EA043762B1 - CRYSTAL STRUCTURE OF GREMLIN-1 AND INHIBITORY ANTIBODY - Google Patents

Description

The invention relates to crystals of human Gremlin-1 protein and human Gremlin-1 protein in complex with an inhibitory antibody. The invention also relates to the structure of human Gremlin-1 (alone or in complex with an antibody) and to the use of these structures in screening agents that modulate the activity of Gremlin-1. The invention also relates to antibodies that bind to the allosteric inhibitory site on Gremlin-1, as well as pharmaceutical compositions and medical uses of such antibodies and agents identified by screening methods.

State of the art

Gremlin-1 (also known as Drm and CKTSF1B1) is a 184 amino acid glycoprotein that is a member of the DAN family of secreted cystine knot-containing proteins (in addition to Cerberus and Dan). In addition to its demonstrated proangiogenic function, Gremlin binds and inhibits the signaling ability of BMP-2, 4, and 7, possibly through agonism of VEGFR2. Gremlin-1 plays an important role in the developmental process in which it is vital, in the formation of kidneys and limb buds. Due to these vital functions, homozygous Gremlin knockout is embryonic lethal in mice.

In adulthood, elevated Gremlin levels are associated with idiopathic pulmonary fibrosis and pulmonary arterial hypertension, in which BMP-2, 4, and 7 signaling is reduced with increased TGF-β levels. In both diabetic and chronic allograft nephropathy, Gremlin-1 expression correlated with fibrosis scores.

Elevated levels of Gremlin are also associated with scleroderma, diabetic nephropathy, and colorectal cancer. Gremlin-1 has been shown to activate cancer cell invasion and proliferation and is thought to play a role in the development of sarcomas and carcinomas of the cervix, lung, ovary, kidney, breast, colon and pancreas.

To date, there are a number of problems associated with the study of Gremlin-1, while there is a lack of general understanding of the nature of Gremlin-1 (and its partner Gremlin-2). The biology of BMPs is complex and there is high homology between species. Gremlin-1 is a difficult protein to work with, and suitable tools and reagents are lacking to study its biology. Obtaining Gremlin-1 is also not a simple process; Proteins with cysteine knots are known to be difficult to produce, and the free cysteine in Gremlin-1 exacerbates this problem. Gremlin-1 is difficult to express, let alone purify. Until now, there was no information about the structure of this protein and there is very little information about this protein in the literature.

The essence of the invention

The term Gremlin-1 as used in the present invention generally refers to the sequence listed in the UniProt database as O60565 (SEQ ID NO:1). The term Gremlin-1 may also refer to the Gremlin-1 polypeptide, which:

(a) contains or consists of the amino acid sequence of SEQ ID NO:1 with or without an N-terminal signal peptide, i.e. may contain or consist of the mature peptide sequence of SEQ ID NO:21; or (b) is a derivative having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence of SEQ ID NO:1 with or without an N-terminal signal peptide (SEQ ID NO:21), while retaining the activity of Gremlin-1, such as the amino acid sequence of SEQ ID NO:20.

(c) is a variant thereof, such variants typically retaining at least about 60, 70, 80, 90, 91, 92, 93, 94, or 95% identity with SEQ ID NO:1 (or SEQ ID NO:20 or 21) (or even about 96, 97, 98 or 99% identity). In other words, such variants may retain from about 60 to about 99% identity to SEQ ID NO:1, preferably from about 80 to about 99% identity from SEQ ID NO:1, more preferably from about 90 to about 99% identity from SEQ ID NO:1 and most preferably from about 95 to about 99% identity with SEQ ID NO:1.

The options are described below.

As discussed below, residue numbers are generally indicated based on the sequence of SEQ ID NO:1. However, one skilled in the art can easily extrapolate the residue numbering to the derived sequence or variant identified above. If residue numbers are indicated, the invention also covers these residues in a variant or derivative sequence.

The authors of the present invention obtained crystals of human Gremlin-1 in crystalline form alone and in complex with an antibody called Ab7326 (Fab fragments). Crystallization of Gremlin-1 allowed the identification of putative residues in the BMP-binding site. In addition, crystallization with Ab 7326, which is an allosteric inhibitory antibody, allowed the identification of residues in the antibody epitope. Antibodies that bind this epitope may be effective as therapeutic agents for the treatment of diseases associated with Gremlin-1.

Accordingly, the present invention relates to a Gremlin-1 crystal.

The present invention also relates to the structure of human Gremlin-1, defined by the coordinates in table. 1.

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In addition, the invention relates to:

a machine-readable storage medium that contains data storage material encoded with machine-readable data defined by the Gremlin-1 structure coordinates in Table. 1 or coordinates defining homologues of the structure;

application of the Gremlin-1 structure, defined by the coordinates in table. 1, as a structural model;

agents identified using the structural model;

a method for screening agents that modulate Gremlin-1 activity, including the following steps:

(a) identification of the ligand-binding site from the structural coordinates in the table. 1;

(b) identifying candidate agents that interact with at least a portion of the ligand binding site; and (c) obtaining or synthesizing said agent;

an agent that modulates the activity of Gremlin-1, identified by screening;

an antibody that binds to an epitope on Gremlin-1 containing at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, wherein residue numbering is based on SEQ ID NO:1;

an anti-Gremlin-1 antibody that contains heavy chain complementarity determining region (HCDR) sequences contained in the heavy chain variable region (HCVR) of SEQ ID NO:10 or 12, and/or light chain complementarity determining region (LCDR) sequences contained in light chain variable region (LCVR) SEQ ID NO:11 or 13;

an anti-Gremlin-1 antibody that contains at least one HCDR sequence selected from SEQ ID NO: 3, 4, 5 and 6, and/or at least one LCDR sequence selected from SEQ ID NO: 7, 8 and 9 ;

an isolated polynucleotide encoding antibodies;

an expression vector carrying this polynucleotide;

a host cell containing the vector;

a method for producing an antibody, comprising culturing a host cell under conditions allowing the production of the antibody, and recovering the produced antibody;

a pharmaceutical composition containing an antibody;

an antibody or pharmaceutical composition for use in a method of therapeutic treatment of a human or animal body;

a method of treating or preventing renal fibrosis, such as diabetic nephropathy, idiopathic pulmonary fibrosis, pulmonary arterial hypertension, angiogenesis and/or cancer, comprising administering a therapeutically effective amount of an antibody or pharmaceutical composition to a patient in need thereof.

Brief description of drawings

In fig. 1 (Table 1) presents the structural data for the crystallography of Gremlin-1.

In fig. Figure 2 shows the structure of human Gremlin-1. Ribbons representing each monomer are shown in different shades of gray, fingers 1 and 2 (F1 and F2) are labeled along with the wrist (wrist) regions (w) and cystine knots (CK). Cysteines that form disulfide bonds are shown as black bars.

In fig. 3 shows a sequence alignment of human Gremlin-1 and mouse Gremlin-2 (PRDC). Residues marked with an asterisk are important for BMP binding, and residues forming key interface contacts in the dimer are marked with squares.

In fig. Figure 4 shows a surface rendering highlighting hydrophobic BMP-binding residues. The monomers are shown in two shades of gray, and the six key residues involved in BMP binding are shown in white.

In fig. Figure 5 shows an overlay of human Gremlin-1 and mouse Gremlin-2. The top image shows a ribbon with two proteins aligned. The bottom image details the amino acids involved in BMP binding in rod form (mouse Gremlin-2 shown in white and human Gremlin-1 in black).

In fig. 6A shows the inhibitory effects of full-length Gremlin-1 in an Id1 reporter gene activity assay in Hek cells.

In fig. 6B shows the inhibitory effects of truncated Gremlin-1 in an Id1 reporter gene activity assay in Hek cells.

In fig. 7 shows the percentage of signal recovery for immunization-derived antibodies in the Hek-Id1 reporter gene activity assay.

In fig. Figure 8 shows the percentage of signal recovery for library-derived antibodies in the Hek-Id1 reporter gene activity assay.

In fig. Figure 9 shows the results of the Hek-Id1 reporter gene activity assay with titers of human Gremlin (Fig. 9A) and mouse Gremlin (Fig. 9B) and the effect of antibody 7326 (shown as antibody PB376) on restoring BMP signaling.

In fig. 10 shows a structural model of the Gremlin-Fab complex indicating possible BMP-binding regions and the Fab epitope.

In fig. Figure 11 shows a surface rendering depicting each Gremlin-1 monomer in two shades of gray, the six key residues identified by mutagenesis that are involved in BMP binding (shown in black), and all surface residues within 6A of these six key residues.

In fig. Figure 12 shows the results of assessing right ventricular systolic pressure (RVP). The effect of anti-Gremlin 1 antibodies on RPV was assessed in normoxia-hypoxia/SU5416-treated C57B1/6 mice. The effects of anti-Gremlin 1 (n=8), IgG1 control antibody (n=6), PBS (n=2), imatinib (n=8) on the development of pulmonary arterial hypertension (PAH) were determined in female C57B1/6 mice. SU5416 (20 mg/kg) was administered subcutaneously every day for three days after inducing chronic normobaric hypoxia (10% O ₂ ) or normoxia for 21 days. The SDPV was determined and the mean SDPV ± SEM was calculated.

*P<0.05; **P<0.01; ***P<0.005; ****P<0.001 by one-way ANOVA.

In fig. Figure 13 shows the results of assessing mean systemic arterial pressure (SBP). The effect of anti-Gremlin 1 antibodies on SBP was assessed in C57B1/6 hypoxic/SU5416 mice treated with anti-Gremlin 1 (n=4), control IgG1 antibody (n=4), imatinib (n=4), and C57B1/ 6 normoxic/SU5416 mice treated with anti-Gremlin 1 (n=4), control IgG1 antibody (n=4), and MAP ± SEM plotted after 21 days.

*P<0.05; **P<0.01; ***P<0.005; ****P<0.001 by one-way ANOVA.

In fig. Figure 14 shows the results of assessing right ventricular hypertrophy. The effect of anti-Gremlin 1 antibodies on right ventricular hypertrophy (RV/LV+S) was assessed in C57B1/6 hypoxic/SU5416 mice. The effects of anti-Gremlin 1 (n=8), control IgG antibody (n=6), PBS (n=2), imatinib (n=8) on the development of pulmonary arterial hypertension (PAH) were determined in female C57B1/6 mice. SU5416 (20 mg/kg) was administered subcutaneously every day for three days after inducing chronic normobaric hypoxia (10% O2) or normoxia for 21 days. The SDPV was determined and the mean SDPV ± SEM was calculated.

*P<0.05; **P<0.01; ***P<0.005; ****P<0.001 by one-way ANOVA.

In fig. Figure 15 presents a histological assessment of the muscularization of pulmonary vessels. The effect of anti-Gremlin 1 antibodies was assessed. Paraffin-embedded lung sections obtained from mice treated with anti-Gremlin 1 (n=6), control IgG antibody (n=6), or imatinib (n=6) were stained for smooth muscle actin (aSMA). to assess the degree of muscle activity and von Willebrand factor (vWF) to identify endothelial cells using immunohistochemistry. Lung sections were digitized using a Nanozoomer virtual microscopy system (Hamamatsu, Welwyn Garden City, UK), and more than 40 vessels per group were scored as unmuscularized, partially, or fully muscularized by independent blinded observers. The means ± SEM for each modality group of each vessel were plotted. Representative images of aSMA- and vWF-stained paraffin-embedded lung sections from IgG1 normoxia groups; IgG1 hypoxia/SU5416; Imatinib Hypoxia/SU5416; anti-Gremlin 1 hypoxia/SU5416.

*P<0.05; **P<0.01; ***P<0.005; ****P<0.001 by one-way ANOVA.

Brief description of the sequence list

SEQ ID NO:1 shows the sequence of human Gremlin-1, including the N-terminal signal sequence of 24 amino acids (Uniprot ID O60565).

SEQ ID NO:2 shows the sequence of the truncated human Gremlin-1 used in crystallography, including the N-terminal tag.

SEQ ID NO:3 shows HCDR1 Ab 7326 (Chothia).

SEQ ID NO:4 shows HCDR1 Ab 7326 (Kabat).

SEQ ID NO:5 shows HCDR2 Ab 7326 (Kabat).

SEQ ID NO:6 shows HCDR3 Ab 7326 (Kabat).

SEQ ID NO:7 shows LCDR1 Ab 7326 (Kabat).

SEQ ID NO:8 shows LCDR2 Ab 7326 (Kabat).

SEQ ID NO:9 shows LCDR3 Ab 7326 (Kabat).

SEQ ID NO:10 shows the heavy chain variable region of Ab7326 (Option 1).

SEQ ID NO:11 shows the light chain variable region of Ab7326 (Option 1).

SEQ ID NO:12 shows the heavy chain variable region of Ab7326 (Option 2).

SEQ ID NO:13 shows the light chain variable region of Ab7326 (Option 2).

