Gremlin-1 inhibitors for the treatment of fractures or bone defects

Abstract

The present invention relates to methods for treating bone fractures or bone defects. The present invention discloses the effective use of anti-gremlin-1 antibodies for accelerating the healing and bridging of bone tissue in segmental cleft defects; and demonstrates that inhibitors of gremlin-1 activity can provide improved therapies for treating or preventing nonunion of fractures.

Description

[0001] The present invention relates to methods for treating bone fractures or bone defects. The present invention discloses the effective use of anti-gremlin-1 antibodies for accelerating the healing and bridging of bone tissue in segmental cleft defects; and demonstrates that inhibitors of gremlin-1 activity can provide improved therapies for treating or preventing nonunion of fractures. Background of the Invention

[0003] A fracture is a break or break in bone tissue and can be the result of a traumatic injury, such as a fall or collision, but can also occur as a result of disease affecting the integrity of the bone.

[0004] Unstable fractures heal through the process of endochondral ossification, which begins with blood clot formation or hematoma. This is coupled with the immune response of regulating immune cells and the surrounding skeletal stem cell population. Subsequently, through the action of various growth factors (including transforming growth factor β (TGFβ) (Cho et al., 2002), fibroblast growth factor (FGF) (Schmid et al., 2009) and bone morphogenetic protein (BMP) (Yu et al., 2010)), the hematoma is replaced by mineralized cartilage callus. Through the action of osteoclasts and osteoblasts, the mineralized callus is replaced by woven bone. The final remodeling stage involves the replacement of woven bone by lamellar bone. Depending on the patient's age and disease state, the completion of this process can take many years.

[0005] Typically, fractures are treated clinically by stabilization (via the use of supports such as splints, casts, or braces). In extreme cases involving complex fractures, surgical intervention may be required and involves the use of internal and external fixators directly attached to the bone. Even with these measures, in approximately 10% of patients, the tissue repair process is still insufficient (Einhorn et al., 2014), which leads to delayed bone union (failure to achieve union 6 months after fracture) or nonunion. Nonunion is defined as incomplete union within 9 months, and combined with radiographic features associated with fracture union that are not observed in three consecutive months (Buza et al., 2016). Current surgical techniques for repairing nonunion fractures and critical bone defects are often limited in terms of the quantity and quality of available materials. Commonly used treatments involve autologous or allogeneic transplants, but these bring additional risks of donor site morbidity (Goulet et al., 1997) and infection (Bostrom et al., 2005), respectively.

[0006] Bone defects are bone loss caused by trauma or disease.

[0007] There is currently a large unmet medical need for improved treatment of bone fractures and bone defects.It is therefore an object of the present invention to provide new methods for treating bone fractures or bone defects.

[0008] The present invention provides a gremlin-1 activity inhibitor for use in treating bone fractures or bone defects. The present invention discloses the effective use of anti-gremlin-1 antibodies for accelerating the healing and bridging of bone tissue in segmental cleft defects; and demonstrates that a gremlin-1 activity inhibitor can provide an improved therapy for treating or preventing nonunion of fractures.

[0009] Description of the invention

[0010] Unless defined otherwise, scientific and technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.All publications mentioned herein are incorporated herein by reference.

[0011] It will be appreciated that any of the embodiments described herein may be combined.

[0012] The present invention provides a gremlin-1 activity inhibitor for use in treating bone fractures or bone defects. The present invention also provides the use of the gremlin-1 activity inhibitor in preparing a medicament for treating bone fractures or bone defects. The present invention further provides a method for treating bone fractures or bone defects, comprising administering a therapeutically effective amount of the gremlin-1 activity inhibitor.

[0013] Gremlin-1 (also known as Drm and CKTSF1B1) is a 184 amino acid glycoprotein that forms part of the DAN family of cysteine ​​knot secreted proteins (particularly together with Cerberus and Dan). Gremlin binds to and inhibits the ability of BMP-2, 4 and 7 to conduct signals, along with documented pro-angiogenic effects, possibly through stimulation of VEGFR2. The major role of Gremlin-1 is in development, where it is crucial during kidney formation and during limb bud formation.

[0014] Bone morphogenetic protein (BMP) signaling is known to control endochondral bone formation, of which Gremlin 1 (GREM1) is one of the natural antagonists of this pathway through 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 enhanced 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 the overall knockout is embryonic lethal in the BL6 background, 49% of the pups survived more than 24 hours after birth in the C57BL / 6 / FVB mixed genetic background, and although developmental skeletal defects are present in large numbers, elevated bone formation rates can be observed (Canalis et al., 2012). Despite the knowledge of this developmental function of GREM1, there is no data suggesting that inhibition of this protein alone will enhance postnatal fracture repair. Indeed, although endochondral bone formation is the primary mechanism of skeletogenesis during the embryonic stage, the mechanisms regulating cell recruitment are distinct processes when compared to postnatal fracture repair (Ferguson et al., 1999). The role of inflammation has been implicated as a key factor in adult bone repair, and therefore developmental factors controlling the skeletogenesis process cannot simply be extrapolated to postnatal repair mechanisms.

[0015] The term "Gremlin-1" used in the present invention typically has the sequence shown in UniProt entry O60565 (SEQ ID NO: 1). The term "Gremlin-1" may also refer to a Gremlin-1 polypeptide that:

[0016] (a) comprises or consists of the amino acid sequence of SEQ ID NO: 1 with or without an N-terminal signal peptide, i.e., may comprise or consist of the mature peptide sequence shown in SEQ ID NO: 21; or

[0017] (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 (shown in SEQ ID NO: 21), which retains the activity of Gremlin-1, such as the sequence of SEQ ID NO: 20,

[0018] (c) variants thereof, such variants typically retain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or 95% identity (or even about 96%, 97%, 98% or 99% identity) to SEQ ID NO: 1 (or SEQ ID NO: 20 or 21). In other words, such variants may retain about 60% to about 99% identity to SEQ ID NO: 1, suitably about 80% to about 99% identity to SEQ ID NO: 1, more suitably about 90% to about 99% identity to SEQ ID NO: 1, and most suitably about 95% to about 99% identity to SEQ ID NO: 1. Variants are further described below.

[0019] As discussed further below, residue numbers are typically referenced based on the sequence of SEQ ID NO: 1. However, a skilled person can easily extrapolate residue numbers to derivative or variant sequences, as discussed above. When citing residue numbers, the present invention also includes these residues on the variant or derivative sequence.

[0020] The inventors of the present invention have crystallized human Gremlin-1 alone and in complex with an antibody (Fab fragment) called Ab 7326. Crystallization of Gremlin-1 allowed the determination of putative residues in the BMP binding site. Further, crystallization with Ab7326 (which is an allosteric inhibitory antibody) allowed the determination of residues in the antibody epitope (WO 2018 / 115017A2). Antibodies that bind to this epitope have the potential to be therapeutic agents in the treatment of fractures or bone defects.

[0021] Gremlin-1 activity inhibitors

[0022] The gremlin-1 activity inhibitor according to the present invention is an agent that reduces or blocks the activity of gremlin-1. The inhibitor according to the present invention can partially or completely inhibit the activity of gremlin-1. The inhibitor used in the present invention includes but is not limited to inhibitors that can bind to gremlin-1 or to nucleic acid molecules encoding gremlin-1 or can inhibit the expression of gremlin-1. Such inhibitors can be (but are not limited to) proteins, polypeptides, peptides, peptide mimetics, nucleic acids (e.g., DNA, RNA, antisense RNA and siRNA), carbohydrates, lipids and small molecules.

[0023] In one embodiment, the inhibitor of gremlin-1 activity is an anti-gremlin-1 antibody or a functionally active fragment, variant or derivative thereof.

[0024] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. Antibodies or immunoglobulins typically refer to glycoproteins comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or V L ) and light chain constant regions. The heavy and light chain variable regions contain the binding domain that interacts with the antigen. H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).

[0025] The constant regions of the antibodies may mediate the binding of the immunoglobulin 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.

[0026] The antibody used in the present invention can be a monoclonal antibody or a polyclonal antibody, and typically will be a monoclonal antibody. The antibody used in the present invention can be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human antibody or a humanized antibody or any antigen binding portion thereof.

[0027] Polyclonal antibodies can be produced by conventional methods, such as immunizing a suitable animal with the antigen of interest. Blood can then be taken from the animal and the immunoglobulin fraction purified.

[0028] Antibodies against Gremlin-1 can be obtained by administering the polypeptide to an animal, such as a non-human animal, where immunization of the animal is necessary, using well-known and conventional experimental protocols, see, for example, Handbook of Experimental Immunology, DM Weir (ed.), Volume 4, Blackwell Scientific Publishers, Oxford, England, 1986. Many warm-blooded animals, such as rabbits, mice, rats, sheep, goats, cattle, camels, llamas or pigs can be immunized. However, rabbits, mice and rats are generally the most suitable.

[0029] Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4: 72) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R Liss, Inc., 1985).

[0030] Antibodies for use in the present invention can also be produced by using the single lymphocyte antibody method, which is produced by cloning and expressing immunoglobulin variable region cDNAs produced from single lymphocytes selected for the production of specific antibodies, which is described, for example, by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO 92 / 02551; WO 2004 / 051268; and WO 2004 / 106377.

[0031] Antibodies for use in the present invention can also be produced using various phage display methods known in the art, and 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 1879-18), Burton et al. (Advances in Immunology, 1994, 57: 191-280); and WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401; and US 5,698,426, US 5,223,409, US 5,403,484, US 5,580,717, US 5,427,908, US 5,750,753, US 5,821,047, US5,571,698, US 5,427,908, US 5,516,637, US 5,780,225, US 5,658,727, US 5,733,743 and US 5,969,108.

[0032] The antibody of complete human is such those antibodies, wherein the variable region and constant region (when present) of both heavy chain and light chain are all people's origin, or are substantially identical with the sequence of people's origin, but not necessarily from identical antibody.The example of complete human antibody can include the antibody produced by the phage display method described above, for example, and the antibody produced by the mouse that wherein murine immunoglobulin variable region gene and optionally constant region gene have been replaced by its people's counterpart, for example, as described generally in EP 0546073, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US 5,770,429, EP 0438474 and EP 0463151.

[0033] Alternatively, the antibody according to the present invention can be produced by a method comprising: immunizing a non-human mammal with a Gremlin-1 immunogen; obtaining an antibody preparation from the mammal; and obtaining a monoclonal antibody recognizing Gremlin-1 therefrom.

