4):355-367). End coordinates were validated with Molprobity (Chen et al. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica D66:12-21). A summary of the refinement model statistics is given in Table 2 above.

Example 2 Gremlin-1 Remains Binding to BMP

As discussed above, gremlin-1 belongs to a family of bone morphogenic protein (BMP) antagonist proteins in a subgroup known as the DAN family. Within the DAN family, gremlin-1 shares the greatest homology with gremlin-2 (PRDC).

, разрешенная в примере 1, имеет много признаков, общих с опубликованной структурой гремлина-2 мыши (Nolan et al. (2013), Structure, 21, 1417-1429). Общий фолдинг очень похож: две копии гремлина-1 образуют антипараллельный нековалентный димер, расположенный в изгибе. Каждый мономер имеет характерное расположение «палец-запястье-палец» с мотивом цистинового узла в направлении к концу «пальца», противоположном «запястью». Идентичность последовательности между белками составляет от 52% до 67% в последовательности, видимой в двух структурах. Наиболее высоко консервативная область находится на обширной поверхности раздела димера, где все ключевые контактные остатки сохранены на 100%.Human gremlin-1 structure 2.7

, resolved in Example 1, has many features in common with the published mouse gremlin-2 structure (Nolan et al. (2013), Structure, 21, 1417-1429). The overall folding is very similar: two copies of gremlin-1 form an anti-parallel non-covalent dimer located in the fold. Each monomer has a characteristic "finger-wrist-finger" arrangement with a cystine knot motif towards the end of the "finger" opposite the "wrist". Sequence identity between proteins ranges from 52% to 67% in the sequence seen in the two structures. The most highly conserved region is found on the extensive dimer interface, where all key contact residues are 100% conserved.

Residues involved in the binding of BMP 2, 4 and 7 to murine gremlin-2 (PRDC) and DAN (NBL1) have been identified using mutagenesis (Nolan et al. (2013), Structure, 21, 1417-1429 and Nolan et al (2014) J Biol Chem 290, 4759-4771). The predicted BMP binding epitope includes a hydrophobic region spanning both monomers on the convex surface of the dimer. Using mutagenesis, six residues were identified; Trp72, Phe96, Tyr98, Phe104, Tyr105 and Phe117 and are 100% conserved in human gremlin-1 (numbering is based on the murine gremlin-2 sequence numbering). The degree of homology extends to the arrangement of side chains that adopt the same conformation in both proteins.

The amino acid numbering used in the Gremlin PDB file corresponds to the numbering in the published mouse gremlin-2 structure based on structural alignment. This allows for the same comparison of amino acids when describing structures. However, for clarity, key residues identified as being important in BMP binding are shown below, numbered based on the PDB file and the UniProt file of SEQ ID NO: 1 in parentheses:

Trp72 (93), Phe96 (117), Tyr98 (119), Phe104 (125), Tyr105 (126) and Phe117 (138).

In both mouse gremlin-2 and human gremlin-1, the hydrophobic binding epitope of BMP is partially located under the alpha helix formed by the N-terminal residues of each protein. A BMP binding model has been proposed whereby the N-terminus can be bent to expose the full BMP binding surface (Nolan et al. (2013), Structure, 21, 1417-1429). In the present assay, N-terminal residues were removed from the structures of human gremlin-1 and mouse gremlin-2 prior to surface imaging to reveal the similarity of BMP binding surfaces on each protein.

от тех, которые мутировали, на поверхности гремлина-1, обнаружена большая область гремлина-1, на которую потенциально может быть нацелено терапевтическое средство. Этот более обширная область охватывает следующие аминокислоты человеческого гремлина-1:The literature describes mutagenesis of only six residues that affect BMP binding. It is possible that the actual BMP epitope covers a large surface area including adjacent amino acids. Selecting all the remainders, within 6

from those that mutated on the surface of gremlin-1, a large area of gremlin-1 was found that could potentially be targeted by a therapeutic agent. This broader scope covers the following human gremlin-1 amino acids:

Asp92-Leu99

Arg116-His130

Ser137-Ser142

Cys176-Cys178

(the numbering is based on the numbering of SEQ ID NO: 1).

Combining published information with information about the crystal structure of human gremlin-1, regions of human gremlin-1 have been identified that can be considered as a potential route for therapeutic intervention blocking its interaction with BMP.

Example 3 Analysis of the reporter gene Hek Id 1.

The basis

Hek Id1 reporter gene assay uses reporter cells of clone 12 Hek293-Id1. This cell line was stably transfected with transcription factor Id1. Id1 is a transcription factor in the BMP signaling pathway. Gremlin is known to bind to BMPs, preventing binding to their receptors by decreasing the luciferase signal from the reporter gene. Therefore, using this reporter assay, it is possible to screen for anti-gremlin antibodies and identify antibodies that block the interaction of gremlin with BMP. In these cells, restoration of the luciferase signal is observed if this interaction is blocked.

Method

Clone 12 cells were cultured in DMEM containing 10% FCS, 1x L-glutamine and 1x NEAA. The cells were also cultured in the presence of hygromycin B (200 μg/ml) to ensure that the cells did not lose Id1 gene expression. Cells were analyzed in DMEM containing 0.5% FCS, 1x L-glutamine and 1x NEAA. Hygromycin B is not required for the short period of time that the cells are in the assay.

Cells were washed with PBS, removed using cell dissociation buffer, centrifuged and counted before seeding at 5×10 ⁴ /well in 70 μl (density 7.14×10 ⁵ /ml). The plates used were 96-well, sterile, poly-D-lysine-coated, white, opaque plates. The cells were placed in a thermostat for about 3-4 hours to settle. BMP heterodimers were reduced to 200 μg/ml in 4 mM HCl. BMP was diluted to 10 µg/mL in assay medium using a glass vial to obtain a new working stock stock.

In a polypropylene plate, gremlin-1 was diluted 1:2 to obtain an 8-point dose-response curve with a maximum final dose of 1 μg/mL.

An additional volume of 20 μl medium per well was added and the plates were incubated at 37° C. for 45 minutes.

BMP prepared at 100x was added to all wells except those containing only cells. All wells were made up to 60 µl with assay medium and incubated for another 45 min at 37°C.

After incubation, 30 µl of the sample was transferred to a well of the assay plate and incubated for 20-24 hours before measuring the luminescence signal.

The Cell Steady Glo reagent was thawed in advance at room temperature. The assay plates were cooled to room temperature for about 10-15 minutes before adding the reagent. The luciferase signal was detected by adding the Cell Steady Glo reagent (100 µl) for 20 min on a shaker at room temperature and measuring the luminescence using the Synergy 2 cell titer protocol.

The maximum signal was generated from wells containing BMP and the minimum signal was generated from wells containing only cells.

results

Full-length and truncated forms of gremlin-1 were tested in the Hek-Id1 reporter gene assay to confirm blocking activity against BMP4/7.

