Antigen-binding protein

Abstract

The present invention provides antigen-binding proteins that specifically bind to BMP1, TLL1, and / or TLL2. Pharmaceutical compositions containing the antigen-binding proteins are also provided. The antigen-binding proteins and pharmaceutical compositions described herein can be used to treat diseases associated with fibrotic conditions or disorders, as well as to promote muscle growth and improve muscle function.

Description

Technical Field

[0001] Cross-reference of related applications This application claims the priority of U.S. Provisional Application No. 63 / 059,387, filed on July 31, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is also hereby incorporated by reference in its entirety. The above ASCII copy created on July 6, 2021, is named PU66960_SL.txt and has a size of 265,335 bytes.

[0003] Field of Invention The present invention relates to antigen-binding proteins that specifically bind to BMP1, TLL1, and / or TLL2, as well as pharmaceutical compositions thereof and uses thereof. The present invention also relates to pharmaceutical compositions containing the above antigen-binding proteins and uses thereof.

Background Art

[0004] Fibrous collagen is a vital part of the extracellular matrix that helps maintain tissue integrity and the cellular microenvironment for normal physiological function. Collagen I-III, the major isoforms of the fibrous collagen protein family, are synthesized as procollagen precursors containing N-terminal and C-terminal propeptides. Procollagen undergoes post-translational modification by proline hydroxylation and is secreted into the perivascular space for further processing. Subsequently, the N-terminal propeptide of collagen is cleaved by proteinases of the ADAMTS (A Distintegrin And Metalloproteinase with ThromboSpondin repeats) family, and the C-terminal propeptide is processed by metalloproteinases of the toroid family, including BMP1 (bone morphogenetic protein 1), TLL1 (tolloid-like 1), and TLL2 (tolloid-like 2) (Hopkins, DR et al., Matrix Biology, 2007, 26, 508-523). The cleavage of both the N-terminal and C-terminal propeptides further matures the collagen, leading to crosslinking at lysine residues and the formation of insoluble fibrous structures (Shoulders, MD et al., Annual Review of Biochemistry, 2009, 78, 929-958).

[0005] While the BMP1, TLL1, and TLL2 proteins are encoded by separate genes, this family also includes BMP1 isoforms that include multiple BMP1 isoforms obtained from alternative splicing of the same gene product (see, for example, Takahara, K., et al., The Journal of Biological Chemistry, 1994, 269, 32572-32578; and Cvetjeticanin, B. et al., Medical Hypotheses, 2014, 83, 656-658). The first discovered form of BMP1 is called BMP-1-1 or BMP1-1. Other BMP1 isoforms encoded by splice variant RNA transcripts have been described at the transcriptional level and are represented by a series of suffixes, e.g., BMP-1-2, BMP-1-3, BMP-1-4, BMP-1-5, BMP-1-6, and BMP-1-7 (e.g., Wozney et al., Science (1988), 242: 1528-1534; Kessler et al., Science, (1996) 271: 360-362; Li et al., Proc. Natl. Acad. Sci. USA (1996), 93: 5127-5130; Janitz et al., J. Mol. Med. (1998), 76: 141-146; Takahara et al., J. Biol. Chem. (1994), 269: See 32572-32578; and Ge and Greenspan, Birth Defect Res. (2006), 78: 47-68).

[0006] Furthermore, several BMP1 isoforms have been confirmed at the protein level to circulate in the blood of patients with various diseases and healthy individuals (see, for example, International Patent Publication Nos. WO2008 / 011193 and WO2013 / 163479, and Grgurevic et al., J. Am. Soc. Nephrol. (2011), 21:681-692). Furthermore, the role of BMP1 in procollagen processing leading to fibrosis and scar tissue in various diseases, as well as the discovery of blood profiles comprising individual BMP1 isoforms in patients with various diseases, made BMP1 an attractive target for the development of novel therapies (see, for example, WO2008 / 011193;WO2013 / 163479;Grgurevic et al., J. Am. Soc. Nephrol. (2011), 21:681-692, Cvetjeticanin, B. et al., Medical Hypotheses, 2014, 83, 656-658; and Turtle et al., Expert Opin. Ther. Patents (2004), 14(8): 1185-1197).

