Inhibitors and their uses

Abstract

The present invention relates to an inhibitor of IGFBP5 and / or at least one of its receptors, which has an antifibrotic effect, a method for producing the same, a pharmaceutical composition containing the inhibitor, and use of the same. In particular, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to human IGFBP5 and / or at least one of its receptors.

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of IGFBP5 and / or at least one of its receptors, which has an antifibrotic effect, a method for producing the same, a pharmaceutical composition containing the inhibitor, and use of the same. In particular, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to human IGFBP5 and / or at least one of its receptors. [Background technology]

[0002] IGFBP5 Insulin-like growth factor binding proteins (IGFs) constitute a family of six binding proteins that regulate the bioavailability of insulin-like growth factors (IGFs). Among them, insulin-like growth factor binding protein 5 (IGFBP5) is the most conserved member of the IGFBP family (Schneider et al., Journal of Endocrinology 172, 423-440 (2002)). It binds to IGFs with high affinity and forms a ternary complex with IGF-1 or -2 and the acid-labile subunit (ALS) (Baxter et al., Am J Physiol Endocrinol Metab. 278: E967-E976 (2000)).

[0003] In addition to its ability to regulate IGF availability, IGFBP5 has also been shown to have IGF-independent functions (Schneider et al., Journal of Endocrinology 172, 423-440 (2002)). Indeed, it can promote fibrosis directly by inducing the expression of extracellular matrix (ECM) genes and indirectly by increasing the levels of other growth factors with profibrotic activity, such as connective tissue growth factor (CTGF), resulting in further increased ECM production. In addition to regulating ECM and growth factor genes, IGFBP5 also increases the expression of lysyl oxidase (LOX), an enzyme responsible for matrix cross-linking, making ECM more resistant to proteolysis. Furthermore, IGFBP5 can bind to ECM components and protect them from degradation, thereby promoting ECM accumulation and fibrosis and altering tissue structure and function. Furthermore, IGFBP5 promotes its own expression, creating a positive feedback loop (Nguyen et al., Frontiers in Endocrinology. 9: 601 (2018)). IGFBP5 is a key mediator of fibrosis, upstream of known profibrotic factors, similar to transforming growth factor-β (TGF-β) signaling. All of these data strongly suggest that IGFBP5 exerts its profibrotic activity by directly inducing the expression of most ECM and profibrotic genes. Finally, whereas most drugs target only a single profibrotic factor downstream of the cascade, we were able to inhibit the entire pathway by targeting IGFBP5, resulting in the downregulation of several profibrotic factors and ECM genes.

[0004] To the best of the inventors' knowledge, there are no commercially available monoclonal antibodies against IGFBP5 that are capable of inhibiting the profibrotic activity of IGFBP5 and other deleterious effects on target tissues / cells.

[0005] IGFBP5 in idiopathic pulmonary fibrosis Idiopathic pulmonary fibrosis (IPF) is a non-neoplastic chronic pulmonary syndrome characterized by abnormal accumulation of fibroblasts / myofibroblasts and progressive abnormal remodeling of the lung parenchyma, with subsequent scarring and disruption of its structure and function. Despite significant recent advances, current treatment regimens are not very promising, with median survival rates of less than three years from the date of diagnosis (Sureshbabu et al., Pulmonary Medicine. 517687 (2011)). A key event in IPF is the abnormal proliferation and migration of fibroblasts into the alveolar space after lung injury. Under normal circumstances, fibroblasts are important for wound healing and connective tissue generation, but their function in fibrotic lungs is dysregulated, leading to the formation of fibroblastic lesions composed of highly proliferative fibroblasts, certain immune cells, and excessive ECM protein deposition. The results of these processes are disproportionate levels of scar tissue, altered alveolar architecture, changes in lung epithelial structure, stiffening of functional lung tissue, loss of lung gas exchange function, and dramatically reduced blood oxygenation (Coward et al. Ther. Adv. Respir. Dis. 4, 367-388 (2010)).

[0006] IGFBP5 has been shown to be increased in the extracellular environment when primary fibroblasts are cultured from fibrotic lung tissue of patients with IPF, and IGFBP5 is deposited in the ECM produced by these cells.

[0007] Moreover, adenovirus-mediated expression of IGFBP5 or addition of recombinant IGFBP5 leads to increased ECM production by normal adult primary lung fibroblasts (Pilewski et al. Am J Pathol 166(2):399-407 (2005)).

[0008] Interestingly, a transgenic mouse model of skin fibrosis has also been observed to utilize IGFBP5 overexpression to promote a profibrotic environment leading to fibroblast activation, myofibroblast transformation, and increased ECM deposition (Yasuoka et al., Am. J. of Pathology 169(5):1633-42. (2006)). Using this model, it was shown that IGFBP5 exerts its profibrotic effects in an IGF-independent manner (Yasuoka et al., Plos One 9(2) 2014).

[0009] IGFBP5 in scleroderma Systemic sclerosis (SSc) is a rare, systemic autoimmune connective tissue disease characterized by vasculopathy, immune dysregulation, and progressive fibrosis, primarily affecting the skin, gastrointestinal tract, lungs, heart, and kidneys (Henes et al., Haematologica. (2020)). It is associated with high disease-related mortality, and the main causes of death are related to cardiac, pulmonary, and renal damage. Effective treatments to prevent or halt the progression of fibrosis in SSc and other fibrotic diseases remain lacking (Tyndall et al., Ann Rheum Dis. 69(10):1809-1815 (2010)).

[0010] Excessive fibrosis of the skin and internal organs results from fibroblast proliferation and excessive production of extracellular matrix (ECM) (Henes et al., Haematologica. (2020)). Fibrotic changes lead to the destruction of normal tissue architecture in the skin and other organs. IGFBP5 is involved in the early stages of fibrosis and appears to be a causative event in ECM production, suggesting its involvement in the development of fibrosis in SSc. Indeed, IGFBP5 expression at the mRNA and protein levels has been shown to be increased in vitro in primary fibroblasts cultured from affected skin of patients with SSc compared with skin from healthy twins. IGFBP5 has also been demonstrated to induce collagen and fibronectin (FN1) production from fibroblasts and fibroblast / myofibroblast transdifferentiation in vitro and in vivo (Yasuoka et al., American Journal of Pathology 169(5):1633-42. (2006)). Furthermore, in vivo overexpression of IGFBP5 using a replication-deficient adenovirus induced skin fibrosis in mice, including increased dermal thickness and collagen bundle thickness (Henes et al., Haematologica. (2020)). Increased expression of α-smooth muscle actin (α-SMA) and vimentin in skin fibroblasts was also evident upon overexpression of IGFBP5.

[0011] IGFBP5 is overexpressed in SSc and IPF (Nguyen XX, Muhammad L, Nietert PJ and Feghali-Bostwick C (2018) Front. Endocrinol. 9:601.doi: 10.3389 / fendo.2018.00601), suggesting that strategies to inhibit IGFBP5 function may be effective for ameliorating fibrosis. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2002 / 020565 [Patent Document 2] US20040132028 [Patent Document 3] U.S. Patent Application Publication No. 2003 / 0157108 [Patent Document 4] US2004 / 0093621 [Patent Document 5] WO2003 / 011878 [Patent Document 6] U.S. Patent No. 6,602,684 [Patent Document 7] US2005 / 0123546 [Patent Document 8] WO1997 / 30087 [Patent Document 9] WO1998 / 58964 [Patent Document 10] WO1999 / 22764 [Patent Document 11] U.S. Patent No. 7,521,541 [Patent Document 12] U.S. Patent No. 7,083,784 [Patent Document 13] U.S. Patent No. 8,323,962 [Patent Document 14] U.S. Patent No. 5,215,534 [Patent Document 15] U.S. Patent No. 9,248,242 [Patent Document 16] U.S. Patent No. 9,427,531 [Patent Document 17] U.S. Patent No. 9,566,395 [Patent Document 18] U.S. Patent No. 5,075,109 [Patent Document 19] U.S. Patent No. 4,452,775 [Patent Document 20] U.S. Patent No. 4,667,014 [Patent Document 21] U.S. Patent No. 4,748,034 [Patent Document 22] U.S. Patent No. 5,239,660

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Non-licensed literature

[0013] [Non-licensed document 1] J. Mol. Biol. 332, 489~503 (2003) [Non-licensed document 2] PNAS 100(4), 1700~1705 (2003) [Non-licensed document 3] J. Mol. Biol. 369, 1015~1028 (2007)

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[0014] Therefore, there is a need for potent and specific IGFBP5 inhibitors, particularly neutralizing antibodies. [Means for solving the problem]

[0015] In the present invention, it has surprisingly been found that disruption, neutralization or inhibition of the IGFBP5 axis, including IGFBP5 and any of its receptors, has beneficial effects on fibrosis.