Percent inhibition in the dose-response assay was calculated based on the maximum and minimum signals in the assay and data fitted using a 4-parameter logistic model. The IC ₅₀ value was calculated at the inflection point of the curve.

Table 3
Efficacy Results for Full Length Gremlin-1 and Truncated Gremlin-1 in the Hek-Id1 Reporter Gene Assay Hek-Id1 reporter gene analysis N Geometric mean (nM) 95% CI (or range where N=<4) Full Size Gremlin-1 2 1.6 1.3-1.9 Truncated Gremlin-1 2 1.7 1.1-2.5

Conclusion

Gremlin-1 was able to inhibit the BMP 4/7 signaling pathway in the Hek-Id1 reporter gene assay.

Example 4 Preparation of anti-gremlin-1 antibody

Anti-gremlin-1 antibodies were generated by immunization with purified gremlin-1 as described in Example 1 and by panning the library. The library was created in situ as a naive human library with V regions amplified from donated blood.

Immunization resulted in 26 different gremlin-1 binding antibodies from the first round of immunization. These antibodies have been scaled up and purified for testing in screening assays.

25 human-mouse cross-reactive antibodies from the library were panned using recombinant human gremlin from R&D Systems. 10 antibodies were selected for scaling and isolated as scFv for testing in screening assays.

Example 5 Screening for anti-gremlin-1 antibodies

Antibodies were screened using the Hek-Id1 reporter gene assay described in Example 3 and measuring SMAD phosphorylation. SMAD 1, 5 and 8 are phosphorylated in the BMP signaling pathway. Therefore, gremlin-1 inhibitors increase SMAD phosphorylation.

SMAD phosphorylation assay was performed on A549 cells or human lung fibroblasts. Phosphorylation levels were determined using MSD.

results

In the analysis of the reporter gene Hek-Id1, there were no visible hits with antibodies obtained by immunization (with a 10-fold excess of antibodies tested against the BMP4/7 heterodimer). The results are shown in FIG. 1.

In contrast, a number of antibodies derived from the library were able to recover the signal in the Hek-Id1 reporter gene assay (50-fold excess of antibodies with 50% dose of gremlin) (FIG. 2). Of these, Ab2416 and Ab2417 contained high levels of endotoxin. Ab7326 maintained blocking capacity at 10-fold excess and 80% inhibition of gremlin-1 concentration.

Additional results are shown in Fig. 3A (human gremlin) and 3B (mouse gremlin). These figures show the titration of Ab7326 (designated PB376) to 15 nM. Ab7326 has been shown to restore BMP signaling when blocked by human (IC ₅₀ 1.3 nM) or mouse (IC ₅₀ 0.2 nM gremlin). The antibody functions as both human and mouse IgG1.

Full length mouse and human IgG1 sequences are shown below. For the synthesis of full-length mouse and human IgG1 proteins, the Ab7326 variable regions obtained from the library were re-cloned into vectors containing the corresponding antibody constant domains.

Because Ab7326 was derived from a naive human library where the Ab was cloned as scFvs, to reclone the 7326 variable regions as full-length Ab or Fab, it was necessary to perform PCR amplification of VH and VK using primer/degenerate primer pools. The PCR amplified products were then digested and cloned simultaneously into mouse and human vectors. Since VH and VK were amplified with primer/degenerate primer pools, two forms of products were obtained differing by one amino acid residue resulting from slightly different primer annealing during the PCR process.

The two variant heavy chain variable regions differed by one amino acid at position 6 and the two variant light chain variable regions differed by one amino acid at position 7, as shown below:

- heavy chain variable region variant 1 contains glutamic acid (E) at position 6;

- heavy chain variable region variant 2 contains glutamine (Q) at position 6;

- light chain variable region variant 1 has a serine (S) at position 7;

- variant 2 of the light chain variable region contains a threonine (T) at position 7.

Mouse full length IgG1 heavy chain variant 1 (SEQ ID NO: 14)

QVQLVESGAE VKKPGATVKI SCKVS GYTFT DYYMH WVQQA PGKGLEWMG L VDPEDGETIY AEKFQG RVTI TADTSTDTAY MELSSLRSED TAVYYCAT DA RGSGSYYPNH FDY WGQGTLV TVSS AKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCGCKP CICTVPEVSS VFIFPPKPKD VLTITLTPKV TCVVVDISKD DPEVQFSWFV DDVEVHTAQT QPREEQFNST FRSVSELPIM HQDWLNGKEF KCRVNSAAFP APIEKTISKT KGRPKAPQVY TIPPPKEQMA KDKVSLTCMI TDFFPEDITV EWQWNGQPAE NYKNTQPIMD TDGSYFVYSK LNVQKSNWEA GNTFTCSVLH EGLHNHHTEK SLSHSPGK

Mouse full-length IgG1 heavy chain version 1 (SEQ ID NO: 15)

DIVMTQSPDS LAVSLGERAT INC KSSQSVL YSSNNKNYLA WYQQKPGQPP KLLIY WASTR ES GVPDRFSG SGSGTDFTLT INSLQAEDVA VYFC QQYYDT PT FGQGTRLE IK RTDAAPTV SIFPPSSEQL TSGGASVVCF LNNFYPKDIN VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC

Mouse full length IgG1 heavy chain variant 2 (SEQ ID NO: 28)

QVQLVQSGAE VKKPGATVKI SCKVS GYTFT DYYMH WVQQA PGKGLEWMG L VDPEDGETIY AEKFQG RVTI TADTSTDTAY MELSSLRSED TAVYYCAT DA RGSGSYYPNH FDY WGQGTLV TVSS AKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCGCKP CICTVPEVSS VFIFPPKPKD VLTITLTPKV TCVVVDISKD DPEVQFSWFV DDVEVHTAQT QPREEQFNST FRSVSELPIM HQDWLNGKEF KCRVNSAAFP APIEKTISKT KGRPKAPQVY TIPPPKEQMA KDKVSLTCMI TDFFPEDITV EWQWNGQPAE NYKNTQPIMD TDGSYFVYSK LNVQKSNWEA GNTFTCSVLH EGLHNHHTEK SLSHSPGK

Mouse full length IgG1 heavy chain variant 2 (SEQ ID NO: 29)

DIVMTQTPDS LAVSLGERAT INC KSSQSVL YSSNNKNYLA WYQQKPGQPP KLLIY WASTR ES GVPDRFSG SGSGTDFTLT INSLQAEDVA VYFC QQYYDT PT FGQGTRLE IK RTDAAPTV SIFPPSSEQL TSGGASVVCF LNNFYPKDIN VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC

Human full-length IgG1 heavy chain version 1 (SEQ ID NO: 30)