SEQ ID NO:14 shows the full-length heavy chain of mouse IgG1 Ab7326 (option 1).

SEQ ID NO:15 shows the full length murine IgG1 light chain of Ab7326 (Option 1).

SEQ ID NO:16 shows the full-length heavy chain of human IgG1 Ab7326 (option 2).

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SEQ ID NO:17 shows the full length human IgG1 light chain Ab7326 (Option 2).

SEQ ID NO:18 shows the Fab heavy chain of murine Ab7326 (Option 1).

SEQ ID NO:19 shows the light chain of Fab murine Ab7326 (option 1).

SEQ ID NO:20 shows the sequence of the truncated human Gremlin-1 used in crystallography, without the N-terminal tag.

SEQ ID NO:21 shows the sequence of mature Gremlin-1 (SEQ ID NO:1 without signal peptide).

SEQ ID NO:22 shows the heavy chain of human IgG4P (option 1).

SEQ ID NO:23 shows the light chain of human IgG4P (option 1).

SEQ ID NO:24 shows human IgG1 heavy chain DNA (Option 1).

SEQ ID NO:25 shows human IgG1 light chain DNA (Option 1).

SEQ ID NO:26 shows human IgG4P heavy chain DNA (option 1).

SEQ ID NO:27 shows human IgG4P light chain DNA (Option 1).

SEQ ID NO:28 shows the full length murine IgG1 heavy chain (Option 2).

SEQ ID NO:29 shows the full length murine IgG1 light chain (Option 2).

SEQ ID NO:30 shows the full-length human IgG1 heavy chain (option 1).

SEQ ID NO:31 shows the full length human IgG1 light chain (Option 1).

SEQ ID NO:32 shows Fab heavy chain (Option 2).

SEQ ID NO:33 shows the Fab light chain (Option 2).

SEQ ID NO:34 shows the heavy chain of human IgG4P (option 2).

SEQ ID NO:35 shows the light chain of human IgG4P (option 2).

Detailed Description of the Invention

Crystal structure of Gremlin-1.

The present invention relates to the structural coordinates of human Gremlin-1. Full coordinates are shown in Fig. 1 (Table 1).

The present invention also provides a human Gremlin-1 crystal consisting of space group C2 with unit cell dimensions a=84.55 A, b=107.22 A and c=77.09 A.

The present invention also provides a Gremlin-1 crystal complexed with an antibody, more specifically a heavy chain Fab of SEQ ID NO:18 and a light chain of SEQ ID NO:19.

The present invention also provides a computer-readable storage medium that contains data storage material encoded with computer-readable data defined by the Gremlin-1 structure coordinates in Table 1. 1 or coordinates defining homologues of the structure.

The invention provides for the use of structural data in table. 1 and computer readable storage medium, as a structural model for Gremlin-1. Such a structural model can be used to screen agents that interact with Gremlin-1. Screening can be high-throughput screening.

An agent that interacts with Gremlin-1 is usually an agent that binds with Gremlin-1. Agents that interact with Gremlin-1 can modulate Gremlin-1 activity. An inhibitory modulating agent can affect any of the functions of Gremlin-1, but usually reduces the binding of Gremlin-1 to BMPs (BMP 2/4/7). Gremlin-1 is a negative regulator of BMPs, so decreased binding increases signaling through BMPs. An activating modulating agent may enhance the binding of Gremlin-1 to BMP.

BMP binding and signaling can be detected by any method known in the art. For example, the examples in the present invention describe a SMAD phosphorylation assay. Phosphorylation of SMAD1, 5 and 8 occurs during BMP signaling. Therefore, increased SMAD phosphorylation can be used as an indicator of increased BMP signaling, which may reflect decreased binding to Gremlin-1.

The examples also describe the analysis of Id1 reporter gene activity, the Id1 gene being a target gene of the BMP signaling pathway. Therefore, the acceleration of signal recovery in this assay can also be used to determine whether an agent inhibits Gremlin-1 binding to BMP.

The agent mentioned herein can be any molecule that has the potential to interact with Gremlin-1, but is preferably a small molecule or antibody.

The invention also relates to a method for screening agents that modulate the activity of Gremlin-1, including the following steps:

(a) identification of the ligand-binding site from the structural coordinates in the table. 1;

(b) identifying candidate agents that interact with at least a portion of the ligand binding site; and (c) obtaining or synthesizing said agent.

The ligand binding site can be any putative site on Gremlin-1 that interacts with the protein (ligand). The ligand binding site is typically a BMP binding site. As shown in the Examples, the present inventors have identified a putative BMP binding site based on the crystal structure of Gremlin-1. This binding site contains the following amino acids: Trp93, Phe117, Tyr119, Phe125, Tyr126 and Phe138, where residue numbering is based on SEQ ID NO:1.

Therefore, the screening method of the invention may involve identifying agents that react with one or more of these residues, preferably at least 2, 3, 4 or all 6 of these residues.

The interaction of the agent with protein residues can be determined by any suitable method known in the art, such as determining the distance between the residue and the agent by X-ray diffraction analysis (typically less than 6 or less than 4A). As discussed in the examples below, the region of Gremlin-1 that can be targeted by a therapeutic agent may include the amino acids Asp92-Leu99, Arg116-His130, Ser137-Ser142, Cys176-Cys178. They are within 6A of the amino acids mutated on the surface of Gremlin-1.

Steps (a) and (b) of the screening method are typically performed in silico, and the agent can be prepared and synthesized by any method known in the art.

In one embodiment, the present invention provides an antibody that binds to an epitope on Gremlin-1 containing at least one residue selected from: Trp93, Phe117, Tyr119, Phe125, Tyr126 and Phe138, wherein the residue numbering corresponds to SEQ ID NO: 1. The present invention also provides an antibody that binds to an epitope comprising all of the residues Trp93, Phe117, Tyr119, Phe125, Tyr126 and Phe138.

The invention also relates to the use of this Gremlin-1 BMP-binding region for the generation of (potentially inhibitory) antibodies. For example, the invention relates to an antigen containing at least one (preferably all) of the above residues, which can be used to generate antibodies.

Instead of interacting with the BMP-binding site of Gremlin-1, the agent may have an allosteric effect. As used herein, the agent binds away from the normal binding site and is still capable of modulating Gremlin-1 activity, for example through induced conformational changes in the protein. Therefore, the structural model and screening method of the invention can also be used to identify allosteric modulators of Gremlin-1.

The antibody Ab 7326 of the invention has been found to have an allosteric effect. The epitope of this antibody contains the following residues: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, with residue numbering based on SEQ ID NO:1. Accordingly, the screening method of the invention may include identifying agents that react with at least 1, 2, 3, 4, 5 or all 9 of these residues. Such agents can then be tested, for example, using the assays described in the examples, to inhibit BMP binding. Preferably, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 are located on one Gremlin-1 monomer, and Ile131 is located on another Gremlin-1 monomer (Gremlin-1 dimers bind to BMP dimers).

Once again, it should be noted that the invention also includes an antigen containing at least one (preferably all) of these residues for the production of antibodies to Gremlin-1.

As already indicated above, agents can be identified as interacting with these residues by any suitable method known in the art. The agent is preferably a small molecule or antibody.

Antibodies.

The present invention relates to antibodies that bind to Gremlin-1.

The term antibody as used herein includes full-length antibodies and any antigen-binding fragment (ie, antigen-binding portion) or individual chains thereof. An antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FR).

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

The antibody of the invention may be a monoclonal antibody or a polyclonal antibody and is typically a monoclonal antibody. An antibody of the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanized antibody, or an antigen-binding portion of any of these. To produce both monoclonal and polyclonal antibodies, an experimental animal that is a non-human mammal such as a goat, rabbit, rat or mouse is typically used, but the antibody can also be produced using other species.

Polyclonal antibodies can be produced by conventional methods, such as immunization of a suitable animal with the antigen of interest. Subsequently, the animal's blood can be collected and the IgG fraction can be purified.

Antibodies to Gremlin-1 can be obtained when immunization of an animal is necessary by administering the polypeptides to an animal, for example a non-human animal, according to well known and traditional protocols, see, for example, Handbook of Experimental Immunology, D. M. Weir (ed .), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals can be immunized, such as rabbits, mice, rats, sheep, cows, camels or pigs. However, mice, rabbits, pigs and rats are generally the most suitable.

Monoclonal antibodies can be produced by any method known in the art, for example, the hybridoma method (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma method, the human B cell hybridoma method (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies of the invention can also be produced by methods of generating antibodies from individual lymphocytes by cloning and expressing immunoglobulin variable region cDNA molecules derived from individual lymphocytes selected for the production of specific antibodies, for example, by the methods described in Babcook, J. et al., 1996, Proc ., Natl. Academician Sci. USA 93 (15):7843-78481; WO 92/02551; WO 2004/051268 and WO 2004/106377.

Antibodies of the present invention can also be produced by various phage display methods known in the art, including those described by Brinkman et al. (J. Immunol. Methods, 1995, 182:41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997, 187, 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US 5698426; 5223409; 5403484; 5580717; 5427908; 5750753; 5821047; 5571698; 5427908; 5516637; 5780225;5658727;5733743 and 5969108.

Fully human antibodies are those in which the variable regions and constant regions (if present) of both the heavy and light chains are of human origin or are substantially identical to sequences of human origin, but are not necessarily derived from the same antibody. Examples of fully human antibodies may include antibodies produced, for example, by the phage display methods described above, and antibodies produced in mice in which the mouse immunoglobulin variable and, optionally, constant region genes are replaced with human counterparts, for example, as described generally in the EP 0546073, US 5545806, US 5569825, US 5625126, US 5633425, US 5661016, US 5770429, EP 0438474 and EP 0463151.

Alternatively, an antibody of the invention can be produced by a method comprising: immunizing a non-human mammal with a Gremlin-1 immunogen; obtaining an antibody preparation from said mammal; isolating monoclonal antibodies from it that recognize Gremlin-1.

The antibody molecules of the present invention may comprise a complete antibody molecule having full-length heavy and light chains, or a fragment or antigen-binding portion of an antibody. The term antigen-binding portion of an antibody refers to one or more antibody fragments that retain the ability to selectively bind an antigen. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody. Antibodies and fragments thereof and antigen binding portions thereof may be, without limitation, Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi-, tri- or tetravalent antibodies, Bis-scFv, diabodies, tribodies, tetrabodies and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23 (9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2 (3), 209217). Methods for creating and producing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments useful 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 FabdAb fragments described in international patent application WO 2009/ 040562. Multivalent antibodies may contain multiple specificities or may be monospecific (see, for example, WO 92/22853 and WO 05/113605). These antibody fragments can be prepared by conventional methods known to those skilled in the art, and the fragments can then be screened for their utility in the same manner as intact antibodies.

The constant region domains of the antibody molecule of the present invention, if present, can be selected taking into account the intended function of the antibody molecule and, in particular, effector

- 6 043762 functions that may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG, or IgM domains. In particular, in cases where the antibody molecule is intended for therapeutic use and the effector functions of the antibody are required, human IgG constant region domains, especially the IgG1 and IgG3 isotypes, can be used. Alternatively, the IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and the effector functions of the antibody are not required.

An antibody of the invention may be produced, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from an animal (eg, mouse) that is transgenic or transchromosomal for the immunoglobulin genes of interest, or a hybridoma derived therefrom , (b) antibodies isolated from a host cell transformed to express an antibody of interest, such as transfectomes, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies produced, expressed, generated or isolated by any other means that involve splicing immunoglobulin gene sequences with other DNA sequences.

The antibody of the invention may be a human antibody or a humanized antibody.

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

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

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

The term human antibody derivatives refers to any modified form of a human antibody, such as a conjugate of the antibody with another agent or antibody.

The term humanized antibody is intended to refer to CDR-grafted antibody molecules in which CDR sequences derived from the germ line of other mammalian species, such as mouse, are grafted onto human framework sequences. Additional modifications to the framework region may be introduced into human framework sequences.

As used herein, the term CDR-grafted antibody molecule refers to an antibody molecule in which the heavy and/or light chain contains one or more CDRs (including, if necessary, one or more modified CDRs) derived from a donor antibody (e.g., a mouse or rat monoclonal antibodies) grafted into the variable region framework of the heavy and/or light chain of an acceptor antibody (eg, a human antibody). For review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more specificity-determining residues from any of the CDRs described above are transferred to the human antibody framework. in the present invention (see, for example, 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 the present invention are transferred into the human antibody framework. In another embodiment, only the specificity-determining residues from each of the CDRs described above in the present invention are transferred into the human antibody framework.

In the case of grafting CDRs or specificity-determining residues, any suitable variable region acceptor framework sequence can be used, taking into account the class/type of donor antibody from which the CDRs are derived, including mouse, primate and human frameworks. Accordingly, the CDR-grafted antibody of the present invention has a variable domain containing human acceptor framework regions as well as one or more CDRs or specificity-determining residues described above. Thus, in one embodiment, a neutralizing CDR graft antibody is provided in which the variable domain comprises human acceptor framework regions and non-human donor CDRs.