[0007] Excessive production of extracellular matrix (ECM) proteins, including collagen, can lead to fibrotic pathologies in various organs or tissues, which may be associated with increased tissue rigidity, parenchymal tissue replacement, abnormal electrical conductivity, sclerosing wound healing (e.g., infarction and burns), and / or abnormal intercellular interactions. For example, acute and chronic heart disease (e.g., heart failure, arrhythmia, hypertrophic cardiomyopathy) and myocardial infarction (Lopez, B. et al., Circulation, 2010, 121, 1645-1654; Ho, CY, et al., New England Journal of Medicine, 2010, 363, 552-563; Kostin, S. et al., Cardiovascular Research, 2002, 54, 361-379; See, F. et al., Current Pharmaceutical Design, 2005, 11, 477-487; Cvetjeticanin, B. et al. Medical Hypotheses, 2014, 83, 656-658), chronic obstructive pulmonary disease ("COPD") (Salazar, LM, et al., Lung, 2011, 189, 101-109), cirrhosis and non-alcoholic steatohepatitis ("NASH") (Bataller, R., et al., Journal of Clinical Investigation, 2005, 115, 209-218), idiopathic pulmonary fibrosis (Chakraborty, S, et al., Expert Opin Investig Drugs, 2014, 23, 893-910), collagen fibrosis, e.g., systemic lupus erythematosus, rheumatoid arthritis and scleroderma (Eckes, B., et al., J Mol Med, 2014, 92, 913-924), muscular dystrophy (e.g., Serrano, AC, et al., Experimental Cell Research, 2010, 316, 3050-3058; Klingler, W., et al., Acta Myoligica, XXXI, 2012, 184-195), chronic kidney disease (Liu, Y., Nature Reviews Nephrology, 2011, 7, 684-696), acute kidney injury (Molitoris, B., The Journal of Clinical Investigation, 2014, 124, 2355-2363; Venkatachalam, MA et al., Am J Physiol Renal Physiol 298: F1078-F1094, 2010), diabetic nephropathy (Sun, YM, et al., Biochemical and Biophysical Research Communications, 2013, 433, 359-361), keloids, wound healing, adhesions, hypertrophy and other scarring (e.g., scarring associated with burns, surgery and other traumas) (Meier K., et al., Expert Opinion on Emerging Drugs, 2006, 11, 39-47; Malecaze, F., et al., Investigative Opthalmology and Visual Science, 2014, 55, 6712-6721; van der Weer, W. et al., Burns, 2009, 35, 15-29) as well as stroke, multiple sclerosis and spinal cord injury (Fernandez-Klett, F. and Piller, J. Brain Pathology, 2014, 24, 404-13; Rimar, D. et al., Arthritis & Rheumatology, Vol. 66, No. 3, March 2014, Patients with 726-730) consistently exhibit increased fibrosis and collagen production. Therefore, reducing excessive collagen production and maturation by targeting the BMP1, TLL1, and / or TLL2 pathways may be an effective therapeutic strategy for treating fibrotic conditions such as these diseases. This is supported by recent published studies using pharmacological agents that inhibit BMP1, TLL1, and / or TLL2 activity in small animal cardiac and renal disease models (Grgurevic, L, et al.).Journal of the American Society of Nephrology, 2011, 21, 681-692; He, W., et al., Proceedings of the National Academy of Sciences, 2010, 107, 21110-21115; Cvetjeticanin, B. et al., Medical Hypotheses, 2014, 83, 656-658; International Patent Publications WO2008 / 011193 and WO2013 / 163479).