[0034] The antibody molecule used in the present invention may comprise a complete antibody molecule having a full-length heavy chain and a light chain or a fragment or antigen-binding portion thereof. The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retains the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. The antibody and its fragment and antigen-binding portion may be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, a single domain antibody (e.g., VH or VL or VHH), scFv, a bivalent, trivalent or tetravalent antibody, a double-scFv, a double-chain antibody, a three-chain antibody, a four-chain antibody, and an epitope-binding fragment of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing and preparing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include Fab and Fab' fragments described in International Patent Applications WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171, and Fab-dAb fragments described in International Patent Application WO 2009 / 040562. Multivalent antibodies may contain multispecificity, or may be monospecific (see, e.g., WO 92 / 22853 and WO 2005 / 113605). These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments may be screened for usefulness in the same manner as intact antibodies.

[0035] In one example, the functionally active antibody fragment for use in the present invention is Fab, Fab', F(ab')2, Fv or scFv.

[0036] In view of the effector functions that may be required, the constant region domains of the antibody molecules used in the present invention can be selected, if present. For example, the constant region domains can be human IgA, IgD, IgE, IgG or IgM domains. In particular, when antibody effector functions are required, human IgG constant region domains, especially IgG1 and IgG3 isotypes, can be used. Alternatively, when antibody effector functions are not required, IgG2 and IgG4 isotypes can be used. In one example, the isotype is IgG4P, as described by Angal S. et al., Mol Immunol, Vol 30 (1), pp. 105-108, 1993.

[0037] The antibodies for use in the present invention can be prepared, expressed, generated or isolated by recombinant means, such as (a) isolating the antibodies from an animal that is transgenic or transchromosomal for the immunoglobulin genes of interest or a hybridoma prepared therefrom, (b) isolating the antibodies from a host cell transformed to express the antibody of interest (e.g., from a transfectoma), (c) isolating the antibodies from a recombinant combinatorial antibody library, and (d) preparing, expressing, generating or isolating the antibodies by any other means involving splicing of immunoglobulin gene sequences into other DNA sequences.

[0038] The antibody used in the present invention can be a human antibody or a humanized antibody. The term "human antibody" used in this article is intended to include such an antibody, which has a variable region in which the framework region and the CDR region are all derived from human germline immunoglobulin sequences. Further, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibody used in the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" used in this article is not intended to include such an antibody, wherein the CDR sequence derived from the germline of another mammalian species (such as a mouse) has been transplanted onto a human framework sequence.

[0039] Such human antibodies can be human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals (e.g., transgenic mice) having a genome comprising human heavy chain transgenes and light chain transgenes fused with immortalized cells.

[0040] Human antibodies can be prepared by in vitro immunization of human lymphocytes and subsequent transformation of the lymphocytes with Epstein-Barr virus.

[0041] The term "derivative" refers to any modified form of the antibody, such as a conjugate of the antibody with another agent or effector molecule.

[0042] The effector molecule may comprise a single effector molecule, or two or more such molecules, which are connected in such a way that a single part of the antibody for use in the present invention is formed. In the case of an antibody fragment connected to an effector molecule, it is desired to obtain, this can be prepared by standard chemical or recombinant DNA procedures, wherein the antibody fragment is directly or by a coupling agent connected to the effector molecule. The technology for conjugating such effector molecules to antibodies is well known in the art (see, Hellstrom et al., Controlled Drug Delivery, 2nd edition, Robinson et al., ed., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62: 119-58; and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular chemical procedures include those described, for example, in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 2003 / 031581. Alternatively, where the effector molecule is a protein or polypeptide, the linkage may be achieved using recombinant DNA procedures, for example as described in WO 86 / 01533 and EP 0392745.

[0043] The effector molecule may increase the in vivo half-life of the antibody, and / or reduce the immunogenicity of the antibody, and / or enhance the 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.

[0044] The term "humanized antibody" is intended to refer to a CDR-grafted antibody molecule in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0045] As used herein, the term "CDR-grafted antibody molecule" refers to an antibody molecule in which the heavy and / or light chains comprise one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g., a murine or rat monoclonal antibody) that are grafted into the heavy and / or light chain variable region framework of a recipient antibody (e.g., a human antibody). For review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, rather than transferring the entire CDR, only one or more of the specificity determining residues from any one of the CDRs described above in this article are transferred to a human antibody framework (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity determining residues from one or more of the CDRs described above in this article are transferred to a human antibody framework. In another embodiment, only the specificity determining residues from each of the CDRs described above in this article are transferred to a human antibody framework.

[0046] When transplanting CDR or specificity determining residue, consider the category / type of the donor antibody that described CDR is derived from, can use any suitable acceptor variable region framework sequence, comprise mouse, primate and people's framework region.Suitably, the CDR-grafting type antibody used in the present invention has such variable domains, it comprises people's acceptor framework region and one or more in CDR or specificity determining residue described above.Therefore, in one embodiment, provide such neutrality CDR-grafting type antibody, wherein variable domains comprises people's acceptor framework region and non-human donor CDR.

[0047] Examples of human frameworks 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 and light chains. Alternatively, human germline sequences can be used; these are available, for example, at http: / / www.vbase2.org / (see Retter et al., Nucl. Acids Res. (2005) 33 (Suppl. 1), D671-D674).

[0048] In the CDR-grafted antibodies for use in the present invention, the recipient heavy and light chains do not necessarily need to be derived from the same antibody, and may, if desired, comprise composite chains having framework regions derived from different chains.

[0049] In addition, in the CDR-grafted antibodies used in the present invention, the framework region does not need to have those identical sequences with the recipient antibody. For example, uncommon residues can be changed to residues that occur more frequently for that kind of recipient chain category or type. Alternatively, the selected residues in the recipient framework region can be changed so that they correspond to the residues found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be maintained at the minimum necessary for restoring the affinity of the donor antibody. The experimental scheme for selecting the residues in the recipient framework region that may need to be changed is set forth in WO 91 / 09967.

[0050] Those skilled in the art will also appreciate that antibodies can undergo a variety of post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the antibody. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and aspartic acid deamidation. A common modification is the loss of carboxyl terminal basic residues (e.g., lysine or arginine), which is attributed to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705: 129-134, 1995).

[0051] In one embodiment, the antibody heavy chain comprises a CH1 domain, and the antibody light chain comprises a CL domain (κ or λ).

[0052] Biomolecules, such as antibodies or fragments, contain acidic and / or basic functional groups, thereby giving the molecule a positive or negative net charge. The amount of the total "observed" charge will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure, and the environmental conditions of the molecule. The isoelectric point (pi) is the pH at which a particular molecule or surface does not carry a net charge. In one embodiment, an antibody or fragment according to the present disclosure has an isoelectric point (pi) of at least 7. In one embodiment, the 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. The isoelectric point of the antibody or fragment can be predicted using a program such as **ExPASY http: / / www.expasy.ch / tools / pi_tool.html (see Walker, The Proteomics Protocols Handbook, Humana Press (2005), 571-607).

[0053] The antibody for use in the present invention may comprise at least one, at least two or all three heavy chain CDR sequences (HCDR1 / HCDR2 / HCDR3, respectively) of SEQ ID NOs: 4 to 6. These are the HCDR1 / HCDR2 / HCDR3 sequences of the Ab7326 antibody of the Examples, which were determined using the Kabat methodology.

[0054] The Kabat and Chothia methods (and other techniques) for determining CDR sequences are well known in the art. CDR sequences can be determined using any suitable method, and in the present invention, although Kabat is typically used, other techniques may also be used. In the present case, SEQ ID NO:3 represents the Ab7326 HCDR1 sequence, as determined by using the combined Chothia & Kabat definition.

[0055] The antibody for use in the present invention may comprise at least one, at least two or all three light chain CDR sequences (LCDR1 / LCDR2 / LCDR3, respectively) of SEQ ID NOs: 7 to 9. These are the LCDR1 / LCDR2 / LCDR3 sequences of Ab7326 by using Kabat methodology.

[0056] In one embodiment, the antibody comprises at least the HCDR3 sequence of SEQ ID NO:6.

[0057] Typically, the antibody comprises at least one heavy chain CDR sequence selected from SEQ ID NOs: 4 to 6 and at least one light chain CDR sequence selected from SEQ ID NOs: 7 to 9. The antibody may comprise at least two heavy chain CDR sequences selected from SEQ ID NOs: 4 to 6 and at least two light chain CDR sequences selected from SEQ ID NOs: 7 to 9. Typically, the antibody comprises all three heavy chain CDR sequences (HCDR1 / HCDR2 / HCDR3, respectively) of SEQ ID NOs: 4 to 6 and all three light chain CDR sequences (LCDR1 / LCDR2 / LCDR3, respectively) of SEQ ID NOs: 7 to 9. The antibody may be a chimeric antibody, a human antibody or a humanized antibody.

[0058] The antibody may comprise a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12 (HCVR of Ab7326 variants 1 and 2). The antibody may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13 (LCVR of Ab7326 variants 1 and 2). The antibody preferably comprises a heavy chain variable region sequence of SEQ ID NO: 10 or 12 and a light chain variable region sequence of SEQ ID NO: 11 or 13 (particularly a HCVR / LVCR pair of SEQ ID NO: 10 / 11 or 12 / 13).

[0059] Ab7326 variants 1 and 2 differ in a single amino acid in the heavy chain variable region and a single amino acid in the light chain variable region as follows:

[0060] Heavy chain variable region variant 1 has glutamic acid (E) at position 6 (SEQ ID NO: 10),

[0061] Heavy chain variable region variant 2 has glutamine (Q) at position 6 (SEQ ID NO: 12),

[0062] Light chain variable region variant 1 has serine (S) at position 7 (SEQ ID NO: 11),

[0063] • Light chain variable region variant 2 has a threonine (T) at position 7 (SEQ ID NO: 13).

[0064] Thus, in one embodiment, the antibody comprises a heavy chain variable region (HCVR) sequence of SEQ ID NO:10, wherein the glutamic acid residue at position 6 is substituted with a glutamine residue (E6Q), wherein the residue numbering is according to SEQ ID NO:10.

[0065] In one embodiment, the antibody comprises a heavy chain variable region (HCVR) sequence of SEQ ID NO:12, wherein the glutamine residue at position 6 is substituted with a glutamic acid residue (Q6E), wherein the residue numbering is according to SEQ ID NO:12.