Examples of human scaffolds that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM

- 7 043762 can be used for heavy chain, REI can be used for light chain, and EU, LAY and POM can be used for both heavy and light chains. Alternatively, human germline sequences can be used; they are available, for example, at:

http://www.vbase2.org/ (See Retter et al., Nucl. Acids Res. (2005) 33(Suppl. 1), D671-D674).

In the CDR-grafted antibody of the present invention, the acceptor heavy and light chains need not be derived from the same antibody and may optionally contain composite chains having framework regions derived from different chains.

In addition, in the CDR-grafted antibody of the present invention, the framework regions do not need to have exactly the same sequence as the acceptor antibody. For example, unusual residues may be replaced by more commonly occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions can be modified to correspond to a residue at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in acceptor framework regions that may need to be changed is set out in WO 91/09967.

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

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

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

Antibodies that bind to an epitope disclosed herein may include at least one, at least two, or all three heavy chain CDR sequences of SEQ ID NO:4-6 (HCDR1/HCDR2/HCDR3, respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the Ab 7326 antibody of the examples, determined by the Kabat method.

The Kabat and Chothia methods for determining CDR sequences are well known in the art (as are other methods). CDR sequences can be determined by any suitable method, and in the present invention, although the Kabat method is generally used, other methods can also be used. In this case, SEQ ID NO:3 represents the sequence of HCDR1 Ab 7326, determined by the combined method of Chothia & Kabat.

Antibodies of the invention may contain at least one, at least two or all three light chain CDR sequences of SEQ ID NO:7-9 (LCDR1/LCDR2/LCDR3, respectively). They are the LCDR1/LCDR2/LCDR3 sequences of Ab 7326 determined by the Kabat method.

The antibody preferably contains at least the sequence HCDR3 SEQ ID NO:6.

Typically, the antibody contains at least one heavy chain CDR sequence selected from SEQ ID NOs: 3-5 and at least one light chain CDR sequence selected from SEQ ID NOS 7-9. The antibody may contain at least two heavy chain CDR sequences selected from SEQ ID NO:3-5 and at least two light chain CDR sequences selected from SEQ ID NO:7-9. The antibody typically contains all three heavy chain CDR sequences of SEQ ID NO:3-5 (HCDR1/HCDR2/HCDR3, respectively) and all three light chain CDR sequences of SEQ ID NO:7-9 (LCDR1/LCDR2/LCDR3, respectively). Antibodies can be chimeric, human or humanized antibodies.

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

- 8 043762 and the light chain variable region sequence SEQ ID NO:11 or 13 (especially the pairs

HCVR/LVCR SEQ ID NO:10/11 or 12/13).

The antibody may contain a heavy chain (H chain) sequence:

full-length murine IgG1 heavy chain variant 1 of SEQ ID NO:14 or full-length murine IgG1 heavy chain variant 2 of SEQ ID NO:28, or full-length human IgG1 heavy chain variant 1 of SEQ ID NO:30, or full-length human heavy chain variant 2 IgG1 with SEQ ID NO:16, or full-length human IgG4P heavy chain variant 1 with SEQ ID NO:22, or full-length human IgG4P heavy chain variant 2 with SEQ ID NO:34, or Fab heavy chain variant 1 with SEQ ID NO:18 , or Fab heavy chain variant 2 of SEQ ID NO:32.

The antibody may contain a light chain (L chain) sequence:

Full Length Mouse IgG1 Light Chain Variant 1 SEQ ID NO:15 or Full Length Mouse IgG1 Light Chain Variant 2 SEQ ID NO:29 or Full Length Human IgG1 Light Chain Variant 1 SEQ ID NO:31 or Full Length Human Light Chain Variant 2 IgG1 with SEQ ID NO:17, or full-length human IgG4P light chain variant 1 with SEQ ID NO:23, or full-length human IgG4P light chain variant 2 with SEQ ID NO:35, or Fab light chain variant 1 with SEQ ID NO:19 , or Fab light chain variant 2 of SEQ ID NO:33.

In one example, the antibody contains a heavy/light chain sequence pair:

variant 1 full-length mouse IgG1 with SEQ ID NO:14/15 or variant 2 full-length mouse IgG1 with SEQ ID NO:28/29, or variant 1 full-length human IgG1 with SEQ ID NO:30/31, or variant 2 full-length human IgG1 with SEQ ID NO:16/17, or Full Length Human IgG4P Variant 1 with SEQ ID NO:22/23, or Full Length Human IgG4P Variant 2 with SEQ ID NO:34/35, or Fab Light Chain Variant 1 with SEQ ID NO:18 /19, or Fab light chain variant 2 of SEQ ID NO:32/33.

Variant forms of the corresponding sequences can be used interchangeably. For example, an antibody may contain a heavy/light chain sequence pair:

heavy chain variant 1/light chain variant 2 of full-length mouse IgG1 with SEQ ID NO:14/29 or heavy chain variant 2/light chain variant 1 of full-length mouse IgG1 with SEQ ID NO:28/15 or heavy chain variant 1/variant 2 full-length human IgG1 light chain with SEQ ID NO:30/17, or heavy chain variant 2/full-length human IgG1 light chain variant 1 with SEQ ID NO:16/31, or heavy chain variant 1/full-length human IgG4P light chain variant 2 with SEQ ID NO:22/35, or heavy chain variant 2/light chain variant 1 of full-length human IgG4P with SEQ ID NO:34/23, or heavy chain variant 1/light chain variant 2 Fab with SEQ ID NO:18/33, or heavy chain variant 2/light chain variant 1 Fab of SEQ ID NO:32/19.

Antibodies can be chimeric, human or humanized antibodies.

The antibody may alternatively be or contain a variant of one of the specific sequences listed above. For example, a variant may be a substitution, deletion, or addition of any of the above amino acid sequences.

A variant antibody may contain 1, 2, 3, 4, 5, up to 10, up to 20, or more (typically up to a maximum of 50) amino acid substitutions and/or deletions in the specific sequences discussed above. Deletion variants may comprise deletion of individual amino acids, deletion of small groups of amino acids, such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as deletion of specific amino acid domains, or other features. Substitution variants typically involve replacing one or more amino acids with the same number of amino acids and are conservative amino acid substitutions. For example, an amino acid may be replaced by 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 properties of the 20 basic amino acids that can be used to select suitable substituents are as follows:

- 9 043762

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar, hydrophilic, charged (-) Pro hydrophobic, neutral Glu polar, hydrophilic, charged (-) Gln polar, hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic, neutral His aromatic, polar, hydrophilic, charged (+) Thr polar, hydrophilic, neutral lie aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral Lys polar, hydrophobic, charged(+) Trp aromatic, hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

Derivatives or variants typically include sequences that replace a naturally occurring amino acid with an amino acid that is a structural analogue of the naturally occurring amino acid. The amino acids used in the sequences may also be derivatized or modified, such as labeled, as long as the function of the antibody is preserved.

The derivatives and variants described above can be produced during antibody synthesis or by modifying it after production, or, in the case of a recombinant antibody, by known methods of site-directed mutagenesis, random mutagenesis, or enzymatic digestion and/or nucleic acid ligation.

The antibody variants may have an amino acid sequence that has greater than about 60 or greater than about 70%, such as 75 or 80%, typically greater than about 85%, such as greater than about 90 or 95% amino acid identity with the amino acid sequences disclosed herein (in particularly with HCVR/LCVR sequences and H- and L-chain sequences). Additionally, the antibody may be a variant that has greater than about 60 or greater than about 70%, such as 75 or 80%, typically greater than about 85%, such as greater than about 90 or 95% amino acid identity to the HCVR/LCVR sequences and sequences The H and L chains disclosed in the present invention, while maintaining the exact CDRs disclosed for these sequences. The variants may have at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the HCVR/LCVR sequences and with the H and L chain sequences disclosed in the present invention (in some cases circumstances, provided that accurate CDRs are maintained).

The variants typically have from about 60 to about 99% identity, from about 80 to about 99% identity, from about 90 to about 99% identity, or from about 95 to about 99% identity. This percentage of amino acid identity may be observed over the entire length of the corresponding SEQ ID NO or a portion of the sequence consisting of, for example, about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

For amino acid sequences, sequence identity refers to sequences that have a declared significance when assessed using the ClustalW program (Thompson et al., 1994, supra) with the following parameters:

parameters for pairwise alignment - method: exact; matrix: RAM; fine for opening a pass: 10.00; penalty for extending the pass: 0.10;

parameters for aligning multiple sequences - matrix: RAM; fine for opening a pass: 10.00; % identity due to delay: 30; penalty for entering an end pass: on; gap spacing: 0; negative matrix: none; penalty for extending the pass: 0.20; penalty for missing a specific balance: on; penalties for missing hydrophilic residue: incl.; hydrophilic wasps

- 10043762 tatki: GPSNDQEKR. The sequence identity for a particular residue is assumed to include identical residues that have simply been changed.

Therefore, antibodies have been proposed that have specific sequences and variants that retain the function or activity of these chains.

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

The term epitope is the region of an antigen that binds to an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have residues that are directly involved in mediating affinity interactions. Epitopes can also be conformational, i.e. consist of nonlinear amino acids. In some embodiments, epitopes may include determinants that are reactive surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in some embodiments, may have specific three-dimensional structural characteristics and/or specific charge.

Using conventional methods known in the art, it is easy to determine whether an antibody binds to the same epitope or competes for binding with a reference antibody. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the invention, binding of the reference antibody to a protein or peptide is performed under saturation conditions. The ability of the test antibody to bind to the protein or peptide is then assessed. If a test antibody is able to bind to a protein or peptide after it has been bound by a reference antibody under saturated conditions, it can be concluded that the test antibody binds to a different epitope than the one to which the reference antibody binds. On the other hand, if the test antibody is unable to bind to the protein or peptide after binding by the reference antibody under saturated conditions, then the test antibody may bind to the same epitope as the epitope to which the reference antibody of the invention is bound.

To determine whether an antibody competes for binding with a reference antibody, the binding method described above is performed in two orientations. In the first orientation, the binding of a reference antibody to a protein/peptide is performed under saturation conditions, followed by an assessment of the binding of the test antibody to the same protein/peptide molecule. In the second orientation, binding of the test antibody to the protein/peptide is performed under saturation conditions, followed by assessment of the binding of the reference antibody to the same protein/peptide. If in both orientations only the first (saturating) antibody is able to bind to the protein/peptide, then it is concluded that the test antibody and the reference antibody are competing for binding to the protein/peptide. As one skilled in the art will appreciate, an antibody that competes for binding to a reference antibody may not necessarily bind to an epitope identical to that to which the reference antibody binds, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

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

Additional routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed the result of binding of the reference antibody to the same epitope, or whether the observed lack of binding is the result of steric blocking (or other phenomenon) . Experiments of this nature can be performed by ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

Antibodies can be tested for binding to Gremlin-1, for example by standard ELISA or Western blotting. The ELISA assay can also be used to screen for hybridomas that

- 11 043762 exhibit positive reactivity with the target protein. The selectivity of antibody binding can also be determined by monitoring the binding of the antibody to cells expressing the target protein, for example, by flow cytometry. Thus, the screening method may include the step of identifying an antibody that is capable of binding Gremlin-1 by performing ELISA or Western blotting or flow cytometry.

Antibodies can selectively (or specifically) recognize Gremlin-1. An antibody or other compound selectively binds or selectively recognizes a protein if it binds with preferential or high affinity to a protein for which it is selective, but substantially does not 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, as discussed above, or is able to discriminate between them. The antibodies of the invention generally recognize human Gremlin-1.

Antibodies may also be cross-reactive with related proteins or with human Gremlin-1 and Gremlin-1 of other species.

By specific (or selective) it is meant 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 in the present invention. Antibody cross-reactivity is considered significant if the antibody binds to another molecule by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , 85, 90 or 100%, is as effective as it binds to the protein of interest. An antibody that is specific (or selective) can bind another molecule to less than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or 20% binding efficiency , with which it binds to the protein of interest. The antibody may bind to the other molecule 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% binding efficiency, with which it binds to the protein of interest.

Antibodies against Gremlin have been described previously. For example, WO 2014/159010 A1 (Regeneron) discloses anti-Gremlin antibodies that inhibit Gremlin-1 activity, with K _D binding affinities ranging from 625 pM to 270 nM at 25°C. Ciuclan et al. (2013) describe a monoclonal antibody to Gremlin-1 with a _KD binding affinity of 5.6 x 10' ¹⁰ M.

The anti-Gremlin-1 antibodies of the invention are allosteric inhibitors of Gremlin-1 activity and bind to a novel epitope distant from the BMP binding site. The antibodies bind to Gremlin-1 with exceptionally high affinity with Kd values <100 pM. Therefore, the antibodies of the invention show a significant improvement over currently available antibodies and are expected to be particularly useful for the treatment of Gremlin-1 mediated diseases.