QVQLVESGAE VKKPGATVKI SCKVS GYTFT DYYMH WVQQA PGKGLEWMG L

VDPEDGETIY AEKFQG RVTI TADTSTDTAY MELSSLRSED TAVYYCAT DA

RGSGSYYPNH FDY WGQGTLV TVSS ASTKGP SVFPLAPSSK STSGGTAALG

CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL

GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLF

PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE

EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP

REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT

TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSLSPGK

Human full-length IgG1 - light chain version 1 (SEQ ID NO: 31)

DIVMTQSPDS LAVSLGERAT INC KSSQSVL YSSNNKNYLA WYQQKPGQPP

KLLIY WASTR ES GVPDRFSG SGSGTDFTLT INSLQAEDVA VYFC QQYYDT

PT FGQGTRLE IK RTVAAPSV FIFPSDEQL KSGTASVVCL LNNFYPREAK

VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE

VTHQGLSSPV TKSFNRGEC

Human full-length IgG1 heavy chain version 2 (SEQ ID NO: 16)

QVQLVQSGAE VKKPGATVKI SCKVS GYTFT DYYMH WVQQA PGKGLEWMG L

VDPEDGETIY AEKFQG RVTI TADTSTDTAY MELSSLRSED TAVYYCAT DA

RGSGSYYPNH FDY WGQGTLV TVSS ASTKGP SVFPLAPSSK STSGGTAALG

CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL

GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLF

PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE

EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP

REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT

TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL

SPGK

Human full-length IgG1 - light chain version 2 (SEQ ID NO: 17)

DIVMTQTPDS LAVSLGERAT INC KSSQSVL YSSNNKNYLA WYQQKPGQPP

KLLIY WASTR ES GVPDRFSG SGSGTDFTLT INSLQAEDVA VYFC QQYYDT

PT FGQGTRLE IK RTVAAPSV FIFPSDEQL KSGTASVVCL LNNFYPREAK

VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE

VTHQGLSSPV TKSFNRGEC

Antibody CDRs were determined using the Kabat method (highlighted in bold in the above sequences). Additional HCDR1 residues using the Chothia determination are in italics. Sequences of constant regions are underlined.

Restoration of p-SMAD signaling by anti-gremlin 1 antibodies is shown in Table 4 below.

Table 4
Recovery of p-SMAD signaling 2417 2418 2419 2481 2482 2483 2484 7326 8427 BMP 2
50 ng/ml 109.1%
+/-2.8% 58.2%
+/-1.9% 32.6%
+/-1.4% 40.4%
+/-0.6% 35.3%
+/-0.8% 43.1%
+/-2.1% 104.0%
+/-2.7% 107.2%
+/-3.5% 51.3%
+/-1.4% BMP 4
25 ng/ml 109.6%
+/-3.0% 71.3%
+/-3.1% 31.7%
+/-1.2% 60.1%
+/-2.2% 54.4%
+/-1.3% 72.5%
+/-2.1% 105.2%
+/-3.3% 110.0%
+/-3.8% 78.2%
+/-2.5% BMP 7
200
ng/ml 111.5%
+/-3.8% 99.5%
+/-3.2% 53.8%
+/-3.4% 64.4%
+/-1.3% 52.3%
+/-1.1% 66.2%
+/-1.2% 105.2%
+/-4.3% 108.0%
+/-3.2% 72.6%
+/-2.5% BMP-2/7
50 ng/ml 119.3%
+/-2.6% 78.6%
+/-3.6% 50.8%
+/-2.7% 53.7%
+/-3.1% 47.6%
+/-1.5% 56.1%
+/-2.5% 120.4%
+/-4.4% 128.5%
+/-2.9% 62.8%
+/-2.5% BMP4/7
50 ng/ml 113.7%
+/-3.1% 78.0%
+/-4.0% 61.4%
+/-4.0% 48.3%
+/-2.1% 41.7%
+/-1.7% 50.8%
+/-1.7% 112.4%
+/-2.5% 127.0%
+/-3.1% 63.3%
+/-2.1%

Results are presented as percentage of SMAD phosphorylation by BMP alone (BMP control). Experiments were performed using lung fibroblasts from patients with idiopathic pulmonary fibrosis. rhGremlin-1 antibodies and anti-gremlin-1 antibodies were pre-incubated for 45 min at room temperature. Then, rhGremlin-1 antibodies and anti-gremlin-1 antibodies with BMP were added to the cells for 30 min.

Table 5 below shows additional results from the SMAD phosphorylation assay where displacement of BMP-2 or BMP4/7 from gremlin-1-BMP complexes by anti-gremlin-1 antibodies was examined. The experiments were again performed using lung fibroblasts from patients with idiopathic pulmonary fibrosis. rhBMP-2 or rhBMP 4/7 were pre-incubated with rhGremlin-1 for 1 h at room temperature. Complexes BMP-2 - or BMP4/7-gremlin-1 were incubated with various concentrations of anti-gremlin 1 antibodies overnight at 4°C. Antibody concentrations represent the final concentration in the tablet.

Table 5
Displacement of BMP-2 or BMP4/7 from gremlin-1-BMP complexes by anti-gremlin-1 antibodies 81.3
mcg/ml 40.6
mcg/ml 20.3
mcg/ml 10.2
mcg/ml 5.1
mcg/ml 2.55
mcg/ml 1.27
mcg/ml 0.63
mcg/ml 2484 BMP 2
50 ng/ml 100.3%
+/-3.5% 98.8%
+/-2.7% 97.0%
+/-2.9% 93.5%
+/-2.6% 86.4%
+/-2.0% 79.9%
+/-1.9% 66.5%
+/-2.8% 54.8%
+/-0.3% 2484 BMP4/7
50 ng/ml 136.4%
+/-4.2% 133.2%
+/-1.0% 121.4%
+/-1.4% 108.1%
+/-4.9% 86.6%
+/-4.4% 74.7%
+/-2.2% 65.8%
+/-0.6% 60.7%
+/-1.5% 7326 BMP 2 50 ng/ml 103.7%
+/-1.1% 101.5%
+/-2.4% 99.4%
+/-3.8% 103.8%
+/-2.4% 100.3%
+/-2.2% 103.2%
+/-4.3% 102.8%
+/-2.8% 97.0%
+/-2.9% 7326 BMP4/7
50 ng/ml 133.7%
+/-0.8% 132.3%
+/-1.8% 130.3%
+/-4.2% 125.6%
+/-10.0% 121.4%
+/-4.2% 120.9%
+/-3.3% 111.1%
+/-2.3% 102.0%
+/-4.5%

The results presented in Table 5 demonstrate that Ab7326 can displace already complexed BMP-2 or BMP4/7 from gremlin-1-BMP complexes. Ab7326 can achieve this displacement at significantly lower concentrations than reference antibody 2484. This indicates that Ab7326 is an allosteric inhibitor, consistent with the applicant's discovery that the binding site for Ab7326 is distal to known BMP binding regions on gremlin. 1. Thus, Ab7326 can access the allosteric binding site even when BMP forms a complex with gremlin-1, resulting in a significant increase in the inhibition of gremlin activity.