[0066] 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 substituted with a threonine residue (S7T), wherein the residue numbering is according to SEQ ID NO:11.

[0067] 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 substituted with a serine residue (T7S), wherein the residue numbering is according to SEQ ID NO:13.

[0068] In one embodiment, the antibody comprises the sequence of SEQ ID NO:3 or 4 as HCDR1, the sequence of SEQ ID NO:5 as HCDR2, the sequence of SEQ ID NO:6 as HCDR3, the sequence of SEQ ID NO:7 as LCDR1, the sequence of SEQ ID NO:8 as LCDR2, and the sequence of SEQ ID NO:9 as LCDR3; and wherein the heavy chain variable region comprises a sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98% or 99% identical) to the sequence of SEQ ID NO:10, and the light chain variable region comprises a sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98% or 99% identical) to the sequence of SEQ ID NO:11.

[0069] In one embodiment, the antibody comprises the sequence of SEQ ID NO:3 or 4 as HCDR1, the sequence of SEQ ID NO:5 as HCDR2, the sequence of SEQ ID NO:6 as HCDR3, the sequence of SEQ ID NO:7 as LCDR1, the sequence of SEQ ID NO:8 as LCDR2, and the sequence of SEQ ID NO:9 as LCDR3; and wherein the heavy chain variable region comprises a sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98% or 99% identical) to the sequence of SEQ ID NO:12, and the light chain variable region comprises a sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98% or 99% identical) to the sequence of SEQ ID NO:13.

[0070] The antibody may comprise the following heavy chain (H-chain) sequence:

[0071] SEQ ID NO: 14, mouse full length IgG1 heavy chain variant 1, or

[0072] SEQ ID NO:28, mouse full length IgG1 heavy chain variant 2, or

[0073] SEQ ID NO: 30, human full length IgG1 heavy chain variant 1, or

[0074] SEQ ID NO: 16, human full length IgG1 heavy chain variant 2, or

[0075] SEQ ID NO: 22, human full length IgG4P heavy chain variant 1, or

[0076] SEQ ID NO: 34, human full length IgG4P heavy chain variant 2, or

[0077] SEQ ID NO: 18, Fab heavy chain variant 1, or

[0078] SEQ ID NO:32, Fab heavy chain variant 2.

[0079] The antibody may comprise the following light chain (L-chain) sequence:

[0080] SEQ ID NO: 15, mouse full length IgG1 light chain variant 1, or

[0081] SEQ ID NO: 29, mouse full length IgG1 light chain variant 2, or

[0082] SEQ ID NO: 31, human full length IgG1 light chain variant 1, or

[0083] SEQ ID NO: 17, human full length IgG1 light chain variant 2, or

[0084] SEQ ID NO: 23, human full-length IgG4P light chain variant 1, or

[0085] SEQ ID NO: 35, human full-length IgG4P light chain variant 2, or

[0086] SEQ ID NO: 19, Fab light chain variant 1, or

[0087] SEQ ID NO:33, Fab light chain variant 2.

[0088] In one embodiment, the antibody comprises the following heavy chain / light chain sequence pairs:

[0089] SEQ ID NO: 14 / 15, mouse full length IgG1 variant 1, or

[0090] SEQ ID NO:28 / 29, mouse full length IgG1 variant 2, or

[0091] SEQ ID NO:30 / 31, human full length IgG1 variant 1, or

[0092] SEQ ID NO: 16 / 17, human full length IgG1 variant 2, or

[0093] SEQ ID NO: 22 / 23, human full length IgG4P variant 1, or

[0094] SEQ ID NO:34 / 35, human full length IgG4P variant 2, or

[0095] SEQ ID NO: 18 / 19, Fab light chain variant 1, or

[0096] SEQ ID NO:32 / 33, Fab light chain variant 2.

[0097] Variant forms of the corresponding sequences can be interchanged. For example, the antibody can comprise the following heavy chain / light chain sequence pairs:

[0098] SEQ ID NO: 14 / 29, mouse full length IgG1 heavy chain variant 1 / light chain variant 2, or

[0099] SEQ ID NO:28 / 15, mouse full length IgG1 heavy chain variant 2 / light chain variant 1, or

[0100] SEQ ID NO:30 / 17, human full length IgG1 heavy chain variant 1 / light chain variant 2, or

[0101] SEQ ID NO: 16 / 31, human full length IgG1 heavy chain variant 2 / light chain variant 1, or

[0102] SEQ ID NO: 22 / 35, human full-length IgG4P heavy chain variant 1 / light chain variant 2, or

[0103] SEQ ID NO: 34 / 23, human full-length IgG4P heavy chain variant 2 / light chain variant 1, or

[0104] SEQ ID NO: 18 / 33, Fab heavy chain variant 1 / light chain variant 2, or

[0105] SEQ ID NO:32 / 19, Fab heavy chain variant 2 / light chain variant 1.

[0106] The antibody may be a chimeric antibody, a human antibody or a humanized antibody.

[0107] Alternatively, the antibody may be or may comprise a variant of one of the specific sequences described above. For example, the variant may be a substitution, deletion or addition variant of any of the above amino acid sequences.

[0108] Variant antibodies may comprise 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 relative to the specific sequences discussed above. "Deletion" variants may comprise the deletion of a single amino acid, the deletion of a small amino acid group (e.g., 2, 3, 4, or 5 amino acids), or the deletion of a larger amino acid region, such as the deletion of a specific amino acid domain or other features. "Replacement" variants typically involve one or more amino acids being replaced with the same number of amino acids and conservative amino acid substitutions. For example, an amino acid may be replaced with an alternative amino acid with 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 of the characteristics of the 20 main amino acids that can be used to select suitable substituents are as follows:

[0109] Table 1 :Amino acid properties

[0110]

[0111] "Derivatives" or "variants" generally include those in which, instead of naturally occurring amino acids, the amino acids appearing in the sequence are structural analogs thereof. The amino acids used in the sequence may also be derivatized or modified, e.g., labeled, provided that the function of the antibody is not significantly adversely affected.

[0112] The derivatives and variants described above may be prepared during synthesis of the antibody or by post-production modification, or when the antibody is in recombinant form, by using known techniques of site-specific mutagenesis, random mutagenesis or enzymatic cleavage and / or ligation of nucleic acids.

[0113] Variant antibodies may have an amino acid sequence having more than about 60%, or more than about 70%, such as 75% or 80%, typically more than about 85%, such as more than about 90% or 95% amino acid identity with the amino acid sequences disclosed herein (particularly HCVR / LCVR sequences and H-chain and L-chain sequences). Further, the antibody may be a variant having more than about 60%, or more than about 70%, such as 75% or 80%, typically more than about 85%, such as more than about 90% or 95% amino acid identity with the HCVR / LCVR sequences and H-chain and L-chain sequences disclosed herein, while retaining the precise CDRs disclosed for these sequences. The variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the HCVR / LCVR sequences and H-chain and L-chain sequences disclosed herein (in some cases, while retaining the exact CDRs).

[0114] Typically, variants retain about 60% to about 99% identity, about 80% to about 99% identity, about 90% to about 99% identity, or about 95% to about 99% identity. This amino acid identity level can be found across the entire length of the relevant SEQ ID NO sequence or over a portion of the sequence, such as across about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0115] In relation to amino acid sequences, "sequence identity" refers to sequences having the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

[0116] Pairwise alignment parameters – Method: Exact, Matrix: PAM, Gap opening penalty: 10.00, Gap extension penalty: 0.10;

[0117] Multiple alignment parameters - Matrix: PAM, Gap Open Penalty: 10.00, % Identity with respect to Delay: 30, Penalty for End Gaps: On, Gap Separation Distance: 0, Negative Matrix: None, Gap Extension Penalty: 0.20, Residue Specific Gap Penalty: On, Hydrophilic Gap Penalty: On, Hydrophilic Residues: GPSNDQEKR. Sequence identities at specific residues are intended to include identical residues that have only been derivatized.

[0118] Thus, antibodies having specific sequences as well as derivatives and variants that retain the function or activity of these chains are provided for use in the present invention.

[0119] As used herein, "derivatives" are intended to include reactive derivatives, such as thiol-selective reactive groups such as maleimide. The reactive groups may be attached to the polymer directly or via a linker segment. It will be appreciated that the residues of such groups will in some cases become part of the product as a linking group between the antibody fragment and the polymer.

[0120] The polymer may be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homopolysaccharide or a heteropolysaccharide.

[0121] Particular optional substituents that may be present on the synthetic polymer include one or more hydroxy, methyl or methoxy groups.

[0122] Particular examples of synthetic polymers include optionally substituted linear or branched polyethylene glycol, polypropylene glycol, polyvinyl alcohol or derivatives thereof, especially optionally substituted polyethylene glycol such as methoxypolyethylene glycol or derivatives thereof.

[0123] Particular naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.

[0124] The size of the polymer can be varied as desired, but will generally be within the range of 500Da to 50000Da, such as 5000 to 40000Da, such as 20000 to 40000Da average molecular weight. In particular, the polymer size can be selected based on the intended use of the product, such as the ability to locate to certain tissues or the ability to extend the circulation half-life (for review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Therefore, for example, in the case where the product is intended to leave the circulation and penetrate tissue, it may be advantageous to use a low molecular weight polymer (for example, with a molecular weight of about 5000Da). For applications where the product is retained in the circulation, it may be advantageous to use a higher molecular weight polymer (for example, with a molecular weight in the range of 20000Da to 40000Da).

[0125] Suitable polymers include polyalkylene polymers, such as polyethylene glycol, or especially methoxypolyethylene glycol or derivatives thereof, especially having a molecular weight in the range of about 15000 Da to about 40000 Da.

[0126] In one example, the antibody used in the present invention is attached to a polyethylene glycol (PEG) moiety. In a particular example, the antibody is an antibody fragment, and the PEG molecule can be attached by any available amino acid side chain or terminal amino acid functional group (e.g., any free amino, imino, thiol, hydroxyl or carboxyl group) located in the antibody fragment. Such amino acids can occur naturally in the antibody fragment, or can be transformed into the fragment using recombinant DNA methods (see, e.g., US 5,219,996; US 5,667,425; WO98 / 25971; WO2008 / 038024). In one example, the antibody molecule is a modified Fab fragment, wherein the modification is to add one or more amino acids to the C-terminus of its heavy chain to allow attachment of effector molecules. Suitably, the additional amino acids form a modified hinge region comprising one or more cysteine ​​residues to which the effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

[0127] The antibody may compete for binding to Gremlin-1 with those defined above in terms of H-chain / L-chain, HCVR / LCVR or CDR sequences, or bind to the same epitope as them. In particular, the antibody may compete for binding to Gremlin-1 with an antibody comprising a HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9, or bind to the same epitope as them. The antibody may compete for binding to Gremlin-1 with an antibody comprising a HCVR and LCVR sequence pair of SEQ ID NO: 10 / 11 or 12 / 13, or a full-length chain of SEQ ID NO: 14 / 15 or 16 / 17, or bind to the same epitope as them.