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

The antibodies of the invention are typically inhibitory antibodies. Gremlin-1 inhibits BMP-2, 4 and 7, so inhibition of Gremlin-1 results in increased signaling through BMPs.

As mentioned above, the examples of the present invention describe two functional assays for screening an antibody capable of inhibiting Gremlin 1, namely the SMAD phosphorylation assay and the Id1 reporter gene activity assay in HEK cells. Typically, the inhibitory antibody restores SMAD phosphorylation and/or restores BMP signaling in an Id1 reporter gene activity assay in Hek cells. Phosphorylation of SMAD can be restored to at least 80, 90 or 100% compared to control BMP. In an Id1 reporter gene activity assay in Hek cells, the inhibitory antibody may have an IC ₅₀ of less than 10 nM, preferably less than 5 nM.

Once a suitable antibody has been identified and selected, the amino acid sequence of the antibody can be determined by methods known in the art. Antibody-encoding genes can be cloned using degenerate primers. The recombinant antibody can be produced using conventional methods.

The present invention also relates to an isolated DNA sequence encoding the heavy and/or light chain variable regions (or full-length H and L chains) of an antibody molecule of the present invention.

A polynucleotide variant may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and/or deletions in the sequences listed in the sequence listing. Typically a variant has 1-20, 1-50, 1-75 or 1-100 substitutions and/or deletions.

Suitable variants may be at least about 70% homologous to a polynucleotide of any of the nucleic acid sequences disclosed in the present invention, typically they are at least about 80 or 90% homologous, and more preferably at least about 95, 97, or 99%. The variants may have at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity. Typically, the identity of the variants is in the range of from about 60 to about 99%, from about 80 to about 99%, from about 90 to about 99%, or from about 95 to about 99%. These levels of homology and identity typically have at least the coding regions of the polynucleotides. Methods for measuring homology are well known in the art, and those skilled in the art will appreciate that in the present context, homology is calculated based on the identity of the nucleic acids. Regions of at least about 15, at least about 30, such as at least about 40, 60, 100, 200, or more contiguous nucleotides (depending on length) may have such homology. Such homology may exist over the entire length of the unmodified polynucleotide sequence.

Methods for measuring homology or identity of polynucleotides are known in the art. For example, the UWGCG package provides the BESTFIT program which can be used to calculate homology (eg using default settings) (Devereux et al (1984) Nucleic Acids Research 12, p.387-395).

The PILEUP and BLAST algorithms can also be used for homology calculations or for sequence alignment (typically using default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290–300; Altschul S., F et al. (1990) J. Mol. Biol. 215:403-10.

Software for performing BLAST analysis is available for public use through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying highly similar sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold T when matched to a word of the same length in the database sequence. The parameter T refers to the threshold value used to evaluate the neighborhood of words (Altschul et al., supra). This initial word similarity value serves as a seed for an initial search to find longer HSPs containing them. Word hits are expanded in both directions along each sequence as long as the cumulative alignment score can increase. Expansion of a match into a word in each direction is stopped when: the cumulative alignment score drops to zero or below due to the accumulation of one or more matching results as negative score residuals; or upon reaching the end of any sequence. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of matching. The default parameters used in the BLAST program are: word length (W)=11, scoring matrix BLOSUM62 (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), alignment (B)= 50, expect(E)=10, M=5, N=4, and compare both threads.

The BLAST algorithm performs statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One of the similarity criteria evaluated by the BLAST algorithm is the least sum of probabilities (P(N)), which indicates the probability of a random pairing between two nucleotide or amino acid sequences.

For example, a sequence is considered similar to another sequence if the smallest sum of probabilities when comparing the first sequence to the second sequence is less than about 1, typically less than about 0.1, preferably less than about 0.01, and most preferably less than about 0.001. For example, the smallest sum of probabilities can range from 1 to 0.001, more often from 0.01 to 0.001.

A homolog may differ from the sequence of the corresponding polynucleotide by less than 3, 5, 10, 15, 20 or more mutations (each of which may be a substitution, deletion or insertion). For example, a homolog may differ by 3-50 mutations, more often by 3-20 mutations. These mutations may be within at least 30, such as at least about 40, 60, or 100 or more contiguous homologue nucleotides.

In one embodiment, the variant sequence may differ from the specific sequences listed in the sequence listing due to degeneracy of the genetic code. The DNA code contains 4 basic nucleic acid residues (A, T, C and G), which are used to write three-letter codons representing the amino acids of the proteins encoded in

- 13 043762 genes of the body. The linear sequence of codons is translated along the DNA molecule into a linear sequence of amino acids in the protein(s) encoded by these genes. The code is highly degenerate because 61 codons encode 20 natural amino acids and 3 codons are stop signals. Thus, most amino acids are encoded by more than one codon—in fact, some are encoded by four or more different codons. Therefore, a variant polynucleotide of the invention may encode the same polypeptide sequence as another polynucleotide of the invention, but may have a different nucleic acid sequence due to the use of different codons to encode the same amino acids.

The DNA sequence of the present invention may comprise synthetic DNA, such as chemically produced DNA, cDNA, genomic DNA, or any combination thereof.

The DNA sequences that encode the antibody molecule of the present invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of an antibody can be synthesized, if desired, from specific DNA sequences or based on corresponding amino acid sequences.

General methods for creating vectors, transfection methods, and culture methods are well known to those skilled in the art. For more details, see Current Protocols in Molecular Biology, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

Also provided is a host cell containing one or more cloning or expression vectors containing one or more DNA sequences encoding an antibody of the present invention. Any suitable host cell/vector system can be used to express DNA sequences encoding the antibody molecule of the present invention. Bacterial, eg E. coli, and other microbial systems may be used, or eukaryotic host cell expression systems, eg mammalian systems, may be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

The present invention also provides a method for producing an antibody molecule of the present invention, comprising culturing a host cell containing a vector of the present invention under conditions suitable for expressing a protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.

Antibody Ab 7326 according to the invention is identified to bind the following Gremlin-1 residues: Ile110 (131), Lys126 (147), Lys127 (148), Phe128 (149), Thr129 (150), Thr130 (151), Arg148 (169), Lys153 (174) and Gln154 (175), where Lys126 (147), Lys127 (148), Phe128 (149), Thr129 (150), Thr130 (151), Arg148 (169), Lys153 (174) and Gln154 (175) are present on one Gremlin-1 monomer, and Ile110 (131) is present on the second Gremlin-1 monomer. Numbering not in parentheses is based on the structure file (and corresponds to the numbering of the mouse Gremlin-2 resulting from the structural alignment). Numbers in parentheses indicate residues according to sequence entry O60565 of SEQ ID NO:1 in the UniProt database. As discussed in the Examples section, these epitope residues were identified by NCONT analysis within 4 A in the Gremlin-1-Fab Ab7326 complex.

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

Antibodies of the invention can also recognize an epitope in which Ile131 is present on another Gremlin-1 monomer that does not contain the other residues indicated.

Although these residues are provided for a specific human Gremlin-1 sequence, one of ordinary skill in the art, using routine techniques, can easily extrapolate the positions of these residues to other corresponding Gremlin sequences (eg, mouse). Therefore, antibodies that bind to epitopes containing corresponding residues in such other Gremlin sequences are also within the scope of the present invention.

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

Antibody epitopes can also be determined using X-ray diffraction analysis. Therefore, the antibodies of the present invention can be assessed by X-ray diffraction analysis of the Gremlin-1-related antibody. Thus, epitopes can be identified, in particular, by identifying residues on Gremlin-1 within 4A of the antibody paratope residue.

- 14 043762

Pharmaceutical compositions, dosages and dosage regimens.

An antibody of the invention or an agent that modulates Gremlin-1, identified by screening methods, can be provided in a pharmaceutical composition. The pharmaceutical composition is typically sterile and typically includes a pharmaceutically acceptable carrier and/or adjuvant.

The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable adjuvant and/or carrier.

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

The pharmaceutical compositions of the invention may include one or more pharmaceutically acceptable salts.

A pharmaceutically acceptable salt refers to a salt that retains the necessary biological activity of the parent compound and does not cause any undesirable toxicological effects. Examples of such salts include acid adducts and base adducts.

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

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

The pharmaceutical compositions of the invention may contain additional active ingredients.

Also included within the scope of the present invention are kits containing the antibodies or modulating agents of the invention and instructions for use. The kit may further contain one or more additional reagents, such as an additional therapeutic or prophylactic agent, as discussed above.

The modulators and/or antibodies of the invention or their formulations or compositions can be administered for prophylactic and/or therapeutic treatment.

In cases of therapeutic use, the compounds are administered to a subject already suffering from a disorder or condition described above in an amount sufficient to cure, alleviate or partially control the condition or one or more symptoms thereof. Such therapeutic treatment may result in a reduction in symptoms of the disease or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a therapeutically effective amount.

In cases of prophylactic use, the compositions are administered to a subject at risk of developing a disorder or condition described above in an amount sufficient to prevent or reduce the likelihood of the effects of the condition or one or more symptoms thereof. An amount sufficient to achieve this is defined as a prophylactically effective amount. Effective amounts for each purpose will depend on the severity of the disease or injury, as well as the weight and general condition of the subject.

The subject to be administered may be a human or a non-human animal. The term non-human animal includes all vertebrate animals, such as mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Usually administered to humans.

The antibody/modulator or pharmaceutical composition of the invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As one skilled in the art will appreciate, the route and/or method of administration will vary depending on the desired results. Examples of routes of administration of the compounds or pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes, e.g.

- 15 043762 by injection or infusion. As used herein, the phrase parenteral administration refers to modes of administration other than enteral and topical administration, and is typically accomplished by injection. Alternatively, the antibody/modulating agent or pharmaceutical composition of the invention can be administered by a non-parenteral route, such as the topical, epidermal or mucosal route. The antibody/modulating agent or pharmaceutical composition of the invention may be formulated for oral administration.

The appropriate dosage of the antibody/modulating agent or pharmaceutical composition of the invention can be determined by a qualified physician. The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response in a particular patient, for a particular composition and route of administration, without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the potency of the specific compositions of the present invention used, route of administration, time of administration, elimination rate of the particular compound used, duration of treatment, other drugs, compounds and/or materials used in combination with the particular compounds used. compositions, age, sex, weight, condition, general health and previous medical history of the patient receiving treatment, and similar factors well known in the medical field.

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

The dosage regimen may be adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single dose may be administered, multiple divided doses over time, or the dose may be proportionally decreased or increased as necessary, depending on the therapeutic situation. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier.

Administration may be in one or more doses. Multiple doses may be administered by the same or different routes to the same or different sites. Alternatively, dosages may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the antagonist in the patient and the required duration of treatment.

As mentioned above, the modulators/antibodies or pharmaceutical compositions of the invention can be administered together with one or more therapeutic agents.

The combined administration of two or more agents can be achieved in several different ways. Both may be administered together in a single composition or may be administered in separate compositions as part of a combination therapy. For example, one may be introduced before, after, or simultaneously with the other.

Therapeutic indications.

The antibodies of the present invention or modulating agents identified by the screening methods of the invention can be used to treat, prevent, or ameliorate any condition associated with Gremlin-1 activity. For example, any condition that is wholly or partly the result of signaling through Gremlin-1. In other words, the invention relates to the treatment, prevention or improvement of conditions mediated or influenced by Gremlin. Such conditions include fibrotic diseases, including renal fibrosis (eg, diabetic nephropathy and chronic allograft nephropathy) and idiopathic pulmonary fibrosis, pulmonary arterial hypertension, angiogenesis and cancer (eg, colorectal cancer).

The following examples are intended to illustrate the invention.

Example 1: Protein expression, purification, refolding and structure determination.

Protein expression and inclusion body preparation.

The truncated human Gremlin-1 coding sequence (SEQ ID NO:20), optimized for expression in E. coli, was cloned into a modified pET32a vector (Merck Millipore) using BamHI/XhoI, creating a vector encoding the Gremlin sequence with an N-terminal tag 6His-TEV (pET-ЪGremlin1).

Expressed sequence:

MGSSHHHHHHSSGENiYRQGSAYPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIH

EEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKK

RVTRVKQCRCISIDLD; SEQ ID NO: 2

- 16 043762 (non-Gremlin remnants of the 6His-TEV tag are in italics). Sequence numbering is based on UniProt 060565 and SEQ ID NO:1.

Plasmid DNA pET-KGremlin1 was used to transform BL21 (DE3) cells. A single ampicillin-resistant colony was picked from an LB/Amp agar plate and used to inoculate 100 ml of LB/Amp starter culture. After shaking (200 rpm) for 16 hours at 37°C, 25 ml of 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 ₆₀₀ value of 3 was reached. Then 20 ml of MOPS + glycerol nutrient mixture (1 M MOPS pH 7.4, 40% glycerol, 0.5% MgSO ₄ , 0.42% MgCl ₂ ), induced with 300 μM IPTG, and additionally incubated at 17°C, 180 rpm for 16 hours. Cells were collected in a centrifuge (4000 g for 20 min at 4°C).