[0016] Disclosed herein are inhibitors of IGFBP5 and / or at least one of its receptors that have antifibrotic activity.

[0017] Antifibrotic activity can be measured by any means known in the art, such as those described herein. An isolated antibody or antigen-binding fragment thereof is considered to have an antifibrotic effect if fibrosis is inhibited by at least 10% when compared to a control condition without the antibody or antigen-binding fragment thereof. Preferably, the inhibitor is considered to have an antifibrotic effect if fibrosis is inhibited by at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, or 90% when compared to a control condition without the antibody or antigen-binding fragment thereof.

[0018] Preferably, the inhibitor of IGFBP5 and / or at least one of its receptors is for use in the treatment and / or prevention of fibrosis or a fibrotic condition.

[0019] More preferably, the inhibitor is a) polypeptides; b) a polynucleotide, or a polynucleotide encoding said polypeptide; c) a vector containing or expressing said polynucleotide; d) a host cell genetically engineered to express said polypeptide or said polynucleotide; e) small molecules; f) a peptide, protein, antibody, antisense oligonucleotide, siRNA, antisense expression vector, or recombinant virus; is selected from the group consisting of:

[0020] More preferably, the inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to human IGFBP5 or at least one of its receptors and has an anti-fibrotic effect.

[0021] Preferably, the inhibitor inhibits, reduces or neutralizes the formation of αSMA induced by profibrotic mediators and / or inhibits, reduces or neutralizes the formation of FN1 induced by profibrotic mediators.

[0022] Preferably, the inhibitor is an isolated antibody or antigen-binding fragment thereof that binds to human IGFBP5 and has anti-fibrotic activity.

[0023] Preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 5; ii. a CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, and 11; and iii. a CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26; and / or b. A light chain variable domain (VL) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36; ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47; and iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60; Includes.

[0024] More preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. A heavy chain variable domain (VH) comprising: i. A CDR1 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 4.1 (Tables 6, 7) and defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 4.1 (Tables 6, 7) and defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 4.1 (Tables 6, 7) and defined using the abysis tool analysis (www.abysis.org); and / or b. A light chain variable domain (VL) comprising: i. A CDR1 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 4.1 (Tables 6, 7) and defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 4.1 (Tables 6, 7) and defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 4.1 (Tables 6, 7) and defined using the abysis tool analysis (www.abysis.org); Includes.

[0025] More preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. A heavy chain variable domain (VH) comprising: i. A CDR1 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 5.1 (Tables 8, 9) and defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 5.1 (Tables 8, 9) and defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 5.1 (Tables 8, 9) and defined using the abysis tool analysis (www.abysis.org); and / or b. A light chain variable domain (VL) comprising: i. A CDR1 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 5.1 (Tables 8, 9) and defined using the abysis tool analysis (www.abysis.org); ii. A CDR2 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 5.1 (Tables 8, 9) and defined using the abysis tool analysis (www.abysis.org); and iii. A CDR3 sequence of an amino acid sequence preferably selected from the group consisting of the sequences shown in Table 5.1 (Tables 8, 9) and defined using the abysis tool analysis (www.abysis.org); Includes.

[0026] More preferably, the isolated antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable domain sequence of the amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75; or b. a light chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90; or c. The heavy chain variable domain of (a) and the light chain variable domain of (b). Includes.

[0027] More preferably, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01, and preferably, the isolated antibody or antigen-binding fragment thereof is antibody B06 or B09.

[0028] More preferably, the isolated antibody or antigen-binding fragment thereof comprises a VH CDR1, CDR2, and CDR3 selected from Table 2 and a VL CDR1, CDR2, and CDR3 selected from Table 3.

[0029] More preferably, the isolated antibody or antigen-binding fragment thereof has an antibody activity of 10 to 10 -8 It has an affinity constant of M or less.

[0030] (a) specifically binds to an epitope on IGFBP5, wherein the epitope is the same as or similar to the epitope recognized by monoclonal antibodies A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01 defined in Tables 2 to 8 (Tables 2 to 12); or (b) cross-competes for binding with monoclonal antibodies A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01 as defined in Tables 2 to 8 (Tables 2 to 12); or (c) exhibits the same or similar binding affinity or specificity, or both, as any of A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G0 as defined in Tables 2 to 8 (Tables 2 to 12); or (d) has one or more biological properties of an antibody molecule described herein, e.g., an antibody molecule selected from any of A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01 as defined in Tables 2 to 8 (Tables 2 to 12); or (e) having one or more pharmacokinetic properties of an antibody molecule described herein, for example, an antibody molecule selected from A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01 as defined in Tables 2 to 8 (Tables 2 to 12); Also disclosed is an isolated antibody or antigen-binding fragment thereof.

[0031] In a preferred embodiment, the isolated antibody or antigen-binding fragment thereof described above comprises: (a) specifically binds to an epitope on IGFBP5, wherein the epitope is the same as or similar to the epitope recognized by monoclonal antibodies A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01 defined in Tables 2 to 8 (Tables 2 to 12); or (b) cross-competes for binding with monoclonal antibodies A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01 as defined in Tables 2 to 8 (Tables 2 to 12); or (c) exhibits the same or similar binding affinity or specificity, or both, as any of A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G0 as defined in Tables 2 to 8 (Tables 2 to 12); or (d) has one or more biological properties of an antibody molecule described herein, e.g., an antibody molecule selected from A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, or G01 as defined in Tables 2 to 8; or (e) has one or more pharmacokinetic properties of an antibody molecule described herein, for example, an antibody molecule selected from A09, B06, C02, D10, F05, B09, B11, C11, C12, D12, E02, E03, E05, F04, and G01 as defined in Tables 2 to 8 (Tables 2 to 12).

[0032] Preferably, the isolated antibody or antigen-binding fragment thereof defined above is a human or humanized antibody, and preferably it is an IgG2 or IgG4 antibody, preferably an IgG2 kappa antibody, an IgG2 lambda antibody, an IgG4 kappa antibody, or an IgG4 lambda antibody.

[0033] Preferably, the inhibitors described above reduce or inhibit the binding of IGFBP5 to at least one of its receptors, preferably the at least one receptor being α2β1 integrin or αvβ6 integrin.

[0034] More preferably, the inhibitor as defined above i) reduces or inhibits extracellular matrix production, and / or ii) reduces or inhibits extracellular matrix deposition, and / or iii) reduces or inhibits collagen and / or fibronectin production in primary lung fibroblasts, and / or iv) reduces or inhibits collagen and / or fibronectin deposition in primary lung fibroblasts. Disclosed herein is an isolated polynucleotide comprising at least one sequence encoding the antibody or antigen-binding fragment thereof as defined above, preferably said polynucleotide is a cDNA.

[0035] Disclosed herein is a vector comprising the polynucleotide defined above, preferably the vector is selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector, and a recombinant expression vector. Disclosed herein is an isolated cell comprising the polynucleotide or vector defined above, preferably the isolated cell is a hybridoma or a Chinese hamster ovary (CHO) cell or a human embryonic kidney cell (HEK293).

[0036] Preferably, the inhibitor, antibody or antigen-binding fragment thereof, or polynucleotide, or vector, or cell as defined above is for use as a medicament, preferably for use in the treatment and / or prevention of fibrosis or fibrotic conditions, preferably scleroderma or pulmonary fibrosis, preferably morphea, fibrosis as a result of graft-versus-host disease (GVHD), keloids and hypertrophic scars, subepithelial fibrosis, endocardial fibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, post-operative scarring, asthma, abnormal wound healing, and fibrosing syndrome, idiopathic interstitial pneumonia (CFA), and idiopathic pulmonary fibrosis (IPF).

[0037] Disclosed herein is a pharmaceutical composition comprising the inhibitor, the isolated antibody or antigen-binding fragment thereof, or polynucleotide, or vector, or cell described above, and a pharmaceutically acceptable carrier, preferably for use in treating fibrosis or a fibrotic condition, and preferably further comprising a therapeutic agent.

[0038] The therapeutic agents can be nintedanib and pirfenidone.

[0039] Disclosed herein are antibodies and antigen-binding fragments thereof that have anti-fibrotic activity.

[0040] In some embodiments, methods of inhibiting fibrosis in vivo or in vitro are disclosed. In additional embodiments, methods of treating fibrosis in a subject are disclosed. In some specific, non-limiting examples, the subject has scleroderma or pulmonary fibrosis.