[0128] An "epitope" is a region of an antigen to which an antibody binds. An epitope can be defined as either structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. An epitope can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope can include a determinant, which is a chemically active surface cluster of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have special three-dimensional structural features and / or special charge characteristics.

[0129] By using conventional methods known in the art, it is easy to determine whether an antibody binds to the same epitope as a reference antibody or competes with the reference antibody for binding. For example, in order to determine whether a test antibody binds to the same epitope as a reference antibody used in the present invention, the reference antibody is allowed to bind to a protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the protein or peptide is evaluated. If the test antibody is able to bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to an epitope different from that of the reference antibody. On the other hand, if the test antibody is unable to bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention.

[0130] In order to determine whether an antibody competes with a reference antibody for binding, the binding methodology described above is performed in two directions. In the first direction, the reference antibody is allowed to bind to the protein / peptide under saturation conditions, and the binding of the test antibody to the protein / peptide molecule is subsequently evaluated. In the second direction, the test antibody is allowed to bind to the protein / peptide under saturation conditions, and the binding of the reference antibody to the protein / peptide is subsequently evaluated. If only the first (saturated) antibody is able to bind to the protein / peptide in both directions, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be appreciated by the technician, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0131] Two antibodies bind to the same or overlapping epitopes if each antibody competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, 75%, 90% or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, the two antibodies have the same epitope if substantially all of the amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody. The two antibodies have overlapping epitopes if some of the amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody.

[0132] Then, additional routine experiments (e.g., peptide mutations and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. Experiments of this type can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0133] The anti-gremlin-1 antibody of the Examples, Ab7326, was found to bind to the following residues of Gremlin-1: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175; wherein Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175 are present on one Gremlin-1 monomer, and Ile131 is present on a second Gremlin-1 monomer. The numbering is based on UniProt entry O60565 of SEQ ID NO: 1. As discussed in the Examples section, these epitope residues were identified by using The following NCONT analysis identified from the Gremlin-1-Ab7326Fab complex.

[0134] Therefore, the antibody used in the present invention can bind to an epitope comprising at least one residue selected from the group consisting of Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175 (residue numbering based on SEQ ID NO: 1). The antibody used in the present invention can bind to an epitope comprising 2, 3, 4, 5, 6, 7, 8, or all 9 residues (preferably, at least 5 residues) of these residues.

[0135] The antibodies for use in the present invention may also recognize an epitope in which Ile131 is present on the Gremlin-1 monomer differently from other residues.

[0136] Although these residues are provided for a specific sequence of human Gremlin-1, the skilled person can extrapolate the positions of these residues to other corresponding Gremlin sequences using conventional techniques. Therefore, antibodies that bind to epitopes containing corresponding residues within these other Gremlin sequences are also provided for use in the present invention.

[0137] To screen for antibodies that bind to a specific epitope, a conventional cross-blocking assay, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scanning mutants, peptide blotting (Reineke (2004) Methods Mol Biol 248: 443-63) or peptide cleavage analysis. In addition, methods 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.

[0138] Antibody epitopes can also be determined by x-ray crystallographic analysis. Thus, antibodies for use in the present invention can be evaluated by x-ray crystallographic analysis of antibodies that bind to Gremlin-1. In particular, the epitopes of antibodies can be determined in this manner by determining the residues in the paratope. The epitope was identified by targeting residues on Gremlin-1 within 1:1.

[0139] The antibodies can be tested for binding to Gremlin-1 by, for example, standard ELISA or Western blotting. ELISA assays can also be used to screen hybridomas that show positive reactivity with the target protein. The binding selectivity of the antibody can also be determined by monitoring the binding of the antibody to cells expressing the target protein. Thus, the screening method can include the step of identifying antibodies capable of binding to Gremlin-1 by performing ELISA or Western blotting or by flow cytometry.

[0140] The antibody 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 is selective, but does not substantially bind or binds with low affinity to other proteins. The selectivity of an antibody can be further studied by determining whether the antibody binds to other related proteins discussed above, or whether it discriminates between them. Antibodies for use in the present invention typically recognize human Gremlin-1.

[0141] The antibodies may also have cross-reactivity to related proteins, or to human Gremlin-1 and to Gremlin-1 from other species.

[0142] By "specific (or selective)", it will 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 herein. The cross-reactivity of an antibody can be considered significant if the antibody binds to other molecules with at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% of the strength with which it binds to the protein of interest. A specific (or selective) antibody can bind to another molecule with less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% of the strength with which it binds to the protein of interest. The antibody may bind to other molecules with 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% of the strength with which it binds to the protein of interest.

[0143] Therefore, antibodies suitable for use in the present invention may have high affinity binding to (human) Gremlin-1. The antibodies may have a dissociation constant (K) of <1 nM, preferably <500 pM. D In one example, the antibody has a dissociation constant (K) of less than 200 pM. D In one example, the antibody has a dissociation constant (K) of less than 100 pM. D A variety of methods can be used to determine the binding affinity of an antibody for its target antigen, such as surface plasmon resonance assays, saturation assays, or immunoassays such as ELISA or RIA, as are well known to those skilled in the art. An exemplary method for determining binding affinity is to use a BIAcore TM The samples were analyzed by surface plasmon resonance on a Biacore 2000 instrument (Biacore AB, Freiburg, Germany) using a CM5 sensor chip as described by Krinner et al. (2007) Mol. Immunol. Feb, 44(5):916-25 (Epub 2006 May 11).

[0144] The anti-Gremlin-1 antibody of the embodiment, Ab7326, is an allosteric inhibitor of Gremlin-1 activity that binds to an epitope away from the BMP binding site (WO 2018 / 115017A2). Ab7326 binds to Gremlin-1 with exceptionally high affinity (Kd value <100 pM), and is expected to be particularly useful for use in the present invention.

[0145] Gremlin-1 activity inhibitors may have an effect on any function of Gremlin-1, but typically reduce the binding of Gremlin-1 to BMPs (BMP 2, 4, and / or 7). Gremlin-1 is a negative regulator of BMPs, and thus reduced binding increases signaling through BMPs.

[0146] BMP binding and signaling can be detected by any method known in the art. The examples of the present application describe two functional assays for testing whether an agent reduces the binding of gremlin-1 to BMP. Example 3 describes an Id1 reporter gene assay, in which the Id1 gene is a target gene for BMP signaling. The increase in signal in this assay can be used to determine whether an agent reduces the binding of Gremlin-1 to BMP. Example 5 describes a SMAD phosphorylation assay. SMAD1, 5, and 8 are phosphorylated after BMP signaling. Therefore, an increase in SMAD phosphorylation can be used to determine whether an agent reduces the binding of Gremlin-1 to BMP.

[0147] 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. The gene encoding the antibody can be cloned using degenerate primers. The antibody can be recombinantly produced by conventional methods.

[0148] Examples of DNA sequences encoding the full-length heavy and light chains of Ab7326 are provided in the Sequence Listing:

[0149] SEQ ID NO: 24 (human IgG1 heavy chain DNA variant 1),

[0150] SEQ ID NO: 25 (human IgG1 light chain DNA variant 1),

[0151] SEQ ID NO: 26 (human IgG4P heavy chain DNA variant 1),

[0152] • SEQ ID NO: 27 (human IgG4 P light chain DNA variant 1).

[0153] Pharmaceutical compositions, dosages and dosing regimens

[0154] The gremlin-1 activity inhibitor for use in the present invention may be provided in a pharmaceutical composition. The pharmaceutical composition will generally be sterile and will typically be contained in a pharmaceutically acceptable carrier and / or adjuvant. The pharmaceutical composition for use in the present invention may additionally contain a pharmaceutically acceptable adjuvant and / or carrier.

[0155] The pharmaceutical composition used in the present invention may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the parent molecule and do not impart any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0156] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier may be suitable for parenteral administration, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral administration, such as by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration, such as topical, epidermal or mucosal administration. The carrier may be suitable for oral administration. Depending on the route of administration, the inhibitor may be coated in a material to protect it from the effects of acid and other natural conditions that may inactivate the inhibitor.

[0157] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions used in the present invention include water, buffered water, and saline. Examples of other carriers include ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it will be desirable to include isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride in the composition.

[0158] Typically, pharmaceutical compositions must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0159] The pharmaceutical compositions for use in the present invention may contain additional active ingredients.

[0160] Also contemplated are kits comprising an inhibitor of gremlin-1 activity and instructions for use in a therapeutic method according to the invention.

[0161] The agents for use in the present invention, or their preparations or compositions, may be administered for therapeutic and / or prophylactic treatment.

[0162] In therapeutic applications, an agent is administered to a subject already suffering from a disease or condition in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of symptoms, or an increase in the frequency or duration of symptom-free periods. An amount sufficient to accomplish this is defined as a "therapeutically effective amount."

[0163] In preventive applications, an agent is administered to a subject at risk for a disorder or condition in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount sufficient to accomplish this is defined as a "prophylactically effective amount." The effective amount for each purpose will depend on the severity of the disease or injury and the weight and general state of the subject.

[0164] The subject for administration can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, dogs, cats, horses, sheep, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0165] The reagent or pharmaceutical composition used in the present invention can be administered by one or more routes of administration using one or more various methods known in the art. As the technician will appreciate, the route and / or mode of administration will vary depending on the desired result. The example of the route of administration for the reagent or pharmaceutical composition used in the present invention includes parenteral route, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous or spinal route of administration, such as by injection or infusion. Alternatively, the reagent or pharmaceutical composition can be administered by non-parenteral route (such as topical, epidermal or mucosal route of administration). The reagent or pharmaceutical composition can be used for oral administration.

[0166] The appropriate dosage of the inhibitory agent or pharmaceutical composition for use in the present invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition for use in the present invention can be varied so as to obtain an amount of the active ingredient that is effective for achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0167] Suitable dosages can be, for example, about 0.01 μg / kg to about 1000 mg / kg of the body weight of the patient to be treated, typically about 0.1 μg / kg to about 100 mg / kg body weight. For example, suitable dosages can be about 1 μg / kg to about 10 mg / kg body weight / day or about 10 μg / kg to about 5 mg / kg body weight / day.