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

Solubilization.

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

Cleaning before refolding.

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

Refolding.

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

After dialysis, the protein was applied to a HiTrap heparin column and eluted using a 20 CV gradient of 0100% heparin elution buffer (20 mM HEPES, 1 M NaCl, pH 7.5). Correctly folded protein eluted at 1 M NaCl, whereas any misfolded protein eluted at lower salt concentrations.

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

Protein characterization was obtained by SDS PAGE (gel shift), the protein was demonstrated to have the desired molecular weight and correct disulfide bond arrangement by liquid chromatography-mass spectrometry (LC-MS), and protein activity was assessed by cell-based assay ( ID1 reporter activity assay).

Defining the structure of Gremlin 1.

Gremlin 1 protein crystals were grown using the hanging drop method by mixing a 6.6 mg/ml solution of Gremlin 1 in 0.1 M citric acid at pH 4.1 M with lithium chloride and 27% polyethylene glycol (PEG) 6000 in a ratio of 1 :1. Before data collection, the crystals were cryoprotected by adding 20% glycerol to the crystallization buffer. Diffraction data were collected on a diamond light source and processed using the XDS program (Kabsch, Wolfgang (2010) Acta Crystallographica Section D 66, 125-132). Diffraction statistics are summarized in the table below. 2.

- 17 043762

table 2

Diffraction Statistics

Diffraction Statistics Wavelength (A) 0.97949 Space group C2 Cell dimensions a=84.55 A, b=107.22 A, c=77.09 A; os=90, 00°, β=120.43°, γ=90, 00° Resolution limit* (A) 26.19-2.72 (2.79-2.72) Completeness (%) 98.5 (99.0) Position multiplicity 3.4 (3.4) I/s i gma 9.6 (2.0) Emerge 0.095 (0.622) Updated statistics Resolution limit (A) 26.19-2.72 Rcryst 0.24 Rfree 0.29 R.m.s.d. bond lengths (A)** 0.013 R.m.s.d. angles (°) 1,782

*values in parentheses refer to the highest resolution shell **R.m.s.d - standard deviation

The Gremlin-1 structure was determined by molecular replacement using the Phaser program (McCoy et al., J Appl Cryst (2007), 40, 658-674) and the Gremlin-1 model available from the proprietary Gremlin-1/Fab complex coordinates. The resulting Gremlin-1 model contained four copies of the Gremlin-1 monomer, organized as two dimers. The model was corrected using the Coot method (Emsley et al. Acta Crystallographica Section D: Biological Crystallography 66 (4), 486-501), and the coordinates were refined using the Refmac method (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 confirmed by the Molprobity method (Chen et al. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica D66:12-21). The results of the updated statistical data of the model are given in table. 2 above.

Example 2. BMP-binding residues on Gremlin-1.

As discussed above, Gremlin-1 belongs to the bone morphogenic protein (BMP) antagonist protein family in a subgroup known as the DAN family. In the DAN family, Gremlin-1 has the greatest homology with Gremlin-2 (PRDC).

The 2.7 A human Gremlin-1 structure described in Example 1 shares many features with the published mouse Gremlin-2 structure (Nolan et al (2013), Structure, 21, 1417-1429). They have a similar overall molecular stacking with two copies of Gremlin-1 forming an antiparallel non-covalent dimer arranged in an arc. Each monomer has a characteristic finger-wrist-finger arrangement with a cystine knot motif toward the end of the wrist, opposite the fingers (Figure 2). The sequence identity between the proteins is 52% and increases to 67% in the sequence visible in the two structures. The most highly conserved region is at the extensive interface in the dimer, at which all key contact residues are 100% conserved (Figure 3).

Residues involved in the binding of BMPs 2, 4 and 7 to mouse Gremlin-2 (PRDC) and DAN (NBL1) were identified by mutagenesis (Nolan et al (2013), Structure, 21, 1417-1429 and Nolan et al (2014 J Biol Chem 290, 4759-4771). The predicted BMP-binding epitope includes a hydrophobic region spanning both monomers on the convex surface of the dimer (Figs. 4 and 5). Six residues were identified by mutagenesis: Trp72, Phe96, Tyr98, Phe 104, Tyr 105 and Phel 17, and are 100% conserved in human Gremlin-1 (numbering based on the mouse Gremlin-2 sequence). The degree of homology extends to the arrangement of the side chains, which have the same conformation in both proteins (Fig. 5).

The amino acid numbering used in the Gremlin PDB file corresponds to the numbering in the published structure of mouse Gremlin-2 resulting from the structural alignment. This

- 18043762 allows for comparable comparison of amino acids when describing structures. However, for clarity, the key residues identified as being involved in BMP binding are listed below, numbered based on the PDB file and UniProt file with SEQ ID NO:1 in parentheses: Trp72 (93), Phe96 (117), Tyr98 (119), Phe104 (125), Tyr105 (126), and Phe117 (138).

In both mouse Gremlin-2 and human Gremlin-1, the hydrophobic BMP-binding epitope is partially covered by an alpha helix formed by the N-terminal residues of each protein. A model for BMP binding has been proposed in which the N-terminus can bend, completely exposing the BMP-binding surface (Nolan et al (2013), Structure, 21, 1417-1429). In fig. 4 N-terminal residues were removed from human and mouse Gremlin-1 structures

Gremlin-2 before surface imaging to reveal the similarity of BMP-binding surfaces on each protein.

Only mutagenesis of six residues that affect BMP binding has been reported in the literature. It is possible that the actual BMP epitope covers a larger surface area that includes adjacent amino acids. By marking on the surface of Gremlin-1 all residues located within 6A of the mutated residues, a larger region of Gremlin-1 is revealed that could potentially be targeted by therapeutics (Fig. 11). This larger region covers the following human Gremlin-1 amino acids:

Asp92-Leu99

Argil6-Hisl30

Serl37-Serl42

Cysl7 6-Cysl78 (numbering based on SEQ ID NO:1).

By summarizing the published information and crystal structure information of human Gremlin-1, regions of human Gremlin-1 have been identified that can be targeted for therapeutic intervention blocking its interaction with BMPs.

Example 3. Analysis of Id1 reporter gene activity in Hek cells.

General information.

The Id1 reporter gene activity assay in Hek cells uses Hek293 clone 12 cells with the Id1 reporter gene. This cell line was stably transfected with the transcription factor Id1. Id1 is a transcription factor in the BMP signaling pathway. Gremlin is known to bind to BMP, preventing BMP from binding to receptors, reducing the luciferase signal from the reporter gene. Therefore, using this reporter gene activity assay, it is possible to screen for antibodies to Gremlin and determine the presence of antibodies that block the interaction of Gremlin with BMP. When this interaction is blocked, these cells exhibit restoration of the luciferase signal.

Way.

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 the cells were still able to express the Id1 gene. Cells were analyzed in DMEM containing 0.5% FCS, 1x L-glutamine and 1x NEAA. During the short time required for cell analysis, hygromycin B is not needed.

Cells were washed in PBS, recovered with cell dissociation buffer, centrifuged and counted before plating at 5 x 10 ⁴ /well in 70 µl (density 7.14 x 10 ⁵ /ml). The dishes used were 96-well, sterile, white plates coated with opaque poly-D-lysine. The cells were placed in an incubator for approximately 3–4 h to allow them to settle. BMP heterodimers were reduced to 200 μg/ml in 4 mM HCl. BMP was diluted to 10 μg/mL in assay medium using a glass vial to create a new stock solution.

Gremlin-1 was diluted 1:2 in a polypropylene dish for an 8-point dose-response curve with a maximum final dose of 1 μg/mL.

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

The prepared 100x BMP was added to all wells except those containing cells only. The volume of all wells was adjusted to 60 μl with assay medium and incubated for an additional 45 min at 37°C.

After incubation, 30 μl of the sample was transferred to a well of an assay plate and incubated for 20–24 h before measuring the luminescent signal.

Cell Steady Glo reagent was thawed in advance at room temperature. The assay plates were cooled to room temperature for approximately 10-15 min before adding the reagent. The luciferase signal was detected by adding Cell Steady Glo reagent (100 μl) for 20 min on a shaker at room temperature and luminescence measurements were performed according to the CellTitre Glo protocol on a Synergy 2 photometer.

- 19 043762

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

Results.

Full-length and truncated forms of Gremlin-1 were tested in the Hek-Id1 reporter gene activity assay to confirm BMP4/7 blocking activity. Results for the full-length protein are shown in FIG. 6A, and the results for the truncated protein are shown in FIG. 6B.

The percentage inhibition obtained from the dose-response analysis was calculated based on the maximum and minimum signals obtained in the analysis and the data obtained by fitting the curve to a 4-parameter logistic model. IC ₅₀ was calculated based on the inflection point of the curve.

Table 3 Efficiency results for full-length Gremlin-1 and truncated ______Gremlin-1 in the Hek-Id1 reporter gene activity assay.______

Hek-Idl reporter gene activity assay N Geometric mean (nM) 95% CI (or range where N=<4) Full size Gremlin 1 2 16 1.3-1.9 Shortened Gremlin 1 2 1.7 1.1-2.5

Conclusion.

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

Example 4. Preparation of antibodies to Gremlin-1.

Antibodies to Gremlin-1 were obtained by immunization and library panning. The library was created in the laboratory as a naive human library with V regions amplified from donor blood.

The first round of immunization resulted in 26 different Gremlin1-binding antibodies. These antibodies were scaled up and purified for screening testing.

human and mouse cross-reactive antibodies from the library were panned using recombinant human Gremlin from R&D Systems. 10 antibodies were selected for scale-up and purified as scFv for screening testing.

Example 5: Anti-Gremlin-1 Antibody Screening.

Antibodies were screened using the Hek-Id1 reporter gene activity assay described in Example 3 and by measuring SMAD phosphorylation. Phosphorylation of SMAD1, 5 and 8 occurs during BMP signaling. Therefore, Gremlin-1 inhibitors increase the level of SMAD phosphorylation.

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

Results.

In the Hek-Id1 reporter gene activity assay, there were no apparent effects provided by antibodies generated by immunization (with a 10-fold excess of antibodies tested against the BMP4/7 heterodimer). The results are shown in Fig. 7.

In contrast, several antibodies obtained from the library were able to restore signal in the Hek-Id1 reporter gene activity assay (50-fold excess antibody with 50% Gremlin dose) (Fig. 8). Of these, Ab2416 and Ab2417 contained high levels of endotoxin. Ab7326 retained blocking potency at 10-fold excess and at a Gremlin-1 concentration providing 80% inhibition.

Additional results are presented in Fig. 9A (human Gremlin) and 9B (mouse Gremlin). These drawings show titers of Ab7326 (designated PB376) up to 15 nM. Ab7326 has been shown to restore BMP signaling blocked by human (IC ₅₀ 1/3 nM) or murine (IC 0.2 nM) Gremlin. This antibody functions as both human and mouse IgG1.

The sequences of full-length human and mouse IgG1 are presented below. To synthesize full-length human and mouse IgG1 proteins, the Ab7326 variable regions obtained from the library were re-cloned into vectors containing the corresponding antibody constant domains.

Because Ab7326 was derived from a naïve human library in which Abs were cloned as scFvs, PCR amplification of VH and VK using primer/degenerate primer pools was necessary to re-cloning the 7326 variable regions as full-length Abs or Fabs. The amplified PCR products were then digested and cloned simultaneously into mouse and human vectors. Since VH and VK were amplified using primer/degenerate primer pools, two variant forms of the products were obtained, differing by one amino acid residue due to slight differences in the primers used for annealing during the PCR process.