[0041] The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures. [Brief explanation of the drawings]

[0042] [Figure 1] Effect of anti-IGFBP5 mAbs on fibroblast-to-myofibroblast transition (FMT) assay with TGF-β1 exposure. Human primary lung fibroblasts from three IPF donors (IPF05, IPF06, and IPF07) were subjected to FMT with 1.25 ng / mL TGF-β1. Anti-IGFBP5 mAbs, B06 (A) and B09 (B), were tested in the FMT assay to check their ability to inhibit αSMA expression. Vehicle 2: Cells treated with 3.6% PBS and 1.25 ng / mL TGF-β1. [Figure 2] Effect of anti-IGFBP5 mAbs on epithelial-to-mesenchymal transition (EMT) assays with TGF-β1 exposure. Human primary bronchial epithelial cells (HBECs) from three IPF donors (IPF05, IPF06, and IPF07) were induced to undergo EMT with 5 ng / mL TGF-β1. Anti-IGFBP5 mAbs B06 (A) and B09 (B) were tested in EMT assays to check their ability to inhibit FN1 expression. Anti-IGFBP5 mAb B06 demonstrated concentration-dependent inhibition of TGF-β1-mediated FN1 expression in tissues from IPF patient donors. Vehicle 2: Cells treated with 3.6% PBS and 5 ng / mL TGF-β1. DETAILED DESCRIPTION OF THE INVENTION

[0043] Unless otherwise specified, terminology is used according to conventional usage in the art.

[0044] The antibody of the present invention specifically binds to human IGFBP5.

[0045] As described herein, these antibodies are collectively referred to as "anti-IGFBP5 antibodies." All such antibodies are encompassed by the description herein. Each antibody can be used alone or in combination in the methods of the present invention.

[0046] By "an antibody that specifically binds to" IGFBP5 is intended that the antibody does not substantially cross-react with another non-homologous human peptide. By "does not substantially cross-react," it is intended that the antibody or fragment has a binding affinity for the non-homologous protein that is less than 10%, more preferably less than 5%, and even more preferably less than 1% of the binding affinity for IGFBP5.

[0047] In various embodiments, an antibody that "specifically binds" to IGFBP5, as used herein, refers to an antibody that specifically binds to IGFBP5, e.g., an antibody that ... -6 Less than M, approximately 5 x 10 -7 Less than M, approximately 3 x 10 -7 Less than M, approximately 2 x 10 -7 Less than M, about 10 -7 Less than M, approximately 9 x 10 -8 Less than M, approximately 8 x 10 -8 Less than M, approximately 7 x 10 -8 Less than M, approximately 6 x 10-8 Less than M, approximately 5 x 10 -8 Less than M, approximately 4 x 10 -8 Less than M, approximately 3 x 10 -8 Less than M, approximately 2 x 10 -8 Less than M, about 10 -8 Less than M, approximately 5 x 10 -9 Less than M, approximately 4 x 10 -9 Less than M, approximately 3 x 10 -9 Less than M, approximately 2 x 10 -9 Less than M, about 10 -9 Less than M or about 5 x 10 -10 K of M D The present invention includes an antibody that binds to human IGFBP5 in the

[0048] The term "antibody" is used herein in its broadest art-recognized sense to include all polypeptides described as antibodies in Sumit G, Wei W, Tsutomu and Satoshi O, Antibodies 2013; 2: 452-500, which is incorporated herein by reference.

[0049] For example, the term "antibody" as used herein includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (antigen-binding fragments), so long as the fragment exhibits the desired antigen-binding activity.

[0050] As used herein, terms such as "antigen-binding fragment" of an antibody or equivalently "antigen-binding portion" of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that comprises a portion of an antibody and specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from intact antibody molecules using, for example, any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding antibody variable and, where appropriate, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, by using chemical or molecular biology techniques to align one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[0051] When using intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different antigen-binding moieties, where each antigen-binding moiety can specifically bind to a separate antigen or a different epitope on the same antigen.

[0052] In certain embodiments, an antigen-binding fragment of an antibody comprises at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may, in various embodiments, consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies may, in various embodiments, comprise homodimers or heterodimers (or other multimers) of any of the configurations of variable and constant domains listed above in non-covalent association with each other (e.g., via disulfide bonds) and / or with one or more monomeric VH or VL domains.

[0053] The term "antigen-binding fragment" of an antibody further includes single domain antibodies.

[0054] Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. In some embodiments, single domain antibodies are derived from the variable domain of an antibody heavy chain from a camelid (also referred to as a nanobody or VHH fragment). In some embodiments, single domain antibodies are spontaneous human heavy chain variable domains (aVH) or VNAR fragments derived from sharks.

[0055] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies and bivalent nanobodies), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0056] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally will contain at least one CDR adjacent to, or in frame with, one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be positioned in any suitable orientation relative to each other. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0057] The term "antibody" as used herein also encompasses ADCs (antibody drug conjugates) and payload-fused antibodies.

[0058] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, including antigen-binding antibody fragments, and scaffold antigen-binding proteins.

[0059] The term "antigen-binding moiety" refers to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. Antigen-binding moieties include antibodies and antigen-binding fragments thereof, such as scFvs, that can specifically bind to an antigen on a target cell. In certain embodiments, an antigen-binding moiety can direct an entity to which it is attached, such as a cell, to a target site. In addition, antigen-binding moieties that can specifically bind to a target cell antigen include binding domains based on scaffold antigen-binding proteins, as defined herein below, such as designed repeat proteins or designed repeat domains, such as designed ankyrin repeat proteins (DARPins) (see, e.g., WO2002 / 020565) or lipocalins (anticalins).

[0060] Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. They can be engineered to bind to different target antigens by randomizing residues in the first α-helix and β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003), and J. Mol. Biol. 369, 1015-1028 (2007), as well as US20040132028.

[0061] In certain embodiments, the antibodies and antigen-binding molecules provided herein are modified to increase or decrease the degree of glycosylation of the antigen-binding portion. Glycosylation variants of the molecule can be conveniently obtained by altering the amino acid sequence to create or remove one or more glycosylation sites. If the antigen-binding molecule contains an Fc region, the carbohydrate attached thereto can be altered. In one aspect, variants of antigen-binding molecules are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylation variants can have improved ADCC function; see, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Additional variants of the antigen-binding molecules of the present invention include those with bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved ADCC function, as described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0062] In certain embodiments, it may be desirable to create cysteine-engineered variants of the antibodies or antigen-binding molecules of the present invention, e.g., "thioMAbs," in which one or more residues of the molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites in the molecule. By replacing these residues with cysteine, reactive thiol groups are consequently positioned at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antigen-binding molecules can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0063] In certain embodiments, the antibodies or antigen-binding molecules provided herein can be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Suitable moieties for derivatization of the antibodies or antigen-binding molecules include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when two or more polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used in therapy under defined conditions.

[0064] In another aspect, a conjugate of an antibody and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, including, but not limited to, a wavelength that does not harm normal cells but heats the nonproteinaceous moiety to a temperature that kills cells in close proximity to the antibody-nonproteinaceous moiety. In another aspect, an immunoconjugate of an antigen-binding molecule provided herein can be obtained. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, such as, but not limited to, a cytotoxic drug.

[0065] The constant region of an antibody is important for the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Therefore, the antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity. In certain embodiments, the constant region is an IgG1, IgG2, IgG3, or IgG4 constant region.

[0066] In various embodiments, the present invention encompasses antibodies with one or more mutations in the hinge, CH2, or CH3 regions, which may be desirable, for example, to improve the yield of a desired antibody form during manufacturing. In some embodiments, for example, the antibodies described herein comprise a human IgG4 constant region. In certain embodiments, the IgG4 constant region has a single amino acid substitution in the hinge region of a human IgG4 hinge that reduced Fab arm exchange (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge.

[0067] In certain embodiments, the antibody comprises one or more mutations in the constant region that increase serum half-life, such as those described in U.S. Pat. Nos. 7,083,784, 8,323,962, and Dall'Aqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Hinton et al., J. Immunology 176:346-356 (2006); Yeung et al., J. Immunology 182:7663-7671 (2009); and Petkova et al., Intn'l Immunology, 18: 1759-1769 (2006), which are incorporated herein by reference in their entireties.

[0068] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies featured in the present invention may, in various embodiments, include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, and in some embodiments, in CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies derived from the germline of another mammalian species, such as a mouse, in which CDR sequences have been grafted onto human framework sequences.

[0069] The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from recombinant, combinatorial human antibody libraries (described further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes, or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, where animals transgenic for human Ig sequences are used, in vivo somatic mutation) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0070] "Isolated antibody," as used herein, refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from the tissue or cell in which it naturally occurs or is produced, is an "isolated antibody." In various embodiments, an isolated antibody also includes an antibody in situ within a recombinant cell. In other embodiments, an isolated antibody is an antibody that has been subjected to at least one purification or isolation step. In various embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0071] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have two or more epitopes. Thus, different antibodies may bind to different areas on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are created by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are created by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes can include carbohydrate, phosphoryl, or sulfonyl moieties on the antigen.

[0072] The anti-IGFBP5 antibodies described herein and useful for the methods featured herein may, in various embodiments, contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains when compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences, for example, from public antibody sequence databases.