[0168] The dosing regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the therapeutic situation. "Dosage unit form" as used herein refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined amount of active agent (calculated to produce the desired therapeutic effect) in association with the required pharmaceutical carrier.

[0169] Administration may be in a single dose or multiple doses. Multiple doses may be administered by the same or different routes and to the same or different locations. Alternatively, the dose may be by sustained release formulation, in which case less frequent administration is required. The dosage and frequency may vary depending on the half-life of the inhibitory agent in the patient and the desired duration of treatment.

[0170] The reagent, preparation or pharmaceutical composition used in the present invention can be co-administered with one or more other therapeutic agents. The combined administration of two or more reagents can be achieved in many different ways. Both can be administered together in a single composition, or they can be administered as a part of a conjoint therapy in a separate composition. For example, one can be administered before, after or simultaneously with another.

[0171] Treatment Indications

[0172] Gremlin-1 activity inhibitors according to the invention are provided for use in treating bone fractures or bone defects. A fracture is a break or break in bone tissue and can be the result of a traumatic injury (e.g. a fall or collision), but can also occur as a result of a disease affecting bone integrity. A bone defect is bone loss caused by trauma or disease.

[0173] The fracture may be a fracture of any bone in the body.

[0174] The bone defect may be a bone defect in any bone of the body.

[0175] In one embodiment, the fracture is a delayed union or nonunion fracture. A delayed union fracture is defined as a fracture that fails to achieve union within 6 months after fracture. A nonunion fracture is defined as incomplete union within 9 months, combined with radiographic features associated with fracture union not observed in three consecutive months. (Einhorn et al., 2014; Buza et al., 2016). Examples of fractures that tend to develop delayed union or nonunion include the tibia, distal radius, femoral neck, and scaphoid.

[0176] In one embodiment, the fracture or bone defect occurs as a result of a disease that affects bone integrity. Examples of diseases that affect bone integrity include, but are not limited to, osteoporosis, osteogenesis imperfecta, diabetes, Paget's disease of bone, rheumatoid arthritis, ankylosing spondylitis, multiple myeloma, primary bone cancer (e.g., osteosarcoma, Ewing's sarcoma, and chondrosarcoma), cancer that metastasizes to bone (e.g., breast cancer, prostate cancer, and lung cancer), diffuse idiopathic skeletal hyperostosis, osteomyelitis, renal disease, Duchenne muscular dystrophy, and thalassemia major.

[0177] Attached photos

[0178] Figure 1 Shown are the percentages of signal recovery in the HEK-ID1 reporter gene assay for antibodies derived from immunization.

[0179] Figure 2 The percentage of signal recovery in the HEK-ID1 reporter gene assay for antibodies derived from the library is shown.

[0180] Figure 3 Results are shown for a HEK-ID1 reporter gene assay with human Gremlin ( Figure 3 A) and mouse Gremlin ( Figure 3 B) Titration of and the effect of antibody 7326 (shown as antibody PB376) in restoring BMP signaling.

[0181] Figure 4 A structural model of the Gremlin-Fab complex is shown, with the possible BMP-binding regions and Fab epitopes highlighted.

[0182] Figure 5 Examination of the area without callus / bone tissue during fracture repair in the acquired X-ray images is shown. The area without tissue within the defect was quantified by using definens image analysis and then compared between the control and anti-gremlin 1 treated groups. The results are presented as mean ± SD of 10 rats / group. *P < 0.05; **P < 0.01; ***P < 0.001, as measured by Mann-Whitney U test.

[0183] Figure 6 Examination of LMB (low mineral bone; newly formed bone) and HMB (high mineral bone; mature bone) within a 3 mm femoral defect is shown. Panel A: 3D μCt analysis of the femoral defect area to detect newly formed bone or mature bone. The percentage of bone volume / tissue volume was measured and all subjects (total) were compared between control and treatment with anti-gremlin 1. Comparisons between control and anti-gremlin 1 treated groups in animals separated into low responders (LR-incomplete bridging) and high responders (HR-complete bridging) were also performed. Results are presented as mean ± SD. *P<0.05; **P<0.01; ***P<0.001, as measured by Mann-Whitney U test. Panel B: Representative μCt illustrating 3D bone volume reappearance in LR and HR groups in control and after anti-gremlin 1 treatment.

[0184] Figure 7 Histomorphometry analysis of femoral defects is shown. The percentage of bone volume / tissue volume (BV / TV (%)), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were compared between the control and anti-gremlin 1 treated groups. The results are presented as mean ± SD of 10 rats / group. *P < 0.05; **P < 0.01; ***P < 0.001, as measured by Mann-Whitney U test.

[0185] Figure 8 Correlation of 3DμCT analysis and 2D histomorphometry analysis of total BV / TV% is shown. LMB (low mineral bone; newly formed bone) and HMB (high mineral bone; mature bone) within the 3mm femoral defect were correlated in both groups and compared with 2D histomorphometry score data (n=20). Pearson scores indicated a significant correlation of BV / TV% between 3DμCT analysis and 2D histomorphometry analysis.

[0186] The following examples illustrate the invention. Example

[0187] Example 1 - Protein expression, purification, refolding and structure determination

[0188] Protein expression and inclusion body preparation

[0189] The truncated human Gremlin-1 coding sequence (SEQ ID NO: 20), which was optimized for expression in E. coli, was cloned into a modified pET32a vector (Merck Millipore) using BamHI / XhoI to generate a vector encoding the Gremlin sequence with an N-terminal 6His-TEV tag (pET-hGremlin1).

[0190] The expressed sequence:

[0191] MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD; SEQ ID NO: 2 (wherein the non-Gremlin residues of the 6His-TEV tag are shown in italics). The sequence numbering is based on UniProtO60565 and SEQ ID NO: 1.

[0192] The pET-hGremlin1 plasmid DNA was used to transform BL21(DE3) cells. A single ampicillin-resistant colony was picked from the LB / Amp agar plate and used to inoculate 100 ml of LB / Amp starter culture. After shaking (200 rpm) at 37°C for 16 hours, 25 ml of the starter culture was used to inoculate 500 mL of 2xTY / Amp medium. The culture was shaken (250 rpm) at 37°C until an OD of 3 was obtained. 600 Subsequently, the culture was supplemented with 20 mL of MOPS + glycerol feed mixture (1 M MOPS pH 7.4, 40% glycerol, 0.5% MgSO4, 0.42% MgCl2), induced with 300 μM IPTG, and further incubated at 17°C, 180 rpm for 16 hours. The cells were harvested in a centrifuge (4,000 g for 20 minutes at 4°C).

[0193] The cell pellet was resuspended in lysis buffer (PBS pH 7.4, 0.35 mg / ml lysozyme, 10 μg / ml DNase, and 3 mM MgCl2) at 4°C and the insoluble fraction was harvested by centrifugation at 3,500 g for 30 minutes at 4°C. The pelleted inclusion bodies were washed three times by resuspending 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 minutes. Two additional washes were performed using wash buffer without Triton X-100.

[0194] Solubilization

[0195] The inclusion bodies were resuspended in denaturation buffer (8 M urea, 100 mM Tris, 1 mM EDTA, 10 mM Na2S4O6, and 100 mM Na2SO3, pH 8.5), stirred at room temperature for 16 h, and clarified by centrifugation at 21,000 g for 15 min.

[0196] Purification before refolding

[0197] The dissolved inclusion bodies were loaded onto a Sephacryl S-200 26 / 60 column (120 mL) equilibrated in 8 M urea, 50 mM MES, 200 mM NaCl, 1 mM EDTA (pH 6.0). The fractions containing Gremlin-1 protein were diluted with 6 M urea, 20 mM MES (pH 6.0) and loaded onto a HiTrap SP HP cation exchange column and eluted with a 1 M NaCl gradient in 10 column volumes (10 CV). The fractions containing purified, denatured hGremlin-1 protein were pooled.

[0198] Refolding

[0199] Denatured purified Gremlin-1 protein was added dropwise to a 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 for 5 days at 4° C. with continuous stirring. After 5 days, Gremlin-1 protein was dialyzed against 20 mM HEPES, 100 mM NaCl (pH 7.5).

[0200] After dialysis, the protein was applied to a heparin HiTrap column and eluted using a gradient of 0-100% heparin elution buffer (20 mM HEPES, 1 M NaCl, pH 7.5) over 20 CV. Correctly folded protein eluted at 1 M NaCl, while any misfolded protein eluted at lower salt concentrations.

[0201] The protein eluted at 1 M NaCl was concentrated and further purified on a S75 26 / 60 column equilibrated with 20 mM Hepes, pH 7.5, 1 M NaCl.

[0202] The protein was characterized by SDS PAGE (migrated in the gel), was shown to have the expected molecular weight and correct disulfide bond arrangement (using liquid chromatography-mass spectrometry (LC-MS)), and was shown to be active in a cellular assay (ID1 reporter gene assay).

[0203] Gremlin 1 Structure determination

[0204] Gremlin 1 protein crystals were grown using the hanging drop method by mixing a 6.6 mg / ml Gremlin 1 solution with 0.1 M citric acid (pH 4), 1 M lithium chloride, and 27% polyethylene glycol (PEG) 6000 in a 1:1 ratio. Crystals were cryoprotected by adding 20% ​​glycerol to the crystallization buffer prior to data collection. Diffraction data were collected at Diamond Light Source and processed using XDS (Kabsch, Wolfgang (2010) Acta Crystallographica Section D 66, 125-132). Diffraction data statistics are summarized in the table below.

[0205] Table 2 :Diffraction data statistics

[0206]

[0207]

[0208] *The values ​​in brackets correspond to the highest resolution shell

[0209] **rmsd root mean square deviation

[0210] The Gremlin-1 structure was solved by molecular replacement using Phaser (McCoy et al., J Appl Cryst (2007), 40, 658-674) and a Gremlin-1 model available from proprietary Gremlin-1 / Fab complex coordinates. The resulting Gremlin-1 model contains four copies of Gremlin 1 monomer organized as two dimers. Model correction was performed with Coot (Emsley et al., Acta Crystallographica Section D: Biological Crystallography 66 (4), 486-501), and the coordinates were refined by using Refmac (Murshudov et al., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallographica Section D: Biological Crystallography. 2011, 67 (Pt 4): 355-367). The final coordinates were validated using Molprobity (Chen et al., (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica D66: 12-21). A summary of the model refinement statistics is shown in Table 2 above.