Example 6 Preparation of the Crystal Structure of Gremlin-1 in Complex with Fab 7326

. Последовательности Fab показаны ниже:The crystal structure of human gremlin-1 in complex with Ab7326 Fab was obtained with a resolution of 2.1

. The Fab sequences are shown below:

Heavy chain: SEQ ID NO: 18

QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTSSSLGTQICVSSSLGTQVTSSSSLGTQICVDSKVKVK

Light chain: SEQ ID NO: 19

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

между гремлином-1 и Fab. Были идентифицированы следующие остатки: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 и Gln175 (нумерация основана на последовательности UniProt SEQ ID NO: 1 (нумерованы как Ile110, Lys126, Lys127, Phe128, Phe128, Arg148, Lys153 и Gln154 в структурном файле, который соответствует нумерации мышиного гремлина-2).The CCP4 NCONT software was then used to identify all contact residues at 4

between Gremlin-1 and Fab. The following residues were identified: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 (numbering based on the sequence of UniProt SEQ ID NO: 1 (numbered as Ile110, Lys126, Lys127, Phe128, Phe128, Arg148, Lys153 and Gln154 in the structure file, which corresponds to the mouse gremlin-2 numbering).

In FIG. 4 shows structural models of the gremlin-Fab complex, where Fab epitope residues are shown relative to BMP binding regions.

Ab7326 is an inhibitory antibody that functions allosterically, ie, it binds to BMP binding regions.

Example 7 Measurement of the binding affinity of the anti-gremlin-1 antibody Ab7326 to gremlin-1.

Method

The affinity of anti-gremlin mIgG for human gremlin-1 was determined by bimolecular interaction analysis using surface plasmon resonance (SPR) technology on a Biacore T200 system, GE Healthcare Bio-Sciences AB. Anti-gremlin mIgG was captured by surface-immobilized anti-mouse Fc and gremlin-1 was titrated over the captured mIGg. Capture ligand (Affinipure F(ab') _2- fragment of goat anti-mouse IgG specific for Fc fragment, 115-006-071, Jackson ImmunoResearch Inc.) was immobilized at 50 μg/ml in 10 mM NaAc, pH 5.0 in flow cell 2 of the CM4 sensor chip by an amine coupling reaction using 600-sec injections of activation and deactivation to a level of ~1600 response units (RU). The working buffer was HBS-EP+ buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% P20 surfactant) with a flow rate of 10 μl/min. The control surface was prepared in flow cell 1 by surface activation and deactivation as for flow cell 2, but without the capture ligand.

The assay buffer was HBS-EP+ plus 150 mM NaCl to give a final NaCl concentration of 300 mM plus 1% CMD40. A 60-sec injection of anti-gremlin mIgG (at 5 μg/ml in running buffer) was passed through flow cells 1 and 2 to obtain a capture level of approximately 100 RU on surface-immobilized anti-mouse IgG, Fc. Recombinant human gremlin-1 was titrated in running buffer from 5 nM (using 2-fold dilutions) and injected through flow cells 1 and 2 at a flow rate of 30 μl/min for 3 min followed by a 5 min dissociation phase. A buffer-only control was also included. The surface was regenerated at a flow rate of 10 μl/min by 60 sec injection of 50 mM HCl, 30 sec injection of 5 mM NaOH, and 30 sec injection of 50 mM HCl.

Kinetic data were determined using Biacore T200 evaluation software.

Affinity measurements were carried out at 25°C.

results

It was found that the binding affinity, presented as the average value of K _D for 5 definitions, was below 100 pM.

Example 8 Inhibition of Gremlin-1 Activity Accelerates Healing and Union in an In Vivo Bone Fracture Repair Model

8.1. Materials and methods

Rat fracture model and drug administration

Long bone segmental defect models have been widely used to study bone healing and regeneration (Sato et al; 2014). In the present study, a 3 mm femoral defect was reproduced in 10-week-old male rats and fixed using an 8-hole PEEK plate (RIS. 602.105, RISystem, Switzerland). The plate was attached to the bone with clamps in the middle of the diaphysis before the bone was drilled and fixed with screws. A 3 mm break gap was created using a 0.44 mm Gigly saw. Defect size/consistency/fixation was closely monitored by X-ray imaging using Faxitron (MX-20-DC5, Faxitron Bioptics LLC, USA), this time point was defined as day 0.

Weekly administration started on Day 1 for 8 weeks as shown in Table 1.

X-rays were then taken during the intravital phase of the study on days 11, 25, 39 and 57 in order to evaluate callus formation and healing progress. Definiens image analysis was used to quantify the area of the boneless defect on the acquired x-rays.

Table 6
Processing groups Group Animal number Treatment Dose Scheme of introduction Time 1 10 Solvent Solvent: 1 ml, s.c. once/week In total 57 days 2 10 anti-gremlin-1-antibody 30 mg/kg, 1 ml, s.c. once/week

Micro-CT analysis of fracture healing

The femur (fractured side) was scanned at 17.2 µm using micro-CT (SkyScan 1076). An area was obtained about 15 mm from the callus with a fracture in the center. The scans were reconstructed using the Skyscan NRECON software (1.7.10) and then the reconstructed sections were further segmented to exclude pins in a defined area of 3 mm calculated from the midpoint of the hip fracture defect. Histomorphometric analysis of the callus of the fracture in 3D was performed using the SkyScan software (v. 1.13.1). The midpoint within 3 mm of the femoral fracture defect was determined, and sections 1.5 mm distal and proximal to the midpoint were segmented for each limb measured. The reconstructed datasets were then binarized and segmented after two defined thresholds, one to detect low mineralization callus (thus quantifying newly formed bone) and the other to detect mature bone. Further segmentation of these data was performed on the femur of animals assigned to the low-response group, based on meeting the criteria for incomplete bridging of the femoral defect, or high-response indicators showing connection at the fracture site.

Histomorphometric fracture analysis

The femur was fixed in 10% neutral buffered formalin for 24 h, dehydrated and placed in methyl methacrylate (MMA) at low temperature. Sections 50 µm thick were stained with toluidine blue to quantify the bone elements of the healing gap defect. Histomorphometric parameters were determined on the cancellous bone at the site of the fracture defect. Measurements were performed using image analysis.

Statistical analysis

Results are presented as means ± SD. Statistical analysis was performed using a two-tailed Mann-Whitney U test with GraphPad Prism software unless otherwise noted.

8.2 Results

An analysis of x-rays obtained at the life stage of the study showed that anti-gremlin-1 antibody accelerated fracture healing in animals, where statistically significant differences between the control and treated groups appeared after 25 days (P<0.05). This effect was evident for the remainder of the study (Fig. 5).