[0073] The present invention, in various embodiments, includes antibodies and methods involving the use of antibodies, and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations").

[0074] Numerous antibodies and antigen-binding fragments can be constructed that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues in the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a different germline sequence from the germline sequence from which the antibody was originally derived). Moreover, the antibody can have any combination of two or more germline mutations in the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments having one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Use of antibodies and antigen-binding fragments obtained in this general manner is encompassed within the present invention.

[0075] The present invention also includes methods involving the use of anti-IGFBP5 antibodies and anti-IGFBP5 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes the use of anti-IL-6R antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0076] The term "bioequivalent," as used herein, refers to a molecule that has similar bioavailability (rate and extent of availability) after administration of the same molar dose under similar conditions (e.g., the same route of administration), such that the effects, both in terms of efficacy and safety, can be expected to be substantially the same as those of the compared molecule. Two pharmaceutical compositions containing an anti-IGFBP5 antibody are bioequivalent if they are pharmaceutically equivalent, meaning that they contain the same amount of active ingredient (e.g., an IGFBP5 antibody) in the same dosage form via the same route of administration and meet the same or comparable standards. Bioequivalence can be determined, for example, by in vivo studies comparing the pharmacokinetic parameters of the two compositions. Parameters commonly used in bioequivalence testing include peak plasma concentration (Cmax) and area under the plasma drug concentration-time curve (AUC).

[0077] In certain embodiments, the present invention relates to antibodies and methods comprising administering to a subject an antibody comprising a heavy chain variable region comprising a sequence selected from the group of SEQ ID NO: 61 to SEQ ID NO: 75 and a light chain variable region comprising a sequence selected from the group of SEQ ID NO: 76 to SEQ ID NO: 90. The disclosure provides pharmaceutical compositions comprising such antibodies and methods of using these compositions.

[0078] In various embodiments, the antibody is administered to a subject in a formulation suitable for intravenous or subcutaneous injection, including suitable carriers, excipients, and other agents to provide improved mobility, delivery, tolerability, etc.

[0079] Injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium customarily used for injections. Aqueous media for injections include, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, and the like, which can be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 20 or 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), and the like. Oily media include, for example, sesame oil, soybean oil, and the like, which can be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, and the like. The injectable preparations prepared in this manner can be filled into appropriate ampoules.

[0080] The antibodies according to the invention can be administered to a subject using any acceptable device or mechanism. For example, administration can be achieved using a syringe and needle, or by a reusable pen and / or autoinjector delivery device. The methods of the invention include the use of multiple reusable pens and / or autoinjector delivery devices to administer the antibodies (or pharmaceutical formulations comprising the antibodies). Examples of such devices include, but are not limited to, the AUTOPEN™ (Owen Mumford, Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX75 / 25™ pen, the HUMALOG™ pen, the HUMALIN70 / 30™ pen (Eli Lilly and Company, Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN IV ... STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany).Examples of disposable pens and / or autoinjector delivery devices that have use in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly & Co.), the SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), the HUMIRA™ pen (Abbott Labs, Abbott Park, IL), the DAI® autoinjector (SHL Group), and any autoinjector featuring PUSHCLICK™ technology (SHL Group).

[0081] In one embodiment, the antibody is administered by a pre-filled syringe. In another embodiment, the antibody is administered by a pre-filled syringe that includes a safety system. For example, the safety system prevents accidental needlestick injuries. In various embodiments, the antibody is administered by a pre-filled syringe that includes the ERIS™ safety system (West Pharmaceutical Services). See also U.S. Patent Nos. 5,215,534 and 9,248,242, which are incorporated herein by reference in their entirety.

[0082] In another embodiment, the antibody is administered by an autoinjector. In various embodiments, the antibody is administered by an autoinjector featuring PUSHCLICK™ technology (SHL Group, Inc.). In various embodiments, the autoinjector is a device that includes a syringe that allows for the administration of a dose of the composition and / or antibody to a subject. See also U.S. Pat. Nos. 9,427,531 and 9,566,395, which are incorporated by reference in their entireties.

[0083] According to the present invention, "subject" means a human subject or a human patient.

[0084] "Fibrosis" is the formation or development of excess fibrous connective tissue in an organ or tissue as a repair or reactive process, as opposed to the formation of fibrous tissue as a normal component of the organ or tissue. It is a major histopathological feature of a variety of clinical conditions. Many organs, including but not limited to the skin, lungs, heart, and gastrointestinal tract, are prone to pathological fibrosis. The present invention relates to inhibitors that specifically target the pathogenesis of fibrosis by targeting the pro-fibrotic mediator IGFBP5, and have "anti-fibrotic activity or effect."

[0085] Fibrosis is defined by the accumulation of fibrous connective tissue (components of the extracellular matrix (ECM), such as collagen and fibronectin) in or around inflamed or injured tissue, which can lead to permanent scarring, organ dysfunction, and ultimately death. Fibrosis is a pathological hallmark of most chronic inflammatory diseases. Fibrosis can affect nearly every tissue in the body (Schruf et al., Respiratory Research (2019) 20:87).

[0086] After activation by profibrotic stimuli, fibroblasts differentiate into a malignant phenotype and secrete excessive amounts of collagen-rich extracellular matrix, thereby constituting the primary pathological fibroblast phenotype (Geng et al. Respiratory Research (2015) 16:124).

[0087] Exemplary fibrotic conditions are scleroderma, idiopathic pulmonary fibrosis, morphea, fibrosis as a result of graft-versus-host disease (GVHD), keloids and hypertrophic scars, as well as subepithelial fibrosis, endocardial fibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, post-surgical scarring, asthma, abnormal wound healing, glomerulonephritis, and fibrosing syndromes.

[0088] "Idiopathic pulmonary fibrosis," also known as idiopathic interstitial pneumonia (CFA), is a chronic, progressive form of lung disease characterized by fibrosis of the lung's supporting structure (the interstitium). By definition, the term is used only when the cause of pulmonary fibrosis is unknown ("idiopathic"). When lung tissue from patients with IPF is examined microscopically by a pathologist, it displays a characteristic set of histologic / pathologic features known as usual interstitial pneumonia (UIP). UIP is characterized by progressive scarring of both lungs involving the lung's supporting structure (the interstitium).

[0089] Inhibiting or treating a disease: Inhibiting a disease, such as fibrosis, means inhibiting or slowing the full development of the disease. In some embodiments, inhibiting a disease means reducing a symptom of fibrosis, such as the formation of scar tissue, or increasing range of motion or reducing pain. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or a pathological condition associated with the disease, such as fibrosis.

[0090] "Scleroderma" is a chronic autoimmune disease characterized by fibrosis (or hardening), vascular changes, and autoantibodies. It has two major forms: a limited systemic sclerosis form, which includes limited scleroderma cutaneously, primarily affecting the hands, arms, and face, but frequently involving pulmonary hypertension. On the other hand, diffuse scleroderma cutaneously (or systemic sclerosis) progresses rapidly and affects large areas of the skin and one or more internal organs, frequently the kidneys, esophagus, heart, and lungs. Both forms of systemic sclerosis can be fatal. Other forms of scleroderma include systemic sclerosis, which involves systemic symptoms but no skin changes, and two localized forms that affect the skin but not internal organs: morphea and linear scleroderma. The disclosed antibodies can be used to treat any form of scleroderma.

[0091] Therapeutic methods and pharmaceutical compositions The antibodies disclosed herein can be used to treat fibrosis that occurs in several disease states. The antibodies disclosed herein can reduce fibrosis that has already occurred, resolve existing fibrosis, and / or reduce the rate or amount of additional fibrosis. In some embodiments, the antibodies are useful for reducing fibrosis during a pathogenic process in a subject.

[0092] Thus, in some embodiments, the method includes administering to a subject a therapeutically effective amount of one or more of the antibodies disclosed herein to reduce fibrosis. Any of the antibodies disclosed herein can be used to reduce fibrosis. In some embodiments, the antibody can be administered as a unit dose.

[0093] Suitable subjects include those with fibrosis of the skin or lungs, although fibrosis of any tissue can be treated using the methods disclosed herein. In one example, the subject has scleroderma. In other examples, the subject has idiopathic pulmonary fibrosis, morphea, fibrosis as a result of graft-versus-host disease (GVHD), keloid or hypertrophic scars, subepithelial fibrosis, endocardial fibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, post-surgical scarring, asthma, abnormal wound healing, glomerulonephritis, and fibrosing syndrome.

[0094] In further embodiments, the method is used to treat systemic forms of sclerosis, such as limited cutaneous scleroderma or diffuse cutaneous scleroderma (systemic sclerosis). The method can be used to treat localized forms of sclerosis, such as morphea and linear scleroderma.