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

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

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

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

light chain variable region variant 2 contains threonine (T) at position 7;

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

QVQLVESGAE VKKPGATVKI : SCKVSGYTFT DYYMHWVQQA PGKGLEWMGL VDPEDGETIY AEKFQGRVTI TADTSTDTAY MELSSLRSED TAVYYCATDA RGSGSYYPNH FDYWGQGTLV TVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCGCKP CICTVPEVSS VFIFPPKPKD VLTITLTTPKV TCVWDISKD DPEVQFSWFV DDVEVHTAQT OPREEQFNST FRSVSELPIM HQDWLNGKEF KCRVNSAAFP APIEKTISKT KGRPKAPQVY TIPPPKEOMA KDKVSLTCMI TDFFPEDITV EWQWNGOPAE NYKNTQPIMD

TDGSYFVYSK LNVQKSNWEA GNTFTCSVLH EGLHNHHTEK SLSHSPGK

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

DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLA WYQQKPGQPP

KLLIYWASTR ESGVPDRFSG SGSGTDFTLT INSLQAEDVA VYFCQQYYDT

PTFGQGTRLE IKRTDAAPTV SIFPPSSEQL TSGGASWCF LNNFYPKDIN

VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE

ATHKTSTSPI VKSFNRNEC

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

QVQLVQSGAE VKKPGATVKI SCKVSGYTFT DYYMHWVQQA PGKGLEWMGL

VDPEDGETIY AEKFQGRVTI TADTSTDTAY MELSSLRSED TAVYYCATDA RGSGSYYPNH FDYWGQGTLV TVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCGCKP CICTVPEVSS VFIFPPKPKD VLTITLTTPKV TCVWDISKD DPEVQFSWFV DDVEVHTAQT OPREEQFNST FRSVSELPIM HQDWLNGKEF KCRVNSAAFP APIEKTISKT KGRPKAPQVY TIPPPKEOMA KDKVSLTCMI TDFFPEDITV EWQWNGOPAE NYKNTQPIMD

TDGSYFVYSK LNVQKSNWEA GNTFTCSVLH EGLHNHHTEK SLSHSPGK

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

DIVMTQTPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLA WYQQKPGQPP

KLLIYWASTR ESGVPDRFSG SGSGTDFTLT INSLQAEDVA VYFCQQYYDT

PTFGQGTRLE IKRTDAAPTV SIFPPSSEQL TSGGASWCF LNNFYPKDIN

VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE

ATHKTSTSPI VKSFNRNEC

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

QVQLVESGAE VKKPGATVKI SCKVSGYTFT DYYMHWVQQA PGKGLEWMGL

VDPEDGETIY AEKFQGRVTI TADTSTDTAY MELSSLRSED TAVYYCATDA

RGSGSYYPNH FDYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG

CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SWTVPSSSL

GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLF

PPKPKDTLM^SRTPEVTCVIOiDVSHEpPEIL^FNWYYDGVE^HNAKTKPRE

- 21 043762

EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDG5 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

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

DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLA WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT INSLQAEDVA VYFCQQYYDT PTFGQGTRLE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLS KAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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

QVQLVQSGAE VKKPGATVKI SCKVSGYTFT DYYMHWVQQA PGKGLEWMGL VDPEDGETIY AEKFQGRVTI TADTSTDTAY MELSSLRSED TAVYYCATDA RGSGSYYPNH FDYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFP AV LQSSGLYSLS SWTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REP QVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

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

DIVMTQTPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLA WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT INSLQAEDVA VYFCQQYYDT PTFGQGTRLE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLS KAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

Antibody CDRs were determined by the Kabat method (shown in bold in the above sequences). Additional HCDR1 residues identified from Chothia are in italics. Constant region sequences are also underlined.

Restoration of p-SMAD signaling by anti-Gremlin 1 antibodies is shown in the table below. 4.

Table 4

Restoring p-SMAD signaling

2417 2418 2419 2481 2482 2483 2484 7326 8427 BMP 2 109.1% 58.2% 32.6% 40.4% 35.3% 43.1% 104.0% 107.2% 51.3% 50 ng/ml +/- +/- +/- +/- +/- +/- +/- +/- +/- 2.8% 1.9% 14% 0.6% 0.8% 2.1% 2.7% 3.5% 1.4% BMP 4 109.6% 71.3% 31.7% 60.1% 54.4% 72.5% 105.2% 110.0% 78.2% 25 ng/ml +/- +/- +/- +/- +/- +/- +/- +/- +/- 3.0% 3.1% 1.2% 2.2% 1.3% 2.1% 3.3% 3.8% 2.5% BMP 7 111.5% 99.5% 53.8% 64.4% 52.3% 66.2% 105.2% 108.0% 72.6% 200 +/- +/- +/- +/- +/- +/- +/- +/- +/- ng/ml 3.8% 3.2% 3.4% 1.3% 1.1% 1.2% 4.3% 3.2% 2.5% VMR-2/7 119.3% 78.6% 50.8% 53.7% 47.6% 56.1% 120.4% 128.5% 62.8% 50 ng/ml +/- +/- +/- +/- +/- +/- +/- +/- +/- 2.6% 3.6% 2.7% 3.1% 1.5% 2.5% 4.4% 2.9% 2.5% VMR4/7 113.7% 78.0% 61.4% 48.3% 41.7% 50.8% 112.4% 127.0% 63.3% 50 ng/ml +/- +/- +/- +/- +/- +/- +/- +/- +/- 3.1% 4.0% 4.0% 2.1% 1.7% 1.7% 2.5% 3.1% 2.1%

Results are presented as a percentage of the level of SMAD phosphorylation by BMP alone (control BMP). Experiments were performed in lung fibroblasts obtained from patients with idiopathic pulmonary fibrosis. Antibodies to rhGremlin-1 and Gremlin-1 were preincubated for 45 min at room temperature. Anti-hGremlin-1 and Gremlin-1 antibodies were then added to BMP-coated cells for 30 min.

In table 5 shows additional results obtained from a SMAD phosphorylation assay that examined the displacement of BMP-2 or BMP4/7 from Gremlin 1-BMP complexes by anti-Gremlin-1 antibodies. Experiments were again performed in lung fibroblasts obtained from patients with idiopathic pulmonary fibrosis. rhBMP-2 or rhBMP 4/7 was preincubated with hGremlin-1 for 1 h at room temperature. BMP-2- or BMP4/7-Gremlin-1 complexes were incubated with various concentrations of anti-Gremlin-1 antibodies overnight at 4°C. Antibody concentrations represent the final concentration in the plate.

Table 5

Replacement of BMP-2 or BMP4/7 from Gremlin 1-BMP complexes with antibodies to Gremlin-1

81.3 µg/ml 40.6 µg/ml 20.3 µg/ml 10.2 µg/ml 5, 1 µg/ml 2.55 µg/ml 1.27 µg/ml 0.63 µg/ml BMP 2 100.3% 98.8% 97. 0% 93.5% 86.4% 79.9% 66.5% 54.8% 2484 50 +/- +/- +/- +/- +/- +/- +/- +/- ng/ml 3.5% 2.7% 2.9% 2.6% 2.0% 1.9% 2.8% 0.3% VMR4/7 136.4% 133.2% 121.4% 108.1% 86.6% 74.7% 65.8% 60.7% 2484 50 +/- +/- +/- +/- +/- +/- +/- +/- ng/ml 4.2% 1.0% 1.4% 4.9% 4.4% 2.2% 0.6% 1.5% BMP 2 103.7% 101.5% 99.4% 103.8% 100.3% 103.2% 102.8% 97.0% 7326 50 +/- +/- +/- +/- +/- +/- +/- +/- ng/ml 1.1% 2.4% 3.8% 2.4% 2.2% 4.3% 2.8% 2.9% VMR4/7 133.7% 132.3% 130.3% 125.6% 121.4% 120.9% 111.1% 102.0% 7326 50 +/- +/- +/- +/- +/- +/- +/- +/- ng/ml 0.8% 1.8% 4.2% 10.0% 4.2% 3.3% 2.3% 4.5%

The results presented in table. 5 show that Ab7326 can displace pre-complexed BMP-2 or BMP4/7 from Gremlin 1-BMP complexes. Ab7326 performs this displacement at much lower concentrations than reference antibody 2484. This indicates that Ab7326 is an allosteric inhibitor, consistent with our finding that the Ab7326-binding site on Gremlin-1 is distant from known BMP-binding regions . Thus, Ab7326 can access the allosteric binding site even when BMP forms a complex with Gremlin-1, resulting in significantly improved inhibition of Gremlin activity.

Example 6. Preparation of the crystal structure of Gremlin-1 in complex with Fab 7326.

The crystal structure of human Gremlin-1 complexed with Fab Ab 7326 was obtained at 2.1 A resolution. Fab sequences are shown below:

Heavy Chain: SEQ ID NO:18

QVQLVE S GAEVKKPGATVKIS CKVS GYT FT DYYMHWVQQAPGKGLEWMGLVDPE DGE TI

YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

Light chain: SEQ ID NO:19

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST

RESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

All contacts within 4 A between Gremlin-1 and Fab were then identified using CCP4 NCONT software. The following residues were identified: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 (numbering based on the sequence SEQ ID NO:1 in the UniProt database (numbered as Ile110, Lys126, Lys127, Phe128, Phe128, Arg148 , Lys153 and Gln154 in the structure file, which corresponds to the numbering of the mouse Gremlin2)).

In fig. 10 shows structural models of the Gremlin-Fab complex, with Fab epitope residues shown relative to the BMP-binding regions.

Ab7326 is an inhibitory antibody that acts allosterically, i.e. binds outside the BMP-binding regions.

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

Way.

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

The assay buffer was HBS-EP+ plus 150 mM NaCl, resulting in 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 obtain a capture level of approximately 100 RU on the anti-mouse IgG-immobilized Fc surface. Recombinant human Gremlin 1 was titrated in 5 nM running buffer (using 2-fold dilutions) and injected through flow cells 1 and 2 at a flow rate of 30 μl/min for 3 min, followed by a 5-min dissociation phase. A control, which was a buffer, was also used. The surface was restored at a flow rate of 10 L/min with 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.

Kinetic characteristics were determined using Biacore T200 software.

Binding affinity measurements were performed at 25°C.

Results.

The binding affinity, expressed as the average KD value of 5 determinations, was found to be below 100 pM.

Example 8. In vivo assessment of the effect of antibodies to Gremlin-1 on chronic hypoxia/SU5416-induced pulmonary arterial hypertension in mice.

Short review.

Imbalance in the TGFe superfamily is an important factor associated with the development of several lung pathologies, including pulmonary arterial hypertension (PAH) (Budd & Holmes Pharm Ther 2012). Gremlin-1 is involved in the development and progression of PAH (Thomas et al AJP 2009; Ciuclan et al AJP 2013). Recent studies have shown that anti-Gremlin-1 antibody can inhibit the development of pulmonary arterial hypertension in a preclinical hypoxia/SU5416-induced PAH model (Ciuclan et al AJP 2013). The present example assessed the effects of anti-Gremlin1 antibodies on hemodynamics and vascular remodeling in a preclinical hypoxia/SU5416-induced PAH mouse model.

Hypoxia/SU5416 resulted in a significant increase in right ventricular systolic pressure (RVS) and right ventricular hypertrophy (Figs. 12 and 15). Administration of the anti-Gremlin-1 antibody resulted in a significant (P < 0.005) decrease in BPPV compared with the IgG1 and PBS control groups. In animals in normoxia, no significant effect of the anti-Gremlin-1 antibody on SDPV was observed (Fig. 12). There was also no effect on systemic pressure (mean arterial pressure or MAP; Fig. 13) or on right ventricular hypertrophy (Fig. 14). Taken together, this study supports the role of Gremlin-1 in the development of pulmonary vascular remodeling and increased RPV in the chronic hypoxia/SU5416 model of PAH and the use of anti-Gremlin-1 antibodies for its treatment.

General information.

Pulmonary hypertension (PH) is a hemodynamic condition in which the pressure measured in the pulmonary artery is elevated. Clinically, this is defined as a resting mean pulmonary arterial pressure (mPAP) > 25 mm Hg. Art., pulmonary vascular resistance (PVR) > 3 Wood units (Wood) and pulmonary wedge pressure < 15 mm Hg. Art. (Badesch et al 2009). The pathological characteristics of PH are multifactorial and include pulmonary arterial pressure, vascular remodeling of small and medium arteries, right ventricular hypertrophy and ultimately right ventricular heart failure (Faber & Loscalzo 2004). The WHO classifies PH into five main categories, including group I. Group I represents pulmonary arterial hypertension, which includes idiopathic PAH and hereditary PAH, as well as associated PAH, which occurs in combination with other complications such as systemic sclerosis (Simonneau et al 2009) . Row

- 24 043762 predisposing mutations are associated with the development of PAH in patients with hereditary and idiopathic PAH, the most predominant of which are mutations in the bone morphogenetic protein receptor, BMPR2 (Budd & Holmes Pharm Ther 2012). In addition, mutations in BMP-activated components of downstream Smad signaling pathways have also been reported in patients with developing PAH (Budd & Holmes Pharm Ther 2012). Recent studies have identified elevated levels of the BMP inhibitor, Gremlin (Gremlin-1 and 2) in patients with PAH (Cahill et al Circ 2012). Consistent with a functional role for Gremlin-1 in the development of PAH, haplo-deficiency of Gremlin-1 increased BMP signaling in the hypoxic mouse lung and resulted in decreased pulmonary vascular resistance by attenuating vascular remodeling (Cahill et al Circ 2012). These observations were also confirmed by Ciuclan and colleagues (AJP 2013), who demonstrated that anti-Gremlin-1 antibody attenuated pulmonary arterial hypertension in mice induced by chronic hypoxia/SU5416. Recent studies have shown that non-genetic mechanisms may contribute to decreased BMPR2 expression in patients with systemic sclerosis and the development of PAH (Gilbane et al AJRCCM). Taken together, imbalances in the BMP superfamily axis can lead to the development of pulmonary pathologies such as PAH.