Micro-CT analysis of final samples showed that treatment with anti-gremlin 1 antibody (30 mg/kg/weekly) resulted in an increase in newly formed bone in the callus at the fracture site (P = 0.06).

The incidence of nonunion fractures in this model without any medical intervention is approximately 60% (Sato et al.; 2014). To test how inhibition of gremlin-1 reduced the rate of nonunion, animals were divided into low response (LR) and high response (HR) responders. Gremlin-1 inhibition resulted in a significant increase in the percentage of bone volume/tissue volume (BV/TV%) within LMB (low mineral bone; newly formed bone) (P < 0.01) and HMB (high mineral bone; mature bone) (P < 0.01) in the low-responder group compared to the control group, indicating progressive recovery of the cohort that may form nonunion.

In addition, there was a trend (not significant) towards an increase in LMB and HMB BV/TV% in the high response rate group to anti-gremlin 1 antibody treatment (Fig. 6A, representative images of LR and HR are shown in Fig. 6B).

Two-dimensional histomorphometric analysis of bone parameters was performed on histological sections of the fracture site (Fig.7). Anti-gremlin 1 antibody treatment significantly increased the percentage of bone volume/tissue volume (BV/TV%) (P<0.05) compared to control. Anti-gremlin 1 antibody significantly increased trabecular number (Tb.N) (P<0.001) and significantly decreased trabecular separation (Tb.Sp) (P<0.01), indicating an increase in trabecular bone from anti-gremlin treatment. 1 antibody.

Correlations were made between the results of two-dimensional histomorphometric analysis and three-dimensional micro-CT analysis by comparing the LMB and HMB groups segmented in the micro-CT analysis and two-dimensional histomorphometric analysis of fracture sections (Fig. 8). Comparisons measured by Pearson's correlation revealed a positive and significant correlation between histomorphometry and micro-CT analysis in the LMB (P < 0.0001) and HMB (P < 0.0001) groups, confirming the validity of the data from each data series.

8.3. Conclusion

Inhibition of gremlin-1 activity using a neutralizing anti-gremlin 1 antibody resulted in accelerated fracture repair, with statistically significant differences between control and treated groups appearing after 25 days (3 antibody doses). In addition, the final analysis of the fracture site showed increased bone formation in animals with a low level of response, which might otherwise have developed nonunion. Therefore, inhibition of gremlin-1 activity is a promising therapy for the prevention or treatment of nonunion fractures and may be of particular value in the treatment of fractures prone to nonunion, such as the tibia, distal radius, femoral neck, and navicular.

Sequence list

SEQ ID NO: 1 (human gremlin-1; Uniprot ID: O60565)

MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

SEQ ID NO: 2 (human truncated gremlin-1 used in crystallography with N-terminal tag)

MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

SEQ ID NO: 3 (Ab7326 HCDR1 combined Kabat & Chothia numbering system)

GYTFTDYYMH

SEQ ID NO: 4 (Ab7326 HCDR1 Kabat)

DYYMH

SEQ ID NO: 5 (Ab7326 HCDR2 Kabat)

LVDPEDGETIYAEKFQG

SEQ ID NO: 6 (Ab7326 HCDR3 Kabat)

DARGSGSYYPNHFDY

SEQ ID NO: 7 (Ab7326 LCDR1 Kabat)

KSSQSVLYSSNNKNYLA

SEQ ID NO: 8 (Ab7326 LCDR2 Kabat)

WASTRES

SEQ ID NO: 9 (Ab7326 LCDR3 Kabat)

QQYYDTPT

SEQ ID NO: 10 (Ab7326 Heavy Chain Variable Region Variant 1)

QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

SEQ ID NO: 11 (Ab7326 Light Chain Variable Region Variant 1)

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

SEQ ID NO: 12 (Ab7326 Heavy Chain Variable Region Variant 2)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

SEQ ID NO: 13 (Ab7326 Light Chain Variable Region Variant 2)

DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

SEQ ID NO: 14 (mouse full-length IgG1 heavy chain version 1)

QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

SEQ ID NO: 15 (mouse full-length IgG1 light chain version 1)

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLRNTKDEYERHNSYTCVEATKTSF

SEQ ID NO: 16 (Human full-length IgG1 heavy chain version 2)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 17 (Human full-length IgG1 light chain version 2)

DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

SEQ ID NO: 18 (Fab heavy chain version 1)

QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTSSSLGTQICVSSSLGTQVTSSSSLGTQICVDSKVKVK

SEQ ID NO: 19 (Fab light chain version 1)

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLTLECSKADYSFEKHKVY

SEQ ID NO: 20 (human truncated gremlin-1 used in crystallography without N-terminal tag)

AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

SEQ ID NO: 21 (mature gremlin-1 sequence of SEQ ID NO: 1 without signal peptide from amino acids 1-21)

KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

SEQ ID NO: 22 (human IgG4P heavy chain version 1)

QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 23 (Human IgG4P light chain version 1)

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

SEQ ID NO: 24 (Human IgG1 heavy chain DNA variant 1)

caagtgcaactggtggaatccggggccgaagtgaaaaagcccggagccactgtgaagatctcttgcaaagtgtccggctacaccttcaccgactattacatgcactgggtccagcaggcacctgggaagggccttgagtggatgggtctggtcgatcccgaggacggcgaaactatctacgccgagaagttccagggtcgcgtcaccatcaccgccgacacttccaccgacaccgcgtacatggagctgtccagcttgaggtccgaggacacagccgtgtactactgcgccacggatgctcggggaagcggcagctactacccgaaccacttcgactactggggacagggcactctcgtgactgtctcgagcgcttctacaaagggcccctccgtgttcccgctcgctccatcatcgaagtctaccagcggaggcactgcggctctcggttgcctcgtgaaggactacttcccggagccggtgaccgtgtcgtggaacagcggagccctgaccagcggggtgcacacctttccggccgtcttgcagtcaagcggcctttactccctgtcatcagtggtgactgtcccgtccagctcattgggaacccaaacctacatctgcaatgtgaatcacaaacctagcaacaccaaggttgacaagaaagtcgagcccaaatcgtgtgacaagactcacacttgtccgccgtgcccggcacccgaactgctgggaggtcccagcgtctttctgttccctccaaagccgaaagacacgctgatgatctcccgcaccccggaggtcacttgcgtggtcgtggacgtgtcacatgaggacccagaggtgaagttcaattggtacgtggatggcgtcgaagtccacaatgccaaaactaagcccagagaagaacagtacaattcgacctaccgcgtcgtgtccgtgctcacggtgttgcatcaggattggctgaacgggaaggaatacaagtgcaaagtgtccaacaagg cgctgccggcaccgatcgagaaaactatctccaaagcgaagggacagcctagggaacctcaagtctacacgctgccaccatcacgggatgaactgactaagaatcaagtctcactgacttgtctggtgaaggggttttaccctagcgacattgccgtggagtgggaatccaacggccagccagagaacaactacaagactacccctccagtgctcgactcggatggatcgttcttcctttactcgaagctcaccgtggataagtcccggtggcagcagggaaacgtgttctcctgctcggtgatgcatgaagccctccataaccactatacccaaaagtcgctgtccctgtcgccgggaaag