[0211] Example 2 - BMP binding residues on Gremlin-1

[0212] As discussed above, Gremlin-1 belongs to a family of bone morphogenetic protein (BMP) antagonist proteins within a subgroup (called the DAN family). Within the DAN family, Gremlin-1 shares the greatest homology with Gremlin-2 (PRDC).

[0213] In Example 1, The human Gremlin-1 structure shares many features with the published mouse Gremlin-2 structure (Nolan et al., (2013), Structure, 21, 1417-1429). The overall fold is very similar, with two copies of Gremlin-1 forming an antiparallel non-covalent dimer arranged in an arch. Each monomer adopts a characteristic finger-wrist-finger arrangement, with the cystine knot motif toward the end of the wrist, opposite the finger. The sequence identity between the proteins is 52%, rising to 67% within the sequences visible in the two structures. The most highly conserved regions are located in the extensive dimer interface, where all key contact residues are 100% conserved.

[0214] Residues involved in BMP2, 4, and 7 binding to mouse Gremlin-2 (PRDC) and DAN (NBL1) have been identified by 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 patch spanning both monomers on the convex surface of the dimer. Six residues were identified by mutagenesis: Trp72, Phe96, Tyr98, Phe104, Tyr105, and Phe117, and they are 100% conserved in human Gremlin-1 (numbering is based on the mouse Gremlin-2 sequence). The degree of homology extends to the arrangement of the side chains, which adopt the same conformation in the two proteins.

[0215] The amino acid numbering used in the Gremlin PDB file matches that in the published mouse Gremlin-2 structure, based on a structural alignment. This enables like-for-like comparisons of amino acids when describing structures. However, for clarity, the key residues identified as playing a role in BMP binding are shown below with numbers based on the PDB file and the UniProt file for SEQ ID NO: 1 (in parentheses): Trp72 (93), Phe96 (117), Tyr98 (119), Phe104 (125), Tyr105 (126), and Phe117 (138).

[0216] In both mouse Gremlin-2 and human Gremlin-1, the hydrophobic BMP binding epitope is partially hidden by an alpha helix formed by the N-terminal residues of each protein. A model for BMP binding has been proposed according to which the N-terminus can bend, exposing the complete BMP binding interface (Nolan et al., (2013), Structure, 21, 1417-1429). In this analysis, the N-terminal residues were removed from the human Gremlin-1 and mouse Gremlin-2 structures before resurfacing to reveal the similarities of the BMP binding surface on each protein.

[0217] The literature describes mutagenesis of only six residues that have an effect on BMP binding. It is likely that the actual BMP epitope covers a larger surface area, including adjacent amino acids. By highlighting the mutated residues on the surface of Gremlin-1, All residues within , revealing a larger region of Gremlin-1 that can potentially be targeted by therapeutic agents. This broader region includes the following amino acids of human Gremlin-1:

[0218] Asp92-Leu99

[0219] Arg116-His130

[0220] Ser137-Ser142

[0221] Cys176-Cys178

[0222] (Numbering is based on SEQ ID NO: 1).

[0223] By combining the published information with the crystal structure information of human Gremlin-1, regions of human Gremlin-1 were identified that offer themselves as potential avenues for therapeutic intervention to block its interaction with BMPs.

[0224] Example 3 - Hek Id1 reporter gene assay

[0225] background

[0226] Hek Id1 reporter gene assay uses clone 12Hek293-Id1 reporter cells. This cell line is stably transfected with Id1 transcription factor. Id1 is a transcription factor in the BMP signal transduction pathway. Known Gremlin binds BMP, preventing the binding to its receptor, thereby reducing the luciferase signal from this reporter gene. Therefore, using this reporter gene assay, it is possible to screen anti-Gremlin antibodies and see if there is any antibody that blocks the interaction of Gremlin and BMP. If there is this interactive blocking, then the recovery of luciferase signal is seen in these cells.

[0227] method

[0228] Clone 12 cells were cultured in DMEM containing 10% FCS, 1x L-glutamine and 1x NEAA. Cells were also grown in the presence of hygromycin B (200 μg / ml) to ensure that cells did not lose Id1 gene expression. Cells were assayed in DMEM containing 0.5% FCS, 1x L-glutamine and 1x NEAA. Hygromycin B was not required for the short time that cells were in the assay.

[0229] The cells were washed in PBS, lifted with cell dissociation buffer, spun down, and counted, and then counted at 5 × 10 4 / well inoculated in 70 μl (7.14×10 5 The plates used were white, opaque, poly-D-lysine coated 96-well sterile plates. The cells were allowed to settle in the incubator for approximately 3-4 hours. The BMP heterodimers were reconstituted to 200 μg / ml in 4 mM HCl. BMP was diluted to 10 μg / ml in assay medium using a glass vial to give a new working stock.

[0230] Gremlin-1 was diluted 1:2 in polypropylene plates for an 8-point dose response curve with a highest final dose of 1 μg / ml.

[0231] An additional volume of 20 μl of medium was added per well, and the plates were incubated at 37°C for 45 minutes.

[0232] BMP prepared at 100x was added to all wells except wells containing cells only. All wells were made up to 60 μl with assay medium and incubated at 37°C for a further 45 minutes.

[0233] After incubation, 30 μl of sample was transferred to each well of the assay plate and incubated for 20-24 hours before measuring the fluorescence signal.

[0234] Thaw Cell Steady Glo at room temperature beforehand. Allow the assay plate to cool to room temperature for approximately 10-15 minutes before adding the reagent. Detect luciferase signal by adding Cell Steady Glo reagent (100 μl) at room temperature on a shaker for 20 minutes and measure luminescence on a Synergy 2 using the cell titre glo protocol.

[0235] The greatest signal was generated from wells containing BMP, and the smallest signal was generated from wells containing cells alone.

[0236] result

[0237] Gremlin-1 full-length and truncated forms were tested in the Hek-Id1 reporter gene assay to confirm blocking activity against BMP4 / 7.

[0238] The percent inhibition from the dose response assay was calculated based on the maximum and minimum signals in the assay, and the data were fit using a 4-parameter logistic fit. The IC was calculated based on the inflection point of the curve. 50 .

[0239] Table 3 : Efficacy results for full-length Gremlin-1 and truncated Gremlin-1 in the Hek-Id1 reporter gene assay

[0240]

[0241] in conclusion

[0242] Gremlin 1 was able to inhibit BMP 4 / 7 signaling in a Hek-Id1 reporter gene assay.

[0243] Example 4 - Generation of anti-Gremlin-1 antibodies

[0244] Anti-Gremlin-1 antibodies were obtained by immunization with purified gremlin-1 and by library panning as described in Example 1. The library was generated in-house as a naive human library with V-regions amplified from blood donations.

[0245] From the first round of immunization, immunization generated 26 different antibodies that bound Gremlin-1. These antibodies were expanded and purified for testing in screening assays.

[0246] 25 human and mouse cross-reactive antibodies were panned from this library using recombinant human Gremlin from R&D Systems. 10 antibodies were selected for expansion and purification (as scFv) for testing in screening assays.

[0247] Example 5 - Screening of anti-Gremlin-1 antibodies

[0248] The antibodies were screened using the Hek-Id1 reporter gene assay described in Example 3 and by measuring SMAD phosphorylation. Following BMP signaling, SMAD 1, 5, and 8 are phosphorylated. Thus, inhibitors of Gremlin-1 increase SMAD phosphorylation.

[0249] SMAD phosphorylation assay was performed on A549 cells or on human lung fibroblasts. Phosphorylation levels were determined using MSD.

[0250] result

[0251] In the Hek-Id1 reporter gene assay, there were no significant hits with the antibodies derived from immunization (antibodies tested were in 10-fold excess for BMP4 / 7 heterodimers). The results are shown in Figure 1 middle.

[0252] In contrast, many of the library-derived antibodies were able to restore signal in the Hek-Id1 reporter assay (50-fold excess of antibody with 50% gremlin dose) ( Figure 2 ). Among these, Ab2416 and Ab2417 contained high levels of endotoxin. Ab7326 maintained blocking ability at a 10-fold excess and 80% inhibition of Gremlin-1 concentration.

[0253] exist Figure 3 Additional results are presented in A (human gremlin) and 3B (mouse gremlin). These figures show titers of Ab7326 (labeled PB376) up to 15 nM. When compared to human (IC of 1.3 nM) 50 ) or mice (0.2 nM Gremlin IC 50 ) was blocked by Ab7326, which was shown to restore BMP signaling. The antibody worked as both human and mouse IgG1.

[0254] The sequences of mouse and human full length IgG1 are presented below.To synthesize mouse and human full length IgG1 proteins, the variable regions of Ab7326 derived from the library were recloned into vectors containing the appropriate antibody constant domains.

[0255] Because Ab7326 comes from a naive human library (where Ab is cloned as scFv), in order to re-clone the 7326 variable regions as full-length Ab or Fab, a pool of primers / degenerate primers is required to PCR amplify VH and VK. The amplified PCR products are then digested and cloned into mouse and human vectors simultaneously. Since VH and VK are amplified by a pool of primers / degenerate primers, two variant forms of the product are obtained, the difference being a single amino acid residue from slightly different primers that anneal during the PCR process.

[0256] The two variant forms of the heavy chain variable region differ by a single amino acid at position 6, and the two variant forms of the light chain variable region differ by a single amino acid at position 7, as shown below:

[0257] Heavy chain variable region variant 1 has glutamic acid (E) at position 6,

[0258] Heavy chain variable region variant 2 has glutamine (Q) at position 6,

[0259] Light chain variable region variant 1 has a serine (S) at position 7,

[0260] • Light chain variable region variant 2 has a threonine (T) at position 7.

[0261] Mouse full length IgG1 - heavy chain variant 1 (SEQ ID NO: 14)

[0262]

[0263] Mouse full length IgG1 - light chain variant 1 (SEQ ID NO: 15)

[0264] Mouse full length IgG1 - heavy chain variant 2 (SEQ ID NO: 28)

[0265]

[0266] Mouse full length IgG1 - light chain variant 2 (SEQ ID NO: 29)

[0267]

[0268] Human full length IgG1 - heavy chain variant 1 (SEQ ID NO: 30)

[0269]

[0270] Human full length IgG1 - light chain variant 1 (SEQ ID NO: 31)

[0271]

[0272] Human full length IgG1 - heavy chain variant 2 (SEQ ID NO: 16)

[0273]

[0274] Human full length IgG1 - light chain variant 2 (SEQ ID NO: 17)

[0275]

[0276] Antibody CDRs were determined using the Kabat method (highlighted in bold in the sequence above). Additional HCDR1 residues using the Chothia definition are italicized. Constant region sequences are underlined.