The purpose of this study was to evaluate the in vivo effect of anti-Gremlin-1 antibody on the development of PAH in the chronic hypoxia/SU5416 mouse model.

Materials and methods.

Reagents.

Imatinib: Science Warehouse 1625-1000.

SU5416: R&D Systems 3037.

aSMA antibody: Dako M085129-2.

VWF antibody: Dako A008202-2.

Biotinylated goat anti-rabbit IgG antibody: Vector Labs BA-1000.

Biotinylated horse anti-mouse IgG antibody, adsorbed rat: Vector Labs BA-2001.

Carboxymethylcellulose: Sigma C5678 419273.

Tween 80: Sigma P1754.

Benzyl alcohol: Sigma 305197; 402834.

Sodium chloride: Sigma S7653; VWR 10241.

VECTASTAIN ABC-AP set: Vector Labs AK-5000.

VECTASTAIN Elite ABC Set: Vector Labs PK-6100.

Normal Horse Serum: Vector Labs, registration number S-2000.

Polysorbate: Sigma 59924.

Experimental protocols.

Animals.

C57B1/6 mice were housed in a pathogen-free facility with free access to food and water on a 12-hour light/dark cycle. This animal research is licensed under the UK Companion Animals (Scientific Procedures) Act 1986.

Hypoxia/SU5416 mouse model of pulmonary arterial hypertension.

Female C57B1/6 mice aged 8-10 weeks were divided into groups (Table 6). Animals in all groups were weighed and injected subcutaneously (s.c.) with 20 mg/kg SU5416 in 100 μl vehicle (0.5% carboxymethylcellulose (CMC); 0.9% sodium chloride (NaCl); 0.4% polysorbate 80; 0 .9% benzyl alcohol in deionized water) as described by Ciuclan et al. AJRCCM 2013. If necessary, a second s.c. was administered. injection as indicated in table. 6, containing: PBS, 30 mg/kg IgG1 or 30 mg/kg anti-Gremlin-1 antibody. At the same time, mice from the Hypoxia + Imatinib group received food injected with imetinib to obtain 100 mg/kg/day. The hypoxia groups of mice were then placed in a chamber under normobaric hypoxia (10% O2) conditions, while mice in the normoxia groups were kept under normoxic (21% O2) conditions in the same room as the chamber.

- 25 043762

Table 6

Treatment groups: C57B1/6 mice were assigned to the treatment groups indicated in the table

Group Quantity Normoxia+PBS 2 Normoxia+IgGl 6 Normoxia + antibody to GREMLIN-1 8 Hypoxia+PBS 2 Hypoxia!IgG 6 Hypoxia!antibody to GREMLIN-1 8 Hypoxia!Imatinib 8

On days 7 and 14, all mice were weighed and given another s.c. dose of 20 mg/kg SU5416. If necessary, mice were given another s.c. injection of PBS, 30 mg/kg IgG1, or 30 mg/kg anti-Gremlin-1 antibody (as listed in Table 6). The anti-Gremlin-1 antibody used in these studies was Ab7326, full-length mouse IgG1 variant 1, as described in Example 5. On day 21, right ventricular systolic pressure (RVS) and mean arterial pressure (MAP) were measured and collected. fabrics.

Right Ventricular Systolic Pressure and Tissue Collection.

Measurements of hemodynamics of RPV and SBP were carried out on animals after three weeks of exposure to hypoxia and appropriate drug therapy, as indicated in Table. 6. Animals were anesthetized with 1.5% isofluorane and placed supine on a heating blanket maintained at 37°C. First, the right jugular vein was isolated and a pressure catheter (Millar mouse SPR-671NR 1.4F pressure catheter, Millar Instruments, UK) was inserted and advanced into the right ventricle to determine RVD. Second, SBP was measured by isolating the left common carotid artery and inserting a pressure catheter. Both RPV and SBP were recorded in a pre-calibrated PowerLab system (ADInstruments, Australia).

Animals were sacrificed with an overdose of isofluorane anesthetic, and whole blood was collected. Whole blood was centrifuged (220 x g; 2 min), and serum was recovered and stored at 80°C. The heart was removed and the weights of the right and left ventricles were recorded. The lungs were perfused with 2.5 ml saline through the right ventricle. The left lung was fixed by filling with 10% formaldehyde before paraffin embedding and sectioning. The right lung was flash frozen in liquid nitrogen and stored at -80°C.

Histology.

Slides were deparaffinized and rehydrated using xylene and an ethanol concentration gradient. Slides were immersed in 0.3% H ₂ O ₂ in methanol for 30 min to retrieve antigens, washed 3 times in PBS and blocked for 1 h in a 1:30 mixture of normal horse serum and PBS. Primary anti-aSMA antibody was added to each slide at a ratio of 1:100 and incubated at 4°C overnight, then washed in PBS for 5 min three times. Biotinylated horse anti-mouse IgG antibody-adsorbed rat secondary antibody was diluted 1:200 and pipetted onto each slide, then incubated for 45 min; then washed in PBS 3 times for 5 min. According to the manufacturer's instructions, alkaline phosphatase with biotin-avidin complex (ABC-AP) was prepared in 30 min and placed on each slide for 30 min; then washed 3 times for 5 minutes. AP substrate was prepared according to the instructions supplied with the kit and pipetted onto each slide and allowed to develop color; then washed 3 times for 5 minutes. Primary anti-vWF antibody was added to each slide at a ratio of 1:100 and incubated at 4°C overnight, then washed in PBS for 5 min three times. Biotinylated goat anti-rabbit IgG secondary antibody was diluted 1:200 and added to each slide for 45 min; then washed in PBS 3 times for 5 min. According to the instructions provided in the kit, ABC was prepared in 30 min and applied to each slide for 30 min; then washed 3 times for 5 minutes. DAB substrate was prepared according to the instructions provided in the kit and pipetted onto each slide and allowed to develop color for ~5-10 min; then washed 3 times for 5 minutes. Slides were counterstained with hematoxylin for ∼40 s, dehydrated, and coverslipped using Pertex. All slides were digitally scanned using a Hamamatsu NanoZoomer 2.0-HT slide scanner (Hamamatsu, Welwyn Garden City, UK).

Data and statistical analysis.

More than 40 vessels, randomly selected from each hypoxia group, were assessed for the degree of muscularization. Vessels were assessed by at least four independent observers: O=unmuscularized; 1=partially muscularized; 2=fully muscularized; and the modal value for each vessel was determined. The percentage of vessels that were completely, partially, or unmuscularized was determined and the mean ± SEM was calculated. To determine significance *P<0.05;

- 26 043762 **P<0.01; ***P<0.005; ****P<0.001 one-way ANOVA was performed.

Results.

Administration of SU5416 (20 mg/kg) after exposure to chronic normobaric hypoxia (10% O ₂ ) resulted in a significant (P < 0.01) increase in BPPV compared with exposure to hopmokcuu/SU5416 for 21 days in both the PBS-only group and and in the control group IgG1 (Fig. 12). There were no significant differences between the IgG1 and PBS groups. The effect of drug treatment on elevated BPPV in mice treated with SU5416 was maintained under hypoxia: imatinib resulted in a significant (P<0.001) reduction in BPPV compared with controls. Anti-Gremlin-1 antibody resulted in a significant (P<0.005) decrease in BPPV compared with IgG1 and PBS controls. In animals kept under normoxic conditions, no significant effect on the RPV of the Gremlin-1 antibody was observed (Fig. 12).

The effect of anti-Gremlin-1 antibody (n=4) or IgG1 vehicle control (n=4) on RPV was assessed under hypoxia and normoxia conditions (Fig. 13). No significant treatment effects were observed on DPJ.

Right ventricular hypertrophy (right ventricle/left ventricle+septum mass) was determined (Fig. 14). Administration of SU5416 (20 mg/kg) after exposure to chronic normobaric hypoxia (10% O ₂ ) led to a significant (P < 0.01) increase in right ventricular hypertrophy (RV/LV+S) compared with normoxia/SU5416 after 21 days as in the PBS-only group and the control IgG1 group (Fig. 14). There was no significant effect of drugs (imatinib or anti-Gremlin 1 antibody) on increased MAP in hypoxic mice treated with SU5416.

The degree of vascular remodeling was assessed by staining paraffin sections of the lungs. Blood vessels were stained for von Willebrand factor (vWF) to identify endothelial cells and smooth muscle actin (aSMA) to assess the degree of muscularization. Images were digitized using a Hamamatsu NanoZoomer 2.0-HT slide scanner, and at least 40 vessels randomly selected from the IgG1 normoxic group and each hypoxic group were assessed for the degree of muscularization. The vessels were assessed by at least four independent observers: 0=nonmuscular; 1=partially muscularized; 2=fully muscularized; and the modal value for each specific vessel and the percentage of non-muscularized, partially and fully muscularized vessels were plotted (Graph 15). Administration of SU5416 (20 mg/kg) after exposure to chronic normobaric hypoxia (10% O2) resulted in a significant increase in fully muscled vessels (P<0.001) and partially muscled vessels (P<0.01) with a significant cumulative decrease in non-muscularized vessels (P<0.001) ). The effect of drug treatment on elevated RPV in SU5416-treated mice was maintained under hypoxia: imatinib resulted in a significant (P<0.001) reduction in fully muscled (P<0.01) vessels. In addition, the anti-Gremlin 1 antibody resulted in significant (P<0.001 and P<0.05, respectively) compared to the IgG1 control hypoxia/SU5416 group (Fig. 15). There was no significant effect of drug treatment (imatinib or anti-Gremlin-1 antibodies) on the percentage of partially muscled vessels compared with the IgG1 hypoxia/SU5416 control group.

Drug treatment (imatinib or anti-Gremlin-1 antibody) resulted in an increase in the percentage of nonmuscularized vessels compared with the IgG1 hypoxia/SU5416-treated control group, but the increase was not significant.

Discussion.

In this example, the effect of anti-Gremlin-1 antibody on the development of PAH was assessed in the chronic hypoxia/SU5416 model. The anti-Gremlin-1 antibody resulted in significant inhibition of SDPV (Fig. 12) and vascular remodeling (Fig. 15), while having no effect on systemic pressure (SBP) (Fig. 13) in the hypoxia/SU5416 mouse model of PAH. There was no significant effect of anti-Gremlin-1 antibody on RPV in normoxic mice treated with SU5416. The effect of the anti-Gremlin-1 antibody is consistent with the observations described by Ciuclan and colleagues (AJP 2013), which showed that antagonism with the anti-Gremlin-1 antibody reduces elevated RPV and vascular remodeling in the chronic hypoxia/SU5416 mouse model of PAH (Fig. 12 and 15). In contrast to Ciuclan and colleagues, this study did not observe a significant effect of anti-Gremlin1 antibody on right ventricular hypertrophy (Fig. 14). However, this study did not observe a significant effect of the control drug imatinib on the development of right ventricular hypertrophy in hypoxic/SU5416 mice. Taken together, this study supports the role of Gremlin-1 in the development of pulmonary vascular remodeling and increased RPV in the chronic hypoxia/SU5416 model of PAH and the use of anti-Gremlin-1 antibodies in its treatment.

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Literature

Badesch DB, Champion NS, Sanchez MA, Hoeper MM, Loyd JE, Manes A, McGoon M, Naeije R, Olschewski H, Oudiz RJ, Torbicki A. Diagnosis and assessment of pulmonary arterial hypertension. J Am Coll Cardiol. 2009 Jun 30; 54(1 Suppl):S55-66. doi: 10.1016/j.jacc.2009.04. 011. Review. PMID:19555859.

Budd DC, Holmes AM. Targeting ΤΟΕβ superfamily ligand accessory proteins as novel therapeutics for chronic lung disorders. Pharmacol Ther. 2012 Sep; 135(3):279-91. doi: 10.1016/j.pharmthera.2012.06.001. Epub 2012 Jun 18.

Cahill E, Costello CM, Rowan SC, Harkin S, Howell K, Leonard MO, Southwood M, Cummins EP, Fitzpatrick SF, Taylor CT, Morrell NW, Martin F, McLoughlin P. Gremlin plays a key role in the pathogenesis of pulmonary hypertension . Circulation. 2012 Feb 21; 125(7):92 0-30. doi: 10.1161/CIRCULATIONAHA.Ill .038125 . PMID:22247494.