[0095] The method can include selecting a subject in need of treatment, e.g., a subject with fibrosis, such as scleroderma, idiopathic pulmonary fibrosis, morphea, keloid scarring, hypertrophic scarring, or subepithelial fibrosis. In an exemplary application, a composition is administered to a subject with fibrosis, e.g., scleroderma, idiopathic pulmonary fibrosis, morphea, keloid scarring, hypertrophic scarring, or subepithelial fibrosis, or any of the disorders listed above, in an amount sufficient to reduce the fibrosis. Amounts effective for this use will depend on the severity of the disease, the patient's overall state of health, and the robustness of the patient's immune system. In one example, a therapeutically effective amount of the compound is an amount that provides either subjective relief or an objectively discernible improvement in symptoms, as noted by a clinician or other qualified observer.

[0096] Provided herein are methods for reducing skin thickness. The methods comprise administering a therapeutically effective amount of an antibody, thereby reducing skin thickness. In another embodiment, provided are methods for reducing pulmonary fibrosis. The methods comprise administering a therapeutically effective amount of an antibody, thereby reducing skin thickness. Any of the antibodies disclosed herein can be used in these methods.

[0097] Provided herein are methods for reducing αSMA or FN1 expression, such as transforming growth factor (TGF)-β-induced αSMA or FN1 expression. The methods include contacting cells with an effective amount of an antibody, thereby reducing αSMA or FN1 expression. The methods can be practiced in vivo or in vitro. In some embodiments, the methods include comparing the amount of αSMA or FN1 expression produced by cells contacted with the antibody to a control. The control can be a standard value or the amount of αSMA or FN1 produced by cells not contacted with the antibody, such as cells contacted with a carrier.

[0098] The antibodies disclosed herein can be administered either locally or systemically by any means known to those skilled in the art, such as intradermal, intrathecal, intramuscular, subcutaneous, intraperitoneal, or intravenous injection, although oral, nasal, transdermal, or anal administration is also contemplated. In one embodiment, administration is by subcutaneous, intradermal, or intramuscular injection. In another embodiment, administration is by intraperitoneal or intrathecal administration. To extend the time that the antibody is available to stimulate a response, the antibody can be provided as an implant, an oily injection, or a particulate system. The particulate system can be a microparticle, microcapsule, microsphere, nanocapsule, or similar particle (see, e.g., Banga, supra).

[0099] For skin treatment, at least a therapeutically effective amount of the antibody can be administered locally to the affected area of ​​the skin in the form of an ointment or the like (R.G.A. Jones and A. Marino, "Targeted localized use of therapeutic antibodies: a review of non-systemic, topical and oral applications", Crit. Rev. Biotechnol. 36(3):506-520 (2016)).

[0100] In one embodiment, the ointment is a completely homogeneous semi-solid topical preparation with an appropriate consistency for easy application to the skin. Such ointments can contain fats, fatty oils, lanolin, petrolatum, paraffin, wax, hard ointments, resins, plastics, glycols, higher alcohols, glycerol, water, or emulsifiers and suspending agents. Using these materials as a base allows the decoy compound to be uniformly mixed. Depending on the base, the mixture can be in the form of an oleaginous ointment, an emulsified ointment, or a water-soluble ointment. Oleaginous ointments use bases such as vegetable and animal oils, waxes, petrolatum, and liquid paraffin. Emulsified ointments are composed of an oily substance and water emulsified with an emulsifier. They can be either oil-in-water (O / W) or water-in-oil (W / O) forms. An oil-in-water (O / W) form can be a hydrophilic ointment. A water-in-oil (W / O) formulation is initially devoid of an aqueous phase and can contain hydrophilic petrolatum and purified lanolin, or it can contain a water-absorbing ointment (which contains an aqueous phase) and hydrated lanolin. A water-soluble ointment can contain a completely water-soluble macrogol base as its major component.

[0101] Pharmaceutically acceptable carriers include petrolatum, VASELINE®, etc., where the petrolatum contains 5% stearyl alcohol, or is petrolatum alone, or petrolatum containing liquid paraffin. Such carriers allow the pharmaceutical composition to be formulated in a suitable form for consumption, such as tablets, pills, sugar-coated drugs, capsules, liquid preparations, gels, ointments, syrups, slurries, and suspensions. When administered locally to cells in an affected area or tissue of interest, a polynucleotide encoding at least one C-terminal endostatin polypeptide or peptide can be administered in a composition containing a synthetic or natural hydrophilic polymer as a carrier. Examples of such polymers include hydroxypropyl cellulose and polyethylene glycol. One or more antibodies can be mixed with the hydrophilic polymer in a suitable solvent. The solvent is then removed by a method such as air drying, and the residue is then formed into a desired shape (e.g., a sheet) and applied to the target site. Preparations containing such hydrophilic polymers have low water content and are therefore easy to store. At the time of use, they absorb water and become a gel, which also facilitates storage. In the case of sheets, the firmness can be adjusted by mixing the polyhydric alcohol with the same hydrophilic polymers as those mentioned above, such as cellulose, starch, and their derivatives, or synthetic polymeric compounds. The resulting hydrophilic sheet can be used. A therapeutically effective amount of one or more antibodies can also be incorporated into bandages and wound dressings.

[0102] For administration by inhalation, the antibodies may conveniently be delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers by use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0103] In some embodiments, the antibody can be administered by inhalation. For example, the antibody can be administered in aerosolized form, such as by use of a nebulizer or a metered dose inhaler. Useful technologies include ultra-miniature pump nebulizers (such as the AEROGEN GO® system), jet nebulizers designed to produce a large fine particle fraction (such as the PARI LC STAR® system), jet nebulizers that produce less shear during nebulization (such as the HUDSON MICROMIST® system), and ultrasonic nebulizers (such as the DeVilbiss ULTRA-NEB® system).

[0104] The antibody can be dissolved in a carrier, such as saline, and nebulized using the device described above. The relevant aerosol can be collected using a NEXT GENERATION IMPACTOR® (NGI) (MSP Corp., Shoreview, MN), which uses a series of aerodynamic stages to separate and collect the aerosol into separate fractions based on droplet size. Because droplet size is the primary determinant of lung deposition location, this device allows for the specific isolation of the portion of the liquid aerosol that will deposit in the small airways and alveoli.

[0105] Aerosol particle size is often expressed in units of mass median aerodynamic diameter (MMAD), a parameter based on particle size, shape, and density. For spherical particles, MMAD is equal to MMD(p<1 / 2>), where MMD is the mass median diameter and r is the bulk density. For non-spherical particles, MMAD is equal to MMD(p / x)<1 / 2>, where X is the shape factor. Thus, particles with actual diameters greater than unit density will have actual diameters smaller than their MMAD.

[0106] The site of particle deposition within the respiratory tract is segmented based on particle size. In one embodiment, particles of about 1 micrometer to about 500 micrometers are used, for example, about 25 micrometers to about 250 micrometers, or about 10 micrometers to about 25 micrometers. In other embodiments, particles of about 1 micrometer to about 50 micrometers are used. For use in metered dose inhalers, for administration to lung particles of less than about 10 micrometers, for example, particles of about 2 micrometers to about 8 micrometers, for example, particles of about 1 micrometer to about 5 micrometers, for example, particles of about 2 micrometers to about 3 micrometers can be used.

[0107] A therapeutically effective amount of an antibody can be administered in a pharmaceutically acceptable carrier. Pharmacologically acceptable carriers (e.g., physiologically or pharmaceutically acceptable carriers) are well known in the art and include, but are not limited to, buffers at physiological pH (e.g., a pH of about 7.0 to about 8.0, or about 7.4). A specific, non-limiting example of a physiologically compatible buffer is phosphate-buffered saline. Other pharmacologically acceptable carriers include osmotic agents, which are particularly suitable for pharmaceutical preparations intended to be applied topically (e.g., to surgical wounds to promote healing).

[0108] The pharmaceutical compositions disclosed herein facilitate the use of at least one antibody to reduce fibrosis, either in vivo or ex vivo. Such compositions may be suitable for delivery of the active ingredient to any suitable subject and may be prepared by methods known per se, such as conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmacologically (e.g., physiologically or pharmaceutically) acceptable carriers and any appropriate auxiliary agents that facilitate processing of the active compound into a pharmaceutically usable preparation. Appropriate formulations depend on the chosen route of administration. Thus, for injection, the active ingredient may be formulated in an aqueous solution. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0109] For oral administration, the active ingredient can be combined with carriers suitable for incorporation into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like. The active ingredient can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Such compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents, such as suspending, stabilizing, and / or dispersing agents.

[0110] Other pharmacological excipients are known in the art.