[0277] Restoration of p-SMAD signaling with anti-Gremlin 1 antibodies is shown in Table 4 below.

[0278] Table 4 : Restoration of p-SMAD signaling

[0279]

[0280] Results are shown as the percentage of SMAD phosphorylation relative to BMP alone (control BMP). Experiments were performed using lung fibroblasts from patients with idiopathic pulmonary fibrosis. rhGremlin-1 and anti-Gremlin-1 antibodies were pre-incubated at room temperature for 45 minutes. Then, rhGremlin-1 and anti-Gremlin-1 antibodies were added to the cells together with BMP for 30 minutes.

[0281] Then, Table 5 shows further results in the SMAD phosphorylation assay, in which the use of anti-Gremlin-1 antibodies to replace BMP-2 or BMP4 / 7 from the Gremlin 1-BMP complex was explored. The experiment was repeated using lung fibroblasts from patients with idiopathic pulmonary fibrosis. rhBMP-2 or rhBMP 4 / 7 were pre-incubated with rhGremlin-1 for 1 hour at room temperature. BMP-2-Gremlin-1 or BMP4 / 7-Gremlin-1 complexes were incubated overnight at 4°C with different concentrations of anti-Gremlin-1 antibodies. The antibody concentrations represent the final concentrations on the plate.

[0282] Table 5 : Using anti-Gremlin-1 antibody to replace BMP-2 or BMP4 / 7 from the Gremlin 1-BMP complex

[0283]

[0284] The results shown in Table 5 demonstrate that Ab7326 can displace already complexed BMP-2 or BMP4 / 7 from the Gremlin1-BMP complex. Ab7326 is able to achieve this displacement at a much lower concentration than the comparative antibody 2484. This provides evidence that Ab7326 is an allosteric inhibitor, which is consistent with our findings that the binding site for Ab7326 is far from the known BMP binding region on gremlin-1. Thus, Ab7326 is able to access the allosteric binding site even when BMP is complexed with gremlin-1, resulting in significantly improved inhibition of gremlin activity.

[0285] Example 6 - Obtaining the Crystal Structure of Gremlin-1 in Complex with 7326 Fab

[0286] by The crystal structure of human Gremlin-1 in complex with Ab7326 Fab was solved at a resolution of 1.3 Å. The Fab sequence is shown below:

[0287] Heavy chain: SEQ ID NO: 18

[0288] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0289] Light chain: SEQ ID NO: 19

[0290] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0291] Then, CCP4 software NCONT was used to identify the interaction between Gremlin-1 and Fab. The following residues were identified: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175 (numbering based on the UniProt sequence of SEQ ID NO: 1) (numbered Ile110, Lys126, Lys127, Phe128, Thr129, Thr130, Arg148, Lys153, and Gln154 in the structure file matching the numbering of mouse Gremlin-2).

[0292] Figure 4 A structural model of the Gremlin-Fab complex is shown, with the Fab epitope residues indicated relative to the BMP binding region.

[0293] Ab7326 is an inhibitory antibody that acts allosterically, ie, it binds away from the BMP binding region.

[0294] Example 7 - Affinity Measurement of Anti-Gremlin-1 Antibody Ab7326 Binding to Gremlin-1

[0295] method

[0296] The affinity of anti-Gremlin mIgG for human Gremlin 1 was determined by biomolecular interaction analysis using surface plasmon resonance (SPR) technology on a Biacore T200 system (GE Healthcare Bio-Sciences AB). Anti-Gremlin mIgG was captured by an immobilized anti-mouse-Fc surface, and Gremlin 1 was titrated on the captured mIgG.

[0297] The capture ligand (affinity purified F(ab')2 fragment of goat anti-mouse IgG, Fc fragment specific, 115-006-071, Jackson ImmunoResearch Inc.) was immobilized on flow cell 2 of a CM4 sensor chip to a level of 1600 response units (RU) at 50 μg / ml in 10 mM NaAc (pH 5.0) using 600 second activation and passivation injections. HBS-EP+ buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the running buffer at a flow rate of 10 μl / min. A reference surface was prepared on flow cell 1 by activating and passivating the surface as for flow cell 2, but omitting the capture ligand.

[0298] The assay buffer was HBS-EP + plus an additional 150 mM NaCl to give a final NaCl concentration of 300 mM plus 1% CMD40. A 60 second injection of anti-Gremlin mIgG (at 5 μg / ml in running buffer) was passed through flow cells 1 and 2 to give a capture level of approximately 100 RU on the immobilized anti-mouse IgG, Fc surface. Recombinant human Gremlin 1 was titrated in running buffer starting from 5 nM (using a 2-fold dilution) and injected through flow cells 1 and 2 for 3 minutes at a flow rate of 30 μl / min, followed by a 5 minute dissociation period. A buffer-only control was also included. The surface was regenerated at a flow rate of 10 μl / min by a 60 second injection of 50 mM HCl, a 30 second injection of 5 mM NaOH, and a 30 second injection of 50 mM HCl.

[0299] Kinetic data were determined using Biacore T200 evaluation software.

[0300] Affinity measurements were performed at 25°C.

[0301] result

[0302] The binding affinity (considered as the average K for 5 determinations) was found D value) is below 100pM.

[0303] Example 8 - Inhibition of gremlin-1 activity accelerates healing and bridging in an in vivo model of fracture repair

[0304] Materials and methods

[0305] Rat fracture model and drug administration

[0306] The long bone segmental defect model has been widely used in the study of bone healing and regeneration (Sato et al., 2014). In this study, a 3mm femoral defect was produced in 10-week-old male rats, and it was stabilized by using an 8-hole PEEK plate (RIS.602.105, RISystem, Switzerland). Before the bone was drilled and fixed with screws, the plate was fixed to the bone with pliers in the middle of the diaphysis. A 3mm fracture gap was produced using a 0.44mm Gillette wire saw. Quality control of defect size / consistency / fixation was performed using Faxitron (MX-20-DC5, Faxitron Bioptics LLC, USA) by X-ray imaging, and this time point was defined as day 0.

[0307] Weekly dosing began on Day 1 and continued for a period of 8 weeks as outlined in Table 1.

[0308] Subsequently, during the in vivo phase of the study, X-ray images were obtained on days 11, 25, 39, and 57 to assess callus formation and healing progress.

[0309] Defined image analysis was used to quantify the area of ​​the defect devoid of bone tissue in the captured X-ray images.

[0310] Table 6 : Treatment group

[0311]

[0312] Micro-CT analysis of fracture healing

[0313] The femur (fracture side) was scanned using micro-CT (SkyScan 1076) at 17.2 μm resolution. An area of ​​approximately 15 mm with the fractured callus in the center was acquired. The scan was reconstructed using Skyscan NRECON software (1.7.10), and the reconstructed slices were then further segmented to exclude the fixation nails at a 3 mm defined area calculated from the midpoint of the femoral fracture defect.

[0314] Histomorphometric analysis of fracture callus in 3D was performed by SkyScan software (v.1.13.1). The midpoint within the 3 mm femoral fracture defect was determined, and slices 1.5 mm distal and proximal to the midpoint were segmented for each limb measured. Subsequently, binarization and segmentation of the reconstructed dataset set were performed according to two defined thresholds (one depicting low mineralized callus (thus quantifying newly formed bone), and the other defining mature bone). Further segmentation of these data was performed for the femurs of animals that were classified as low responders (based on meeting the criteria of incomplete bridging of the femoral defect) or high responders (exhibiting bridging of the fracture site).

[0315] Histomorphometric analysis of fractures

[0316] Femurs were fixed in 10% neutral buffered formalin for 24 hours, dehydrated, and embedded in methyl methacrylate (MMA) at low temperatures. 50 μm thick sections were stained with toluidine blue to quantify the bone component of the healing cleft defects.

[0317] Histomorphometric parameters were measured for trabecular bone at the fracture defect site. Measurements were performed by image analysis.

[0318] Statistical analysis

[0319] Results are presented as mean ± SD. Statistical analysis was performed with GraphPad Prism software by using a two-tailed Mann Whitney U test unless otherwise stated.

[0320] Results

[0321] Analysis of X-ray images obtained during the in vivo phase of the study showed that anti-gremlin-1 antibodies accelerated fracture healing, with the control and treatment groups significantly different after 25 days (P<0.05). This effect was evident for the remainder of the study ( Figure 5 ).

[0322] Micro-CT analysis of periodic samples revealed that treatment with anti-gremlin-1 antibody (30 mg / kg / weekly) resulted in an increase in newly formed bone within the fracture callus site (P=0.06).

[0323] Without intervention, the incidence of nonunion fractures in this model is approximately 60% (Sato et al., 2014). To test whether gremlin-1 inhibition reduces the incidence of nonunion development, animals were classified as low responders (LR) and high responders (HR). Gremlin-1 inhibition resulted in a significant increase in the percentage of bone volume / tissue volume (BV / TV%) in the 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 controls, thus indicating a progressive repair of the group that may develop nonunions.

[0324] Additionally, there was a trend (non-significant) toward increased LMB and HMB BV / TV% in the high responder group in response to anti-gremlin-1 treatment ( Figure 6 A, Representative images of LR and HR are shown in Figure 6 B).

[0325] Two-dimensional histomorphometric analysis of bone parameters was performed on histological sections of the fracture site ( Figure 7 ). Treatment with anti-gremlin-1 antibody significantly increased the percentage of bone volume / tissue volume (BV / TV%) compared to control (P<0.05). Anti-gremlin-1 significantly increased the number of trabeculae (Tb.N) (P<0.001) and significantly decreased the degree of trabecular separation (Tb.Sp) (P<0.01), indicating increased trabecular bone due to treatment with anti-gremlin-1.

[0326] By comparing the LMB and HMB groups classified in μCT analysis and 2D histomorphometry analysis of fracture sections, a cross-correlation was performed between 2D histomorphometry analysis and 3D μCT analysis ( Figure 8). Comparison measured by Pearson correlation revealed positive and significant correlations between histomorphometry and μCT analysis in the LMB group (P<0.0001) and the HMB group (P<0.0001), thus validating the data from each dataset group.