Ciuclan L, Sheppard K, Dong L, Sutton D, Duggan N, Hussey M, Simmons J, Morrell NW, Jarai G, Edwards M, Dubois G, Thomas M, Van Heeke G, England K. Treatment with anti-Gremlin 1 antibody ameliorates chronic hypoxia/SU5416-induced pulmonary arterial hypertension in mice. Am J Pathol. 2013 Nov; 183(5):1461-73. doi: 10.1016/j.ajpath.2013.07.017. PMID:24160323

Ciuclan L, Hussey MJ, Burton V, Good R, Duggan N, Beach S, Jones P, Fox R, Clay I, Bonneau 0, Konstantinova I, Pearce A, Rowlands DJ, Jarai G, Westwick J, MacLean MR, Thomas M Imatinib attenuates hypoxia-induced pulmonary arterial hypertension pathology via reduction in 5-hydroxytryptamine through inhibition of tryptophan hydroxylase 1 expression. Am J Respir Crit Care Med. 2013 Jan 1; 187(1):78-89. doi: 10.1164/room.201206-1028OC. PMID:23087024

Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med. 2004 Oct 14; 351(16):1655-65. Review. PMID:15483284.

Gilbane AJ, Derrett-Smith E, Trinder SL, Good RB, Pearce A, Denton CP, Holmes AM. Impaired bone morphogenetic protein receptor IT signaling in a transforming growth factor-p-dependent mouse model of pulmonary hypertension and in systemic sclerosis. Am J Respir Crit Care Med. 2015 Mar 15; 191 (6): 665-77. doi: 10.1164/room.201408-1464OC.

Simonneau G, Robbins IM, Beghetti M, Channick RN, Delcroix M, Denton CP, Elliott CG, Gaine SP, Gladwin MT, Jing ZC, Krowka MJ, Langleben D, Nakanishi N, Souza R. Updated clinical

- 28 043762 classification of pulmonary hypertension. J Am Coll Cardiol. 2009 Jun 30; 54(1 Suppl): S43-54. doi:

10.1016/j.jacc.2009.04.012. Review. PMID:19555858.

Thomas M, Docx C, Holmes AM, Beach S, Duggan N, England K, Leblanc C, Lebret C, Schindler F, Raza F, Walker C, Crosby A, Davies RJ, Morrell NW, Budd DC. Activin-like kinase 5 (ALK5) mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the progression of experimental pulmonary arterial hypertension induced by monocrotaline. Am J Pathol. Feb 2009; 174(2):380-9. doi: 10.2353/ajpath.2009.080565. Epub 2008 Dec 30.

Sequence Listings SEQ ID NO:1 (Human Gremlin-1; Uniprot ID: 060565) MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSNRRG

M GSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIH EEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKK RVTRVKQCRCISIDLD

SEQ ID NO:3 (Ab 7326 HCDR1 combined no Chothia & Kabat)

GYTFTDYYMH

SEQ ID NO:4 (Ab 7326 HCDR1 Kabat)

DYYMH

SEQ ID NO:5 (Ab 7326 HCDR2 Kabat)

LVDPEDGETIYAEKFQG

SEQ ID NO:6 (Ab 7326 HCDR3 Kabat)

DARGSGSYYPNHFDY

SEQ ID NO:7 (Ab 7326 LCDR1 Kabat)

KS S QSVLYS SNNKNYLA

SEQ ID NO:8 (Ab 7326 LCDR2 Kabat)

WASTRES

SEQ ID NO:9 (Ab 7326 LCDR3 Kabat)

- 29 043762

QQYYDTPT

SEQ ID NO:10 (heavy chain variable region variant 1 of Ab 7326)

QVQLVE S GAEVKKPGATVKIS CKVS GYT FT DYYMHWVQQAPGKGLEWMGLVDPE DGE TI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS S

SEQ ID NO:11 (Ab 7326 light chain variable region variant 1)

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

SEQ ID NO:12 (heavy chain variable region variant 2 of Ab 7326)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS S

SEQ ID NO:13 (Ab 7326 light chain variable region option 2)

DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

SEQ ID NO:14 (Mouse Full Length IgGl Heavy Chain Option 1)

QVQLVE S GAEVKKPGATVKIS CKVS GYT FT DYYMHWVQQAPGKGLEWMGLVDPE DGE TI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQ SDLYT LSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDV LTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL NGKE FKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTC MITDFFPEDIT VEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLS HSPGK

SEQ ID NO:15 (Mouse Full Length IgGl Light Chain Option 1)

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIF PPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERH NSYTCEATHKTSSTSPIVKSFNRNEC

SEQ ID NO:16 (Human Heavy Chain Option 2

- 30 043762 full-length IgGl)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

SEQ ID NO:17 (human full length IgG1 light chain variant 2)

KADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:18 (Fab Heavy Chain Option 1)

QVQLVE S GAEVKKPGATVKIS CKVS GYT FT DYYMHWVQQAPGKGLEWMGLVDPE DGE TI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

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

KADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

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

AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSF YIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

SEQ ID NO: 21 (sequence of SEQ ID NO:1 Mature Gremlin-1 without signal peptide amino acids 1-21)

KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHV TERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPK KFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

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

QVQLVE S GAEVKKPGATVKIS CKVS GYT FT DYYMHWVQQAPGKGLEWMGLVDPE DGE TI

- 31 043762

YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLM ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQK SLSLSLGK

SEQ ID NO:23 (human IgG4P light chain variant 1)

KADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:24 (Human IgGl Heavy Chain DNA Option 1) caagtgcaactggtggaatccggggccgaagtgaaaaagcccggagccactgtgaagat ctcttgcaaagtgtccggctacaccttcaccgactattacatgcactgggtccagcaggcacct gggaagggccttgagtggatgggtctggtcgatcccgaggacggcga aactatctacgccgaga agttccagggtcgcgtcaecatcaccgccgacacttccaccgacaccgcgtaсаtggagctgtc cagcttgaggtccgaggacacagccgtgtactactgcgccacggatgctcggggaagcggcagc tactacccgaaccacttcgactactggggacagggcactctcgtgactgtctcgagcg cttcta caaagggcccctccgtgttcccgctcgctccatcatcgaagtctaccagcggaggcactgcggc tctcggttgcctcgtgaaggactacttcccggagccggtgaccgtgtcgtggaacagcggagcc ctgaccagcggggtgcacacctttccggccgtcttgcagtcaagcggcctttactcc ctgtcat cagtggtgactgtcccgtccagctcattgggaacccaaacctacatctgcaatgtgaatcacaa acctagcaacaccaaggttgacaagaaagtcgagcccaaatcgtgtgacaagactcacacttgt ccgccgtgcccggcacccgaactgctgggaggtcccagcgtctttctgttccctccaaagccga aaga cacgctgatgatctcccgcaccccggaggtcacttgcgtggtcgtggacgtgtcacatga ggacccagaggtgaagttcaattggtacgtggatggcgtcgaagtccacaatgccaaaactaag cccagagaagaacagtacaattcgacctaccgcgtcgtgtccgtgctcacggtgttgcatcagg attggctga acgggaaggaatacaagtgcaaagtgtccaacaaggcgctgccggcaccgatcga gaaaactatctccaaagcgaagggacagcctagggaacctcaagtctacacgctgccaccatca cgggatgaactgactaagaatcaagtctcactgacttgtctggtgaaggggtttaccctagcg acattgccgtggagtgggaatccaacggccag ccagagaacaactacaagactacccctccagt gctcgactcggatggatcgttcttcctttactcgaagctcaccgtggataagtcccggtggcag cagggaaacgtgttctcctgctcggtgatgcatgaagccctccataaccactatacccaaaagt cgctgtccctgtcgccgggaaag

- 32 043762

SEQ ID NO:25 (human IgGl light chain DNA variant 1) gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccac cattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatgg tatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccg cgaatccg gggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcact gcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaa ggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccg acgag cagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcga ggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcacc gagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgact acgagaagcacaaggtgt acgcctgcgaagtgacccaccaggcctgtccagccccgtgaccaa gtccttcaaccggggcgagtgc

SEQ ID NO:26 (Human IgG4P heavy chain DNA variant 1) caagtgcaactggtggaatccggggccgaagtgaaaaagcccggagccactgtgaagat ctcttgcaaagtgtccggctacaccttcaccgactattacatgcactgggtccagcaggcacct gggaagggccttgagtggatgggtctggtcgatcccgaggacggc gaaactatctacgccgaga agttccagggtcgcgtcaecatcaccgccgacacttccaccgacaccgcgtacatggagctgtc cagcttgaggtccgaggacacagccgtgtactactgcgccacggatgctcggggaagcggcagc tactacccgaaccacttcgactactggggacagggcactctctcgtgactgtctcgagc gcttcta caaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgc tctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgcc ctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactcc ctgtcct ccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaa gccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgc cctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccc tgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccga ggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagag gaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctga acggcaaagag tacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccat ctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagag atgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccg tggaatgggagtccaacggccagcccgagaacaacta caagaccaccccccctgtgctggacag cgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaac gtcttctcctgctccgtgatgcacgaggccctgcacaaccactacccagaagtccctgtccc tgagcctgggcaag

- 33 043762

SEQ ID NO:27 (Human IgG4P light chain DNA variant 1) gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccac cattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatgg tatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacc cgcgaatccg gggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcact gcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaa ggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccg ac gagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcga ggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcacc gagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgact acgagaagcacaaggt gtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaa gtccttcaaccggggcgagtgc

SEQ ID NO:28 (Mouse Full Length IgG1 Heavy Chain Variant 2)

LSSSVTV PSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDV LTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL NGKE FKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDIT VEW QWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLS HSPGK

SEQ ID NO:29 (Mouse Full Length IgGl Light Chain Variant 2)

DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIF PPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERH NSYTCEATHKTSSTSPIVKSFNRNEC

SEQ ID NO:30 (Human Full Length IgGl Heavy Chain Option 1)

QVQLVE S GAEVKKPGATVKIS CKVS GYT FT DYYMHWVQQAPGKGLEWMGLVDPE DGE TI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV

- 34 043762

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

SEQ ID NO:31 (human full-length IgG1 light chain variant 1)

KADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

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

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

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

KADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:34 (Human IgG4P heavy chain variant 2)

QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETI YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK

SEQ ID NO:35 (human IgG4P light chain variant 2)

KADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

Claims (18)

1. An anti-gremlin-1 antibody that contains a combination of the sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 with SEQ ID NO:4/5/6/7/8/9 or with SEQ ID NO:3/5/ 6/7/8/9. 2. The antibody according to claim 1, which contains a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12 and/or a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13. 3. The antibody according to claim 2, which contains a pair of sequences HCVR and LCVR with SEQ ID NO: 10/11 or 12/13. 4. The antibody according to claim 3, wherein the sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 consist of SEQ ID NO:4/5/6/7/8/9 or SEQ ID NO:3/5/6/ 7/8/9, and the remaining portions of HCVR and LCVR are at least 95% identical to SEQ ID NO:10, 11, 12 and/or 13. 5. An antibody according to any one of claims 1 to 4, which contains a heavy chain of SEQ ID NO:14, 16, 18, 22, 28, 30, 32 or 34 and/or a light chain of SEQ ID NO:15, 17, 19, 23, 29, 31, 33 or 35. 6. An antibody according to claim 5, which contains a pair of heavy and light chains SEQ ID NO: 14/15, 16/17, 18/19, 22/23, 28/29, 30/31, 32/33 or 34/35 . 7. The antibody according to claim 6, wherein the sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 consist of SEQ ID NO:4/5/6/7/8/9 or SEQ ID NO:3/5/6/ 7/8/9, and the remaining portions of the heavy and light chains are at least 95% identical to SEQ ID NO:14, 15, 16 and/or 17, respectively. 8. An antibody according to any one of claims 1 to 7, which is a chimeric human antibody or a humanized antibody. 9. Antibody according to any one of claims 1 to 8, which is a Fab, modified Fab, Fab', - 35 043762 modified Fab', F(ab')2, Fv, single domain antibody or scFv. 10. An isolated polynucleotide encoding an antibody according to any one of claims 1 to 9. 11. An expression vector carrying the polynucleotide according to claim 10. 12. A host cell containing the vector according to claim 11. 13. A method for producing an antibody according to any one of claims 1 to 9, including culturing the host cell according to claim 12 under conditions allowing the production of the antibody, and recovering the resulting antibody. 14. A pharmaceutical composition containing an antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable adjuvant and/or carrier. 15. Use of an antibody according to any one of claims 1 to 9 for the treatment/prevention of renal fibrosis, such as diabetic nephropathy, idiopathic pulmonary fibrosis or pulmonary arterial hypertension. 16. Use of a pharmaceutical composition according to claim 14 for the treatment/prevention of angiogenesis or cancer. 17. A method of treating or preventing renal fibrosis, such as diabetic nephropathy, idiopathic pulmonary fibrosis or pulmonary arterial hypertension, comprising administering a therapeutically effective amount of an antibody according to claims 1 to 9 or a pharmaceutical composition according to claim 14 to a patient. 18. A method of treating or preventing angiogenesis or cancer, comprising administering a therapeutically effective amount of an antibody according to claims 1 to 9 or a pharmaceutical composition according to claim 14 to a patient.