[0111] Other delivery systems can include sustained-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated administration of the compositions of the present invention as described above, which increases convenience for both the patient and the physician. Many types of release delivery systems are available and known to those skilled in the art. These include polymer-based systems, such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Drug-containing microcapsules of the aforementioned polymers are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymeric systems, such as lipids containing sterols such as cholesterol, cholesterol esters, and fatty acids or neutral lipids, such as mono-, di-, and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to, (a) erosion systems in which at least one C-terminal endostatin polypeptide or polynucleotide encoding the peptide is contained within a matrix in some form, such as those described in U.S. Patent Nos. 4,452,775; 4,667,014; 4,748,034; 5,239,660; and 6,218,371, and (b) diffusion systems in which the active ingredient permeates through a polymer at a controlled rate, such as those described in U.S. Patent Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are suitable for implantation.

[0112] The use of long-term sustained-release implants may be particularly suitable for the treatment of chronic conditions such as sclerosis. As used herein, long-term sustained-release means that the implant is constructed and arranged to deliver therapeutic concentrations of the active ingredient for at least 30 days, preferably 60 days. Long-term sustained-release implants are well known to those skilled in the art and include some of the release systems described above. These systems have been described for use with oligodeoxynucleotides (see U.S. Pat. No. 6,218,371). For in vivo use, nucleic acids and peptides are preferably relatively resistant to degradation (e.g., by endonucleases and exonucleases). Therefore, modifications such as the addition of a C-terminal amide can be used.

[0113] The therapeutically effective amount of an antibody will depend on the antibody used, the subject being treated, the severity and type of affliction, and the method of administration. For example, a therapeutically effective amount of an antibody can vary from about 0.01 μg per kilogram (kg) of body weight to about 1 g per kilogram of body weight, e.g., from about 1 μg per kilogram (kg) to about 5 mg per kilogram of body weight, or from about 5 μg per kilogram to about 1 mg per kilogram of body weight. The exact dose can be easily determined by one skilled in the art based on the potency of the particular compound, the age, weight, sex, and physiological condition of the subject.

[0114] Single or multiple administrations of the composition are administered depending on the dosage and frequency required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, while in another embodiment, it can be applied periodically until a therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate the symptoms or signs of the disease without causing unacceptable toxicity to the subject. Systemic or local administration can be used.

[0115] In a further method, an additional agent is administered. In one embodiment, the administration is sequential. In another embodiment, the additional agent is administered simultaneously with the antibody.

[0116] For the treatment of scleroderma, examples of additional agents that can be used with the antibody include nifedipine, amlodipine, diltiazem, felodipine, or nicardipine. The investigational drug Gleevec is also used to treat scleroderma. Gleevec or other tyrosine kinase inhibitors can be used with the antibodies disclosed herein. Patients with pulmonary scleroderma benefit from oxygen therapy; the C-terminal endostatin polypeptides disclosed herein can be administered in this therapy. For the treatment of skin fibrosis and scleroderma, useful additional agents are d-penicillamine, colchicine, relaxin, steroids, and cyclosporine. In some embodiments, the additional agent is SAR100842 (see Allanore et al., Arthr. Rheum. 70(10):1634-1643 (2018)). C-terminal endostatin polypeptides can also be used in combination with immunosuppressants. Additionally, the antibodies can be used in conjunction with methotrexate, cyclophosphamide, azathioprine, mycophenolic acid, glitazones, endothelin receptor antagonists, or fulvestrant (ICI-182,780).

[0117] The present disclosure is illustrated by the following non-limiting examples. [Example]

[0118] Example 1 Monoclonal antibody development Monoclonal anti-IGFBP5 antibodies were discovered from a naive human phage display library. Nonspecific or cross-reactive antibody phages were first removed from the library by incubating the library in the presence of a) streptavidin beads and BSA, or b) streptavidin beads and BSA, IGFBP1 (R&D, Catalog No. 871-B1), IGFBP2 (R&D, Catalog No. 674-B2), IGFBP3 (R&D, Catalog No. 675-B3), IGFBP4 (R&D, Catalog No. 804-GB), and IGFBP6 (R&D, Catalog No. 876-B6).

[0119] Antibody phages that bound to the negative antigen were excluded from further selection. The depleted library was then selected against target antigen-specific antibodies. First, biotinylated IGFBP5 (R&D, human: catalog no. 875-B5, mouse: catalog no. 578-B5) was added to library preparations a) and b) in the presence of the negative antigen. Antibody phages that bound to the biotinylated target antigen were captured and recovered from the solution using magnetic streptavidin beads. To remove nonspecific or weakly bound antibody phage particles, the beads were washed multiple times with BSA solution (containing 0.05% Tween-20) and BSA. Antigen-specific antibody phages were eluted from the beads by trypsinization and rescued by infection with E. coli. After a short propagation period, the bacteria were superinfected with M13K07 helper phage to induce antibody phage amplification. The amplified phages were used for two additional selection cycles as described above. An overview of the antibody selection strategy is shown in Table 1.

[0120] [Table 1]

[0121] At the end of the discovery process, each selection result was screened for antigen-specific antibodies, and the binding properties of the monoclonal antibody clones were analyzed. For this purpose, 3072 single colonies were used for scFv antibody production. These were then examined for specific antigen binding by ELISA with streptavidin + biotinylated human IGFBP5, streptavidin + biotinylated mouse IGFBP5, and streptavidin + BSA. 842 clones showed cross-reactive binding between human and mouse IGFBP5 with a signal-to-noise ratio of >10. 192 of these ELISA-positive clones were selected for secondary screening. To that end, a second batch of scFv was generated and tested against a broader panel of antigens: streptavidin + biotinylated human IGFBP5, streptavidin + biotinylated mouse IGFBP5, streptavidin, BSA, human IGFBP1, human IGFBP2, human IGFBP3, human IGFBP4, human IGFBP5, and human IGFBP6.

[0122] Seventeen lead antibody clones were identified that showed binding to streptavidin-captured and directly immobilized human and mouse IGFBP5. No binding to human IGFBP1, human IGFBP2, human IGFBP3, human IGFBP4, or human IGFBP6 was detected. DNA sequence analysis of the 17 lead antibody clones revealed the presence of 15 candidates with unique antibody sequences (≥1 amino acid difference in the CDR). The scFv DNA sequences were amplified by PCR and cloned into the YUMAB mammalian scFv-Fc expression vector, resulting in a gene fusion of the scFv with human IgG4 Fc. Bacterial clones with correctly cloned scFv genes were used to isolate transfection-grade DNA. The DNA was used for transient transfection of HEK cells, which produced and secreted the antibody into the culture medium. The antibody was purified by affinity chromatography (protein A) and rebuffered in PBS. Protein concentration was determined by UV / VIS spectroscopy and purity was checked by Coomassie staining. Thirteen of the 15 antibodies could be successfully produced in the scFv-Fc format.

[0123] The sequences of 15 novel anti-IGFBP5 antibodies are reported in Tables 2–8 (Tables 2–12).

[0124] [Table 2]

[0125] [Table 3]

[0126] [Table 4]

[0127] [Table 5]

[0128] CDR definitions are also provided using the annotation tool from http: / / www.abysis.org / based on the complete VH and VL amino acid sequences defined in Tables 4 and 5.

[0129] For example, the VH or VL amino acid sequence of any antibody disclosed herein can be linked to the annotation tool, and Kabat-defined CDR sequences, IMGT, Chothia, AbM, or Contact-defined CDR sequences can be provided. Using the "All, side by side" function provides the defined CDR sequences.

[0130] Table 4.1 shows such sequences related to B06 VH and VL.

[0131] Table 4.1 (Tables 6, 7) B-06 VH and VL CDRs by different definitions sorted by inserting the aaVH (SEQ ID NO: 62) and VL (SEQ ID NO: 77) sequences in the www.abysis.org annotation tool V H

[0132] [Table 6]

[0133] V L

[0134] [Table 7]

[0135] Table 5.1 (Tables 8, 9) shows such sequences related to B09 VH and VL.

[0136] Table 5.1 (Tables 8, 9) B-09 VH and VL CDRs by different definitions sorted by inserting the aaVH (SEQ ID NO: 66) and VL (SEQ ID NO: 81) sequences in the www.abysis.org annotation tool V H

[0137] [Table 8]

[0138] V L

[0139] [Table 9]

[0140] [Table 10A]

[0141] [Table 10B]

[0142] [Table 10C]

[0143] [Table 11A]

[0144] [Table 11B]

[0145] [Table 12]

[0146] Methods for expressing recombinant proteins in CHO cells The corresponding B06 and B09 cDNAs were cloned into the vitoria vector system using conventional (non-PCR-based) cloning techniques. The vitoria vector plasmids were synthetic genes. Plasmid DNA was prepared under low-endotoxin conditions using anion-exchange chromatography. Sequence accuracy was verified by Sanger sequencing (up to two sequencing reactions per plasmid depending on the size of the cDNA).

[0147] Suspension-adapted CHO K1 cells (evitria) were used for production. Seeds were grown in eviGrow medium, a chemically defined, animal-component-free, serum-free medium. Cells were transfected using eviFect, a custom-made, proprietary transfection reagent from evitria. After transfection, cells were grown in eviMake, an animal-component-free, serum-free medium, at 37°C and 5% CO for 7 days. The supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter).