SEQ ID NO: 25 (Human IgG1 light chain DNA variant 1)

gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

SEQ ID NO: 26 (Human IgG4P Heavy Chain DNA Variant 1)

caagtgcaactggtggaatccggggccgaagtgaaaaagcccggagccactgtgaagatctcttgcaaagtgtccggctacaccttcaccgactattacatgcactgggtccagcaggcacctgggaagggccttgagtggatgggtctggtcgatcccgaggacggcgaaactatctacgccgagaagttccagggtcgcgtcaccatcaccgccgacacttccaccgacaccgcgtacatggagctgtccagcttgaggtccgaggacacagccgtgtactactgcgccacggatgctcggggaagcggcagctactacccgaaccacttcgactactggggacagggcactctcgtgactgtctcgagcgcttctacaaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccct ccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag

SEQ ID NO: 27 (Human IgG4P light chain DNA variant 1)

gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

SEQ ID NO: 28 (mouse full-length IgG1 heavy chain version 2)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

SEQ ID NO: 29 (mouse full-length IgG1 light chain version 2)

DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLRNTKDEYERHNSYTCVEATKTS

SEQ ID NO: 30 (Human full-length IgG1 heavy chain version 1)

QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 31 (Human full-length IgG1 light chain version 1)

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

SEQ ID NO: 32 (Fab heavy chain version 2)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVTYVNSSLGKDK

SEQ ID NO: 33 (Fab light chain version 2)

DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

SEQ ID NO: 34 (human IgG4P heavy chain version 2)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 35 (Human IgG4P light chain version 2)

DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLTLECSKADYSFEKHKVY

Links

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Sato K., Watanabe Y., Harada N., Abe S., Matsushita T., Yamanaka K., Kaneko T., & Sakai Y. (2014) Tissue Eng Part C. Methods 20, 1037-1041.

Schmid G. J., Kobayashi C., Sandell L. J. & Ornitz D. M. (2009), Developmental Dynamics: An Official Publication of the American Association of Anatomists 238, 766-774. Fibroblast growth factor expression during skeletal fracture healing in mice.

Yu Y. Y. et al. (2010), Bone 46, 841-851. Immunolocalization of BMPs, BMP antagonists, receptors, and effectors during fracture repair.

--->

Sequence list

<110> UCB Biopharma SPRL

<120> GREMLIN-1 INHIBITOR FOR TREATMENT OF BONE FRACTURE OR BONE DEFECT

<130> PF0117-WO-PCT

<160> 35

<170>PatentIn version 3.5

<210> 1

<211> 184

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 1

Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Leu Leu Leu Leu Leu Gly

1 5 10 15

Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys Lys Gly Ser Gln Gly Ala

20 25 30

Ile Pro Pro Pro Asp Lys Ala Gln His Asn Asp Ser Glu Gln Thr Gln

35 40 45

Ser Pro Gln Gln Pro Gly Ser Arg Asn Arg Gly Arg Gly Gln Gly Arg

50 55 60

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

65 70 75 80

Leu His Val Thr Glu Arg Lys Tyr Leu Lys Arg Asp Trp Cys Lys Thr

85 90 95

Gln Pro Leu Lys Gln Thr Ile His Glu Glu Gly Cys Asn Ser Arg Thr

100 105 110

Ile Ile Asn Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile Pro

115 120 125

Arg His Ile Arg Lys Glu Glu Gly Ser Phe Gln Ser Cys Ser Phe Cys

130 135 140

Lys Pro Lys Lys Phe Thr Thr Met Val Thr Leu Asn Cys Pro Glu

145 150 155 160

Leu Gln Pro Pro Thr Lys Lys Lys Arg Val Thr Arg Val Lys Gln Cys

165 170 175

Arg Cys Ile Ser Ile Asp Leu Asp

180

<210> 2

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 2

Met Gly Ser Ser His His His His His His Ser Ser Gly Glu Asn Leu

1 5 10 15

Tyr Phe Gln Gly Ser Ala Met Pro Gly Glu Glu Val Leu Glu Ser Ser

20 25 30

Gln Glu Ala Leu His Val Thr Glu Arg Lys Tyr Leu Lys Arg Asp Trp

35 40 45

Cys Lys Thr Gln Pro Leu Lys Gln Thr Ile His Glu Glu Gly Cys Asn

50 55 60

Ser Arg Thr Ile Ile Asn Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe

65 70 75 80

Tyr Ile Pro Arg His Ile Arg Lys Glu Glu Gly Ser Phe Gln Ser Cys

85 90 95

Ser Phe Cys Lys Pro Lys Lys Phe Thr Thr Met Met Val Thr Leu Asn

100 105 110

Cys Pro Glu Leu Gln Pro Pro Thr Lys Lys Lys Arg Val Thr Arg Val

115 120 125

Lys Gln Cys Arg Cys Ile Ser Ile Asp Leu Asp

130 135

<210> 3

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 3

Gly Tyr Thr Phe Thr Asp Tyr Tyr Met His

1 5 10

<210> 4

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 4

Asp Tyr Tyr Met His

15

<210> 5

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 5

Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe Gln

1 5 10 15

gly

<210> 6

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 6

Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp Tyr

1 5 10 15

<210> 7

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 7

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 8

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 8

Trp Ala Ser Thr Arg Glu Ser

15

<210> 9

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 9

Gln Gln Tyr Tyr Asp Thr Pro Thr

15

<210> 10

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120

<210> 11

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 11

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 12

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120

<210> 13

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 13

Asp Ile Val Met Thr Gln Thr Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 14

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn

130 135 140

Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val

180 185 190

Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val

195 200 205

Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg

210 215 220

Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu

245 250 255

Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro

260 265 270

Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val

290 295 300

Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe

305 310 315 320

Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile

340 345 350

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

355 360 365

Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp

370 375 380

Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp

385 390 395 400

Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser

405 410 415

Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly

420 425 430

Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210> 15

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 15

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

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

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 16

<211> 454

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

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

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

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

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 17

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 17

Asp Ile Val Met Thr Gln Thr Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys

225

<210> 19

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 19

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 20

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 20

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

1 5 10 15

Val Thr Glu Arg Lys Tyr Leu Lys Arg Asp Trp Cys Lys Thr Gln Pro

20 25 30

Leu Lys Gln Thr Ile His Glu Glu Gly Cys Asn Ser Arg Thr Ile Ile

35 40 45

Asn Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile Pro Arg His

50 55 60

Ile Arg Lys Glu Glu Gly Ser Phe Gln Ser Cys Ser Phe Cys Lys Pro

65 70 75 80

Lys Lys Phe Thr Thr Met Met Val Thr Leu Asn Cys Pro Glu Leu Gln

85 90 95

Pro Pro Thr Lys Lys Lys Arg Val Thr Arg Val Lys Gln Cys Arg Cys

100 105 110

Ile Ser Ile Asp Leu Asp

115

<210> 21

<211> 160

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 21

Lys Lys Lys Gly Ser Gln Gly Ala Ile Pro Pro Pro Asp Lys Ala Gln

1 5 10 15

His Asn Asp Ser Glu Gln Thr Gln Ser Pro Gln Gln Pro Gly Ser Arg

20 25 30

Asn Arg Gly Arg Gly Gln Gly Arg Gly Thr Ala Met Pro Gly Glu Glu

35 40 45

Val Leu Glu Ser Ser Gln Glu Ala Leu His Val Thr Glu Arg Lys Tyr

50 55 60

Leu Lys Arg Asp Trp Cys Lys Thr Gln Pro Leu Lys Gln Thr Ile His

65 70 75 80

Glu Glu Gly Cys Asn Ser Arg Thr Ile Ile Asn Arg Phe Cys Tyr Gly

85 90 95

Gln Cys Asn Ser Phe Tyr Ile Pro Arg His Ile Arg Lys Glu Glu Gly

100 105 110

Ser Phe Gln Ser Cys Ser Phe Cys Lys Pro Lys Lys Phe Thr Thr Met

115 120 125

Met Val Thr Leu Asn Cys Pro Glu Leu Gln Pro Pro Thr Lys Lys Lys

130 135 140

Arg Val Thr Arg Val Lys Gln Cys Arg Cys Ile Ser Ile Asp Leu Asp

145 150 155 160

<210> 22

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

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

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

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

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Lys

450

<210> 23

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 23

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 24

<211> 1362

<212> DNA

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 24

caagtgcaac tggtggaatc cggggccgaa gtgaaaaagc ccggagccac tgtgaagatc 60

tcttgcaaag tgtccggcta caccttcacc gactattaca tgcactgggt ccagcaggca 120

cctgggaagg gccttgagtg gatgggtctg gtcgatcccg aggacggcga aactatctac 180

gccgagaagt tccagggtcg cgtcaccatc accgccgaca cttccaccga caccgcgtac 240

atggagctgt ccagcttgag gtccgaggac acagccgtgt actactgcgc cacggatgct 300

cggggaagcg gcagctacta cccgaaccac ttcgactact ggggacaggg cactctcgtg 360

actgtctcga gcgcttctac aaagggcccc tccgtgttcc cgctcgctcc atcatcgaag 420

tctaccagcg gaggcactgc ggctctcggt tgcctcgtga aggactactt cccggagccg 480

gtgaccgtgt cgtggaacag cggagccctg accagcgggg tgcacacctt tccggccgtc 540

ttgcagtcaa gcggccttta ctccctgtca tcagtggtga ctgtcccgtc cagctcattg 600

ggaacccaaa cctacatctg caatgtgaat cacaaaccta gcaacaccaa ggttgacaag 660

aaagtcgagc ccaaatcgtg tgacaagact cacacttgtc cgccgtgccc ggcacccgaa 720

ctgctgggag gtcccagcgt ctttctgttc cctccaaagc cgaaagacac gctgatgatc 780

tcccgcaccc cggaggtcac ttgcgtggtc gtggacgtgt cacatgagga cccagaggtg 840

aagttcaatt ggtacgtgga tggcgtcgaa gtccacaatg ccaaaactaa gccagagaa 900

gaacagtaca attcgaccta ccgcgtcgtg tccgtgctca cggtgttgca tcaggattgg 960

ctgaacggga aggaatacaa gtgcaaagtg tccaacaagg cgctgccggc accgatcgag 1020

aaaactatct ccaaagcgaa gggacagcct agggaacctc aagtctacac gctgccacca 1080

tcacgggatg aactgactaa gaatcaagtc tcactgactt gtctggtgaa ggggttttac 1140

cctagcgaca ttgccgtgga gtgggaatcc aacggccagc cagagaacaa ctacaagact 1200

acccctccag tgctcgactc ggatggatcg ttcttccttt actcgaagct caccgtggat 1260

aagtcccggt ggcagcaggg aaacgtgttc tcctgctcgg tgatgcatga agccctccat 1320

aaccactata cccaaaagtc gctgtccctg tcgccgggaa ag 1362

<210> 25

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 25

gacattgtga tgacccagtc ccccgattcg cttgcggtgt ccctgggaga acgggccacc 60

attaactgca agagctcaca gtccgtcctg tattcatcga acaacaagaa ttacctcgca 120

tggtatcagc agaagcctgg acagcctccc aagctgctca tctactgggc tagcacccgc 180

gaatccgggg tgccggatag attctccggga tcgggttcgg gcactgactt cactctgact 240

atcaactcac tgcaagccga ggatgtcgcg gtgtacttct gtcagcagta ctacgacacc 300

ccgacctttg gacaaggcac cagactggag attaagcgta cggtggccgc tccctccgtg 360

ttcatcttcc caccctccga cgagcagctg aagtccggca ccgcctccgt cgtgtgcctg 420

ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

tccggcaact ccggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600

gtgacccacc agggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 26

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 26

caagtgcaac tggtggaatc cggggccgaa gtgaaaaagc ccggagccac tgtgaagatc 60

tcttgcaaag tgtccggcta caccttcacc gactattaca tgcactgggt ccagcaggca 120

cctgggaagg gccttgagtg gatgggtctg gtcgatcccg aggacggcga aactatctac 180

gccgagaagt tccagggtcg cgtcaccatc accgccgaca cttccaccga caccgcgtac 240

atggagctgt ccagcttgag gtccgaggac acagccgtgt actactgcgc cacggatgct 300

cggggaagcg gcagctacta cccgaaccac ttcgactact ggggacaggg cactctcgtg 360

actgtctcga gcgcttctac aaagggcccc tccgtgttcc ctctggcccc ttgctcccgg 420

tccacctccg agtctaccgc cgctctgggc tgcctggtca aggactactt ccccgagccc 480

gtgacagtgt ccctggaactc tggcgccctg acctccggcg tgcacacctt ccctgccgtg 540

ctgcagtcct ccggcctgta ctccctgtcc tccgtcgtga ccgtgccctc ctccagcctg 600

ggcaccaaga cctaaccctg taacgtggac cacaagccct ccaacaccaa ggtggaccaag 660

cgggtggaat ctaagtacgg ccctccctgc cccccctgcc ctgcccctga atttctgggc 720

ggaccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat ctcccggacc 780

cccgaagtga cctgcgtggt ggtggacgtg tcccaggaag atcccgaggt ccagttcaat 840

tggtacgtgg acggcgtgga agtgcacaat gccaagacca agcccagaga ggaacagttc 900

aactccacct accgggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960

1020

1080

gagatgacca agaaccaggt gtccctgacc tgtctggtca agggcttcta cccctccgac 1140

attgccgtgg aatgggagtc caacggccag cccgagaaca actacaagac caccccccct 1200

gtgctggaca gcgacggctc cttcttcctg tactctcggc tgaccgtgga caagtcccgg 1260

tggcaggaag gcaacgtctt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgtccct gagcctgggc aag 1353