[0327] 8.3. Conclusion

[0328] Inhibition of gremlin-1 activity using a neutralizing anti-gremlin-1 antibody resulted in accelerated fracture repair, with significant differences evident between the control and treatment groups after 25 days (3 doses of antibody). In addition, regular analysis of the fracture site demonstrated enhanced bone tissue formation in low responder animals that would otherwise have developed a nonunion. Thus, inhibition of gremlin-1 activity is a promising therapy for the prevention or treatment of nonunion fractures and may be of particular value for treating fractures that are prone to developing nonunions, such as the tibia, distal radius, femoral neck, and scaphoid.

[0329] Sequence Listing

[0330] SEQ ID NO: 1 (human Gremlin-1; Uniprot ID: O60565)

[0331] MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0332] SEQ ID NO: 2 (human truncated Gremlin-1 used in crystallography with an N-terminal tag)

[0333] MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRISIDLD

[0334] SEQ ID NO:3 (Ab7326 HCDR1, combined Kabat & Chothia)

[0335] GYTFTDYYMH

[0336] SEQ ID NO:4 (Ab7326 HCDR1, Kabat)

[0337] D Y

[0338] SEQ ID NO:5(Ab7326 HCDR2, Kabat)

[0339] LVDPEDGETIYAEKFQG

[0340] SEQ ID NO:6 (Ab7326 HCDR3, Kabat)

[0341] DARGSGSYYPNHFDY

[0342] SEQ ID NO:7 (Ab7326 LCDR1, Kabat)

[0343] KSSQSVLYSSNNKNYLA

[0344] SEQ ID NO:8 (Ab7326 LCDR2, Kabat)

[0345] WASTRES

[0346] SEQ ID NO:9 (Ab7326 LCDR3, Kabat)

[0347] QQYYDTPT

[0348] SEQ ID NO: 10 (Ab7326 heavy chain variable region variant 1)

[0349] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

[0350] SEQ ID NO: 11 (Ab7326 light chain variable region variant 1)

[0351] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

[0352] SEQ ID NO:12 (Ab7326 heavy chain variable region variant 2)

[0353] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

[0354] SEQ ID NO:13 (Ab7326 light chain variable region variant 2)

[0355] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

[0356] SEQ ID NO:14 (mouse full-length IgG1 heavy chain variant 1)

[0357] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0358] SEQ ID NO:15 (mouse full-length IgG1 light chain variant 1)

[0359] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0360] SEQ ID NO:16 (Human full-length IgG1 heavy chain variant 2)

[0361] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0362] SEQ ID NO:17 (Human full-length IgG1 light chain variant 2)

[0363] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0364] SEQ ID NO:18 (Fab heavy chain variant 1)

[0365] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0366] SEQ ID NO:19 (Fab light chain variant 1)

[0367] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0368] SEQ ID NO:20 (Human truncated Gremlin-1 used in crystallography without an N-terminal tag)

[0369] AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0370] SEQ ID NO:21 (Mature Gremlin-1 sequence of SEQ ID NO:1, lacking the signal peptide of amino acids 1-21)

[0371] KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0372] SEQ ID NO:22 (Human IgG4P heavy chain variant 1)

[0373] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0374] SEQ ID NO:23 (Human IgG4P light chain variant 1)

[0375] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0376] SEQ ID NO:24 (Human IgG1 heavy chain DNA variant 1)

[0377]

[0378] SEQ ID NO:25 (Human IgG1 light chain DNA variant 1)

[0379] gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

[0380] SEQ ID NO:26 (Human IgG4 P heavy chain DNA variant 1)

[0381]

[0382] SEQ ID NO:27 (Human IgG4 P light chain DNA variant 1)

[0383] gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

[0384] SEQ ID NO:28 (Mouse full-length IgG1 heavy chain variant 2)

[0385] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0386] SEQ ID NO:29 (Mouse full-length IgG1 light chain variant 2)

[0387] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0388] SEQ ID NO:30 (Human full-length IgG1 heavy chain variant 1)

[0389] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0390] SEQ ID NO:31 (Human full-length IgG1 light chain variant 1)

[0391] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0392] SEQ ID NO:32 (Fab heavy chain variant 2)

[0393] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0394] SEQ ID NO:33 (Fab light chain variant 2)

[0395] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0396] SEQ ID NO:34 (Human IgG4 P heavy chain variant 2)

[0397] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0398] SEQ ID NO:35 (Human IgG4 P light chain variant 2)

[0399] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0400] References

[0401] Bostrom,M.P.&Seigerman,D.A.(2005),HSS journal:The MusculoskeletalJournal of Hospital for Special Surgery 1,9-18.The clinical use ofallografts,demineralized bone matrices,synthetic bone graft substitutes andosteoinductive growth factors:a survey study.

[0402] Buza,J.A.,3rd&Einhorn,T.(2016),Clinical cases in mineral and bonemetabolism:The Official Journal of the Italian Society of Osteoporosis,Mineral Metabolism,and Skeletal Diseases 13,101-105.Bone healing in 2016.

[0403] Canalis,E.,Parker,K.&Zanotti,S.(2012),J.Cell Physiol 227,269-277.Gremlin1 is required for skeletal development and postnatal skeletalhomeostasis.

[0404] Cho,T.J.,Gerstenfeld,L.C.&Einhorn,T.A.(2002),Journal of Bone andMineral Research:The Official Journal of the American Society for Bone andMineral Research 17,513-520.Differential temporal expression of members ofthe transforming growth factor beta superfamily during murine fracturehealing.

[0405] Einhorn, T. A. & Gerstenfeld, L. C. (2015), Nat. Rev. Rheumatol. 11, 45 - 54. Fracture healing: mechanisms and interventions.

[0406] Ferguson, C., Alpern, E., Miclau, T. & Helms, J. A. (1999), Mechanisms of development 87, 57 - 66. Does adult fracture repair recapitulate embryonic skeletal formation?

[0407] Gazzerro, E. et al., (2005), Endocrinology 146, 655 - 665. Skeletal overexpression of gremlin impairs bone formation and causes osteopenia.

[0408] Gazzerro, E. et al., (2007), J. Biol. Chem. 282, 31549 - 31557. Conditional deletion of gremlin causes a transient increase in bone formation and bone mass.

[0409] Goulet, J. A., Senunas, L. E., DeSilva, G. L. & Greenfield, M. L. (1997), Clinical Orthopaedics and Related Research, 76 - 81. Autogenous iliac crest bone graft. Complications and functional assessment.

[0410] Hsu, DR, Economides, AN, Wang, X., Eimon, PM& Harland, RM(1998), Mol.Cell 1,673-683.

[0411] 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.

[0412] Schmid , GJ , Kobayashi , C , Sandell , LJ & Ornitz , DM ( 2009 ), Developmental Dynamics: An Official Journal of the American Academy of Pediatrics

[0413] Association of Anatomists 238,766-774.Fibroblast growth factor expression during skeletal fracture healing in mice.

[0414] Immunolocalization of BMPs, BMPantagonists, receptors, and effectors during fracture repair. Entertainment <110> UCB Biopharma SPRL <120> Remove the snowflake spell from the gremlin-1 spell <130> PF0117-WO-PCT <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 184 <212> PRT <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 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> PRT <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> PRT <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> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 4 Asp Tyr Tyr Met His 1 5 <210> 5 <211> 17 <212> PRT <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> PRT <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> PRT <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> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 8 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Recombinant sequence <400> 9 Gln Gln Tyr Tyr Asp Thr Pro Thr 1 5 <210> 10 <211> 124 <212> PRT <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> PRT <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> PRT <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> PRT <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> PRT <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> PRT <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> PRT <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 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> PRT <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> PRT <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> PRT <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> PRT <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> PRT <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> PRT <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> PRT <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 gcccagagaa 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 attctccgga 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 cccaggaatc 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 cctggaactc tggcgccctg acctccggcg tgcacacctt ccctgccgtg 540 ctgcagtcct ccggcctgta ctccctgtcc tccgtcgtga ccgtgccctc ctccagcctg 600 ggcaccaaga cctacacctg taacgtggac cacaagccct ccaacaccaa ggtggacaag 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 aaagagtaca agtgcaaggt gtccaacaag ggcctgccct ccagcatcga aaagaccatc 1020 tccaaggcca agggccagcc ccgcgagccc caggtgtaca ccctgccccc tagccaggaa 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 attctccgga 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 cccaggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540 tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600 gtgacccacc agggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657 <210> 28 <211> 448 <212> PRT <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> PRT <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> PRT <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 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> PRT <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> PRT <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> PRT <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> PRT <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> PRT <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 210 215

Claims

1. Use of a gremlin-1 activity inhibitor in the preparation of a medicament for treating bone fractures or bone defects, wherein the inhibitor is an anti-gremlin-1 antibody or a functionally active fragment of the antibody, wherein the antibody or the functionally active fragment of the antibody comprises a sequence of SEQ ID NO: 3 or 4 as HCDR1, a sequence of SEQ ID NO: 5 as HCDR2, a sequence of SEQ ID NO: 6 as HCDR3, a sequence of SEQ ID NO: 7 as LCDR1, a sequence of SEQ ID NO: 8 as LCDR2, and a sequence of SEQ ID NO: 9 as LCDR3.

2. The use according to claim 1, wherein the functionally active fragment of the antibody is Fab, Fab', F(ab')2, Fv or scFv.

3. The use according to claim 1 or 2, wherein the antibody or functionally active fragment of 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. The method according to claim 1 or 2, wherein the antibody or a functionally active fragment of the antibody comprises: a heavy chain variable region comprising a sequence having at least 95% identity to the sequence of SEQ ID NO: 10, and a light chain variable region comprising a sequence having at least 95% identity to the sequence of SEQ ID NO:

11.

5. The use according to claim 1, wherein the bone fracture or bone defect is a delayed union fracture or a non-union fracture.

6. Use according to claim 1, wherein the bone fracture or bone defect is associated with a disease affecting bone integrity.

7. The method according to claim 6, wherein the disease affecting bone integrity is osteoporosis, osteogenesis imperfecta, diabetes, Paget's disease of bone, rheumatoid arthritis, ankylosing spondylitis, multiple myeloma, primary bone cancer, cancer metastasized to the bone, diffuse idiopathic skeletal hyperostosis, osteomyelitis, renal disease, Duchenne muscular dystrophy or thalassemia major.

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