[0148] The antibody was purified using MabSelect™ SuRe™ with Dulbecco's PBS (Lonza BE17-512Q) as the wash buffer and 0.1 M glycine, pH 3.5 as the elution buffer, followed by size exclusion chromatography on a HiLoad Superdex 200 pg column with the final buffer as the running buffer.

[0149] Monomeric nature was determined by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7 μm 7.8×300 mm) with DPBS as the running buffer at 0.8 ml / min.

[0150] Example 2 Affinity measurement Octet BLI-based analysis To evaluate the binding affinity in more detail, affinity measurements were performed using biomolecular interaction analysis performed by the Octet platform (BLItz Systems), a Biolayer Interferometry (BLI) platform. To establish the assay, target monoclonal antibodies (mAbs, two identical antigen-binding moieties, 2 μg / ml in 1% BSA containing 0.05% Tween 20) were immobilized on an AHC biosensor via Fc, and their interactions with the antigens human IGFBP5 (R&D, Cat. No. 875-B5) and mouse IGFBP5 (R&D, Cat. No. 578-B5) at different concentrations were measured.

[0151] Affinity measurements of B06 and B09 full-length mAbs against the target human and mouse IGFBP5 are reported in Table 9.

[0152] [Table 13]

[0153] The newly generated anti-IGFBP5 mAb was 4 × 10 -10 The antibody exhibits good human antigen-binding affinity with a KD of less than M. The antibody also exhibits mouse cross-reactivity. This data confirms that mice can be considered a relevant animal species for testing monoclonal antibodies during preclinical development.

[0154] Example 3 Primary cell-based assays related to fibrosis (IPF) Fibrosis is a continuous tissue repair process characterized by the formation and deposition of a fibrillar collagen-rich extracellular matrix (ECM), leading to progressive remodeling in most tissues and organs. The formation of fibrous scar tissue is essential for restoring and maintaining the integrity of tissues and organs during wound healing after trauma. However, when the scarring mechanism deteriorates after repeated injury, chronic fibrosis results, resulting in a shift from supportive fibrous tissue to scar tissue. This is accompanied by an increase in the number and activity of extracellular matrix-producing cells, which ultimately leads to the destruction of normal tissue / organ architecture and function.

[0155] In the lungs, tissue scarring / fibrosis occurs, for example, in idiopathic pulmonary fibrosis (IPF). Following tissue injury, the immune system is activated, inducing the release of several cytokines and growth factors, such as transforming growth factor beta-1 (TGFβ-1), which signal the tissue to repair itself. Mechanistically, epithelial-to-mesenchymal transition (EMT) is one of the main drivers of fibrosis, and approximately 30% of ECM-producing (myofibroblasts) are derived from epithelial cells through EMT. During EMT, tightly organized epithelial cells lose expression of the tight junction marker E-cadherin and transform into mesenchymal cells by expressing mesenchymal cell markers N-cadherin, vimentin, and fibronectin. Furthermore, these cells acquire migratory potential, allowing them to migrate throughout the tissue. Another important mechanism during fibrosis is the fibroblast-to-myofibroblast transition (FMT), when resident and invasive fibroblast-like cells derived from EMT are activated (e.g., by TGFβ-1) and begin to differentiate into myofibroblasts. Myofibroblasts are a heterogeneous population derived from different progenitors and are characterized by the formation of alpha-smooth muscle actin (αSMA), which contains stress fibers, and the expression and secretion of fibrillar collagens, fibronectin, and additional ECM components. Increased ECM production and accumulation results in permanently damaged fibrous tissue with an abnormal architecture that cannot function properly.

[0156] FMT and EMT in vitro assays are established models for identifying and characterizing antifibrotic compounds (see Weigle et al. J Biol Methods (2019) Vol. 6(2)).

[0157] Fibroblast-to-myofibroblast transition (FMT) assay Human bronchopulmonary fibroblasts obtained from three IPF patients were seeded into 384-well plates (Cat. No. 6057302, Perkin Elmer) at a density of 750 cells / well in 2% FBS-DMEM (DMEM supplemented with 2% fetal bovine serum (Cat. No. 10100-147, Gibco) and 1% penicillin and streptomycin (Cat. No. 15140, Gibco). Two days after seeding, the cells were refreshed with 2% FBS-DMEM. Five days after seeding, cells were refreshed with 0.2% FBS-DMEM (DMEM supplemented with 0.2% fetal bovine serum (catalog no. 10100-147, Gibco) and 1% penicillin and streptomycin (catalog no. 15140, Gibco)), and anti-IGFBP5 mAbs (B06 and B09) were added in an 8-step concentration-response curve using 0.5 LogM dilution steps with a top concentration of 1.1 μM for B06 and 1.5 μM for B09. 0.1% DMSO (positive control), 0.1% DMSO (vehicle control 1; catalog no. D8418, Sigma), and 1 μM ALK5 inhibitor (SB525334; catalog no. S8822-25, Sigma) in 3.6% PBS (vehicle control 2) were used to evaluate assay performance. One hour after addition of anti-IGFBP5 mAb (B06 and B09), DMSO, or PBS, cells were triggered with 1.25 ng / mL TGF-β1 (catalog no. 240-B-010, R&D Systems) to induce fibroblast-to-myofibroblast transition (FMT). Three days after TGF-β1 addition, the cells were fixed with 4% formaldehyde (catalog no. 8187081000, Merck) and stained for α-smooth muscle actin (αSMA) as a marker for myofibroblasts and nuclei (DAPI). To identify αSMA expression, fixed lung fibroblasts were incubated in blocking buffer (PBS containing 5 mg / mL BSA (catalog no. A2153, Sigma) and 0.2% (v / v) Triton X-100 (catalog no. T8787, Sigma)) at room temperature for 1 hour.The cells were then incubated with monoclonal Alexa Fluor 488-labeled anti-αSMA antibody (1 μg / mL; catalog number AB184675, Abcam) at room temperature for 1 hour, washed with PBS, and incubated with 4',6-diamidino-2-enylindole (DAPI, 0.5 μg / mL; catalog number D3571, Life Technologies) and imaged on an In Cell Analyzer 2200 (GE Healthcare). Expression was quantified using IN Cell Developer software (GE Healthcare). αSMA expression is expressed as the staining intensity multiplied by the stained area (D × A level). Cell nuclei were co-stained with DAPI to quantify cell number as an indicator of potential cytotoxicity.

[0158] After analysis, anti-IGFBP5 mAb demonstrated concentration-dependent inhibition of TGF-β1-mediated αSMA expression in IPF patient samples. Moreover, anti-IGFBP5 mAb did not modulate the number of nuclei, suggesting the absence of potential cytotoxic side effects (Figure 1).

[0159] Epithelial to mesenchymal transition (EMT) assay Human bronchial epithelial cells (HBECs) obtained from three IPF patients (IPF05, IPF06, and IPF07) were seeded at a density of 900 cells / well in 384-well plates (Cat. No. 6057302, Perkin Elmer) in KSFM Complete (keratinocyte serum-free medium supplemented with 0.2 ng / mL epidermal growth factor, 25 μg / mL bovine pituitary extract (all from Cat. No. 17005-075, Gibco), 1 μmol / L isoproterenol (Cat. No. I6504, Sigma), and 1% penicillin and streptomycin (Cat. No. 15140, Gibco). Two days after seeding, cells were refreshed with 2% KSFM Complete. Six days after seeding, cells were refreshed with 2% KSFM Complete and anti-IGFBP5 mAbs (B06 and B09) were added in an 8-step concentration-response curve using 0.5 LogM dilution steps with a top concentration of 1.1 μM for B06 and 1.5 μM for B09. 0.1% DMSO (positive control), 0.1% DMSO (vehicle control 1; catalog no. D8418, Sigma), and 1 μM ALK5 inhibitor (SB525334; catalog no. S8822-25, Sigma) in 3.6% PBS (vehicle control 2) were used to evaluate assay performance. One hour after addition of anti-IGFBP5 mAb (B06 and B09), DMSO, or PBS, cells were triggered with 5 ng / mL TGF-β1 (catalog no. 240-B-010, R&D Systems) to induce epithelial-to-mesenchymal transition (FMT). Three days after trigger addition, cells were fixed with 4% formaldehyde (catalog no. 8187081000, Merck) and stained for fibronectin (FN1), a mesenchymal cell marker, and nuclei (DAPI). To identify FN1 expression, fixed HBECs were incubated in blocking buffer (PBS containing 5 mg / mL BSA (catalog no. A2153, Sigma) and 0.2% (v / v) Triton X-100 (catalog no. T8787, Sigma)) at room temperature for 1 hour.The cells were then incubated with monoclonal anti-FN1 antibody (0.125 μg / mL; Catalog No. 610001, Biohit Healthcare) for 1 hour at room temperature, washed with 0.05% Tween-20 in PBS (Cat. No. P1379, Sigma), and incubated with Alexa Fluor 546-labeled donkey anti-mouse secondary antibody (4 μg / mL; Catalog No. A100365, Life Technologies) for 1 hour at room temperature. The plates were then washed with 0.05% Tween in PBS, incubated with 4',6-diamidino-2-phenylindole (DAPI, 0.5 μg / mL; Catalog No. D3571, Life Technologies), and imaged on an In Cell Analyzer 2200 (GE Healthcare). Expression was quantified using IN Cell Developer software (GE Healthcare). FN1 expression is expressed as the staining intensity multiplied by the stained area (D × A level). Co-staining of cell nuclei with DAPI was performed to quantify cell number as an index of potential cytotoxicity.