<210> 27

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 27

gacattgtga tgacccagtc ccccgattcg cttgcggtgt ccctgggaga acgggccacc 60

attaactgca agagctcaca gtccgtcctg tattcatcga acaacaagaa ttacctcgca 120

tggtatcagc agaagcctgg acagcctccc aagctgctca tctactgggc tagcacccgc 180

gaatccgggg tgccggatag attctccggga tcgggttcgg gcactgactt cactctgact 240

atcaactcac tgcaagccga ggatgtcgcg gtgtacttct gtcagcagta ctacgacacc 300

ccgacctttg gacaaggcac cagactggag attaagcgta cggtggccgc tccctccgtg 360

ttcatcttcc caccctccga cgagcagctg aagtccggca ccgcctccgt cgtgtgcctg 420

ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

tccggcaact ccggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600

gtgacccacc agggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 28

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn

130 135 140

Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val

180 185 190

Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val

195 200 205

Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg

210 215 220

Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu

245 250 255

Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro

260 265 270

Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val

290 295 300

Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe

305 310 315 320

Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile

340 345 350

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

355 360 365

Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp

370 375 380

Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp

385 390 395 400

Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser

405 410 415

Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly

420 425 430

Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210> 29

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 29

Asp Ile Val Met Thr Gln Thr Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

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

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 30

<211> 454

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

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

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

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

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 31

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 31

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 32

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys

225

<210> 33

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 33

Asp Ile Val Met Thr Gln Thr Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 34

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Ala Arg Gly Ser Gly Ser Tyr Tyr Pro Asn His Phe Asp

100 105 110

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

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

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

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

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

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Lys

450

<210> 35

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> recombinant sequence

<400> 35

Asp Ile Val Met Thr Gln Thr Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

<---

Claims (16)

1. The use of an inhibitor of gremlin-1 activity for the manufacture of a medicament for the treatment of a bone fracture or bone defect, where the inhibitor is an anti-gremlin 1 antibody or a functionally active fragment thereof, and where the antibody contains the sequence of SEQ ID NO: 3 or 4 for HCDR1, SEQ ID NO: 5 for HCDR2, SEQ ID NO: 6 for HCDR3, SEQ ID NO: 7 for LCDR1, SEQ ID NO: 8 for LCDR2, and SEQ ID NO: 9 for LCDR3. 2. Use according to claim 1, wherein the functionally active antibody fragment is Fab, Fab', F(ab') ₂ , Fv or scFv. 3. Use according to claim 1 or 2, wherein the antibody comprises a heavy chain variable region (HCVR) of SEQ ID NO: 10 and a light chain variable region (LCVR) of SEQ ID NO: 11. 4. Use according to claim 1 or 2, wherein the antibody comprises SEQ ID NO: 3 or 4 for HCDR1, SEQ ID NO: 5 for HCDR2, SEQ ID NO: 6 for HCDR3, SEQ ID NO: 7 for LCDR1, the sequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and where the heavy chain variable region contains a sequence having at least 95% identity with the sequence of SEQ ID NO: 10, and the light chain variable region contains a sequence having at least 95% identity with the sequence of SEQ ID NO: 11 . 5. Use according to any one of claims 1-4, wherein the antibody binds to an epitope on gremlin-1 containing at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, where the numbering of the residues corresponds to the numbering in SEQ ID NO: 1. 6. Use according to any one of the preceding claims, wherein the bone fracture or bone defect is a slow union fracture or a non-union fracture. 7. Use according to any one of the preceding claims, wherein the bone fracture or bone defect is associated with a disease that adversely affects the integrity of the bone. 8. Use according to claim 7, wherein the disease that adversely affects bone integrity is osteoporosis, osteogenesis imperfecta, diabetes, Paget's disease, rheumatoid arthritis, ankylosing spondylitis, multiple myeloma, primary bone cancer, cancer with bone metastases, diffuse idiopathic skeletal hyperostosis, osteomyelitis, renal failure, Duchenne muscular dystrophy and thalassemia. 9. A method of treating a bone fracture or bone defect, comprising administering a therapeutically effective amount of an inhibitor of gremlin-1 activity, wherein the inhibitor is an anti-gremlin 1 antibody or functionally active fragment thereof, and wherein the antibody comprises the sequence of SEQ ID NO: 3 or 4 for HCDR1 , SEQ ID NO: 5 for HCDR2, SEQ ID NO: 6 for HCDR3, SEQ ID NO: 7 for LCDR1, SEQ ID NO: 8 for LCDR2, and SEQ ID NO: 9 for LCDR3. 10. The method of claim 9 wherein the functionally active antibody fragment is a Fab, Fab', F(ab') ₂ , Fv or scFv. 11. The method of claim 9 or 10, wherein the antibody comprises a heavy chain variable region (HCVR) of SEQ ID NO: 10 and a light chain variable region (LCVR) of SEQ ID NO: 11. 12. The method according to claim 9 or 10, where the antibody contains the sequence of SEQ ID NO: 3 or 4 for HCDR1, the sequence of SEQ ID NO: 5 for HCDR2, the sequence of SEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1 , SEQ ID NO: 8 for LCDR2 and SEQ ID NO: 9 for LCDR3; and where the heavy chain variable region contains a sequence having at least 95% identity with the sequence of SEQ ID NO: 10, and the light chain variable region contains a sequence having at least 95% identity with the sequence of SEQ ID NO: 11. 13. The method according to any one of claims 9-12, wherein the antibody binds to an epitope on gremlin-1 containing at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, where the numbering of the residues corresponds to the numbering in SEQ ID NO: 1. 14. The method of any one of the preceding claims, wherein the bone fracture or bone defect is a delayed union fracture or a non-union fracture. 15. The method of any one of the preceding claims, wherein the bone fracture or bone defect is associated with a disease that adversely affects the integrity of the bone. 16. The method of claim 15, wherein the disease that adversely affects bone integrity is osteoporosis, osteogenesis imperfecta, diabetes, Paget's disease, rheumatoid arthritis, ankylosing spondylitis, multiple myeloma, primary bone cancer, cancer with bone metastases, diffuse idiopathic skeletal hyperostosis, osteomyelitis, renal failure, Duchenne muscular dystrophy and thalassemia.

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