[0160] After analysis, anti-IGFBP5 mAb B06 demonstrated concentration-dependent inhibition of TGF-β1-mediated FN1 expression in IPF patient samples from donors IPF05 and IPF06. Furthermore, all anti-IGFBP5 mAbs showed no modulation of nuclei number, suggesting the absence of potential cytotoxic side effects (Figure 2).

[0161] INCORPORATION BY REFERENCE All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0162] equivalent While various specific embodiments have been illustrated and described, the above specification is not intended to be limiting. It will be understood that various modifications can be made without departing from the spirit and scope of the invention. Many modifications will become apparent to those skilled in the art upon review of this specification.

Claims

1. An inhibitor of IGFBP5 having antifibrotic effect and / or antifibrotic activity, wherein the inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to human IGFBP5; a-1. A heavy chain variable domain (VH) comprising: i. the CDR1 sequence of the amino acid sequence of SEQ ID NO: 1; ii. a CDR2 sequence of the amino acid sequence of SEQ ID NO: 6; and iii. A CDR3 sequence of the amino acid sequence of SEQ ID NO: 17; and b-1. A light chain variable domain (VL) comprising: i. a CDR1 sequence of the amino acid sequence of SEQ ID NO: 27; ii. a CDR2 sequence of the amino acid sequence of SEQ ID NO: 39; and iii. A CDR3 sequence of the amino acid sequence of SEQ ID NO: 51; or a-2. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of the amino acid sequence of SEQ ID NO: 2; ii. a CDR2 sequence of the amino acid sequence of SEQ ID NO: 7; and iii. A CDR3 sequence of the amino acid sequence of SEQ ID NO: 13; and b-2. A light chain variable domain (VL) comprising: i. a CDR1 sequence of the amino acid sequence of SEQ ID NO: 28; ii. a CDR2 sequence of the amino acid sequence of SEQ ID NO: 38; and iii. CDR3 sequence of the amino acid sequence of SEQ ID NO: 49 an inhibitor,

2. 10. The inhibitor of claim 1 for use in the treatment and / or prevention of fibrosis and / or fibrotic conditions.

3. The inhibitor of claim 1 or 2, wherein the isolated antibody or antigen-binding fragment thereof has an anti-fibrotic effect.

4. An inhibitor described in any one of claims 1 to 3, which inhibits, reduces or neutralizes the formation of αSMA induced by profibrotic mediators and / or inhibits, reduces or neutralizes the activation of FN1 expression induced by profibrotic mediators.

5. The inhibitor according to any one of claims 1 to 4, which is an isolated antibody or an antigen-binding fragment thereof that binds to human IGFBP5 and has an anti-fibrotic effect.

6. An inhibitor of IGFBP5 having antifibrotic effect and / or antifibrotic activity, wherein the inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to human IGFBP5, and comprises a combination of CDR1-3 of a heavy chain variable domain (VH) and CDR1-3 of a light chain variable domain (VL) as defined in any one of Chothia, AbM, Kabat, Contact, or IMGT; a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 1 ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 2 and, iii. A CDR3 sequence having an amino acid sequence selected from the group consisting of the following sequences: Table 3 and, b. A light chain variable domain (VL) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 4 ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 5 and, iii. A CDR3 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 6 an inhibitor,

7. An inhibitor of IGFBP5 having antifibrotic effect and / or antifibrotic activity, wherein the inhibitor is an isolated antibody or an antigen-binding fragment thereof that binds to human IGFBP5, and comprises a combination of CDR1-3 of a heavy chain variable domain (VH) and CDR1-3 of a light chain variable domain (VL) as defined in any one of Chothia, AbM, Kabat, Contact, or IMGT; a. A heavy chain variable domain (VH) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 7 ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 8 and, iii. A CDR3 sequence having an amino acid sequence selected from the group consisting of the following sequences: Table 9 and b. A light chain variable domain (VL) comprising: i. a CDR1 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 10 ii. A CDR2 sequence of an amino acid sequence selected from the group consisting of the following sequences: Table 11 and iii. A CDR3 sequence having an amino acid sequence selected from the group consisting of the following sequences: Table 12 an inhibitor,

8. a. a heavy chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 and 66; or b. a light chain variable domain sequence of an amino acid sequence selected from the group consisting of SEQ ID NOs: 77 and 81; or c. The heavy chain variable domain of (a) and the light chain variable domain of (b).

8. The inhibitor of claim 1 , comprising:

9. - a heavy chain variable domain sequence of the amino acid sequence set forth in SEQ ID NO: 62 and a light chain variable domain sequence of the amino acid sequence set forth in SEQ ID NO: 77, or - a heavy chain variable domain sequence of the amino acid sequence set forth in SEQ ID NO: 66 and a light chain variable domain sequence of the amino acid sequence set forth in SEQ ID NO: 81 The inhibitor of claim 8, comprising:

10. 10 for human IGFBP5 -8 10. The inhibitor of claim 1, having an affinity constant of less than or equal to M.

11. (a) specifically binds to an epitope on IGFBP5, wherein the epitope is the same as or similar to the epitope recognized by monoclonal antibody B06 or B09; or (b) cross-competes for binding with B06 or B09; or (c) exhibits the same or similar binding affinity or specificity, or both, as either B06 or B09; or (d) an antibody molecule selected from any of B06 and B09; or (e) an antibody molecule selected from any of B06 and B09, B06 has a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 62; a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 77; and two constant regions having the amino acid sequences of SEQ ID NOs: 121 and 122, respectively; B09 has a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 66; a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 81; and two constant regions having the amino acid sequences of SEQ ID NOs: 121 and 122, respectively.

11. The inhibitor of any one of claims 1 to 10, which is an isolated antibody or antigen-binding fragment thereof.

12. 12. The inhibitor of any one of claims 1 to 11, which is a human antibody or a humanized antibody.

13. 13. The inhibitor of any one of claims 1 to 12, which is an IgG2 or IgG4 antibody.

14. 14. An inhibitor according to any one of claims 1 to 13, which reduces or inhibits the binding of IGFBP5 to at least one of its receptors.

15. 15. An inhibitor described in any one of claims 1 to 14, which i) reduces or inhibits the production of extracellular matrix, and / or ii) reduces or inhibits the deposition of extracellular matrix, and / or iii) reduces or inhibits the production of collagen and / or fibronectin in primary lung fibroblasts, and / or iv) reduces or inhibits the deposition of collagen and / or fibronectin in primary lung fibroblasts.

16. 16. An isolated polynucleotide comprising at least one sequence encoding an inhibitor according to any one of claims 1 to 15.

17. 17. The polynucleotide of claim 16, which is a cDNA.

18. 18. An isolated vector comprising the polynucleotide of claim 16 or 17.

19. 20. The vector of claim 18, wherein the vector is selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector, and a recombinant expression vector.

20. 20. An isolated cell comprising a polynucleotide according to claim 16 or 17 or a vector according to claim 18 or 19.

21. 21. The cell of claim 20, which is a hybridoma, a Chinese hamster ovary (CHO) cell, or a human embryonic kidney cell (HEK293).

22. A pharmaceutical composition comprising an inhibitor according to any one of claims 1 to 15, a polynucleotide according to claim 16 or 17, a vector according to claim 18 or 19, or a cell according to claim 20 or 21 for use in the treatment and / or prevention of fibrosis or a fibrotic condition.

23. 23. The pharmaceutical composition of claim 22, wherein the fibrosis or fibrotic condition is selected from scleroderma or pulmonary fibrosis, morphea, fibrosis as a result of graft-versus-host disease (GVHD), keloids and hypertrophic scarring, subepithelial fibrosis, endocardial fibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, post-surgical scarring, asthma, abnormal wound healing, fibrosing syndrome, idiopathic interstitial pneumonia (CFA), and idiopathic pulmonary fibrosis (IPF).

24. 24. The pharmaceutical composition of claim 22 or 23, further comprising a pharmaceutically acceptable carrier and a therapeutic agent.

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