[0008] The toroid family of metalloproteinases (BMP1, TLL1, and TLL2) have substrates other than collagen and may also contribute to promoting the production of ECM proteins. For example, the proform of lysyl oxidase 1 (LOX1) has been shown to be a substrate of BMP1, and cleavage by BMP1 promotes LOX enzyme activity, thereby inducing collagen crosslinking (Uzel, MI, et al., Journal of Biological Chemistry, 2001, 276, 22537-22543). Therefore, BMP1 also plays a role in the development of pathological tissue stiffness through this mechanism, for example, in glaucoma (Tovar-Vidales, T., et al., Investigative Ophthalmology & Visual Science, 2013, 54, 4741-4748) and cardiac diastolic dysfunction (Lopez, B., et al., American Journal of Physiology - Heart and Circulatory Physiology, 2010, 299, H1-H9). Furthermore, it has been shown that TGF-β binding protein (LTBP) can be cleaved by BMP1, enhancing the action of TGF-β and further inducing collagen production (Ge, G., et al., Journal of Cell Biology, 2006, 175, 1 11-120). BMP1-mediated regulation of TGF-β may also play a role in other pathologies, such as controlling cancer cell metastasis and invasion (Wu, X., et al. Oncogene, 2014, 33, 1506-1514). Similarly, BMP1, TLL1, and / or TLL2 activate a broader range of other TGF-β-like molecules, such as BMP2 and 4, by proteolytically processing the proteins they interact with (Hopkins, DR et al., Matrix Biology, 2007, 26, 508-523).The combined action of BMP1 and its various substrates suggests that BMP1, TLL1, and TLL2 are important regulators of tissue ECM production / maturation, and that members of the toroid family of metalloproteinases are particularly effective targets for anti-fibrotic therapeutic interventions.

[0009] BMP1, TLL1, and TLL2 may also influence other biological pathways through further substrate processing. In particular, they may affect muscle biology by promoting myostatin activation. Myostatin is a hormone that negatively regulates muscle growth (Lee, SJ, 2004, Annual Review of Cell & Developmental Biology, 20, 61-86). BMP1 has been shown to cleave the inhibitory propeptide of myostatin, thereby increasing myostatin activity (Wolfman NM, et al., Proceedings of the National Academy of Sciences, 2003, 100, 15842-15846). Knockout of TLL2 in mice showed increased muscle mass, thereby supporting the association between toroidal metalloproteinases and myostatin (Lee, SJ, PLoS one, 2008, 3, e1628). Therefore, inhibitors of BMP1, TLL1, and / or TLL2 may be beneficial in diseases of decreased muscle function or mass, including muscular dystrophy, sarcopenia, and cachexia (e.g., cachexia associated with heart failure, CKD, COPD, cancer, or aging).

[0010] Considering the biology of BMP1, TLL1, and TLL2, they play a crucial role in collagen processing, assembly, and crosslinking, leading to the formation of fibrous collagen networks that maintain tissue integrity and a proper cellular microenvironment. This protein family may also play an important role in the pathogenesis of fibrotic conditions, such as those in the heart, lungs, skeletal muscle, kidneys, liver, skin, vascular system, nervous system, and eyes. Inhibitors of these metalloproteinases may be effective in treating fibrosis-related diseases, such as myocardial infarction, heart failure, cardiac arrhythmias, hypertrophic cardiomyopathy, chronic kidney disease (CKD), post-acute kidney injury, diabetic nephropathy, post-transplant graft function delay, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cirrhosis, and non-alcoholic fatty liver disease. It may offer broad benefits as an antifibrotic agent for the treatment of inflammatory bowel disease (NASH), muscular dystrophy (e.g., Duchenne, Becker, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreyfus types), glaucoma, corneal scarring, keloids, wound healing, adhesions, hypertrophic scarring, and other scarring (e.g., scarring associated with burns, surgery, or other trauma), stroke, collagen vascular diseases (e.g., systemic lupus erythema, rheumatoid arthritis, and scleroderma), spinal cord injury, and multiple sclerosis. Furthermore, based on their effects on myostatin biology, BMP1, TLL1, and TLL2 inhibitors may have further therapeutic applications in muscle diseases, particularly muscular dystrophy (e.g., Duchenne, Becker, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreyfus types), sarcopenia, and cachexia (e.g., cachexia associated with heart failure, CKD, COPD, cancer, or aging). [Overview of the project]

[0011] Summary of the Invention According to a first aspect of the present invention, (a)(i) Any one or combination of CDRH1, CDRH2, CDRH3 in SEQ ID NOs. 7, 22, 40, 54, 67, 82, 96, 110, 124, 138, 152, 166, 180, 194, 207 and 222 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NOs. 8, 21, 39, 53, 68, 81, 95, 109, 123, 137, 151, 165, 179, 193, 208 and 221 of the CDR; or (ii) A variant of the CDR of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 7, 22, 40, 54, 67, 82, 96, 110, 124, 138, 152, 166, 180, 194, 207 or 222, and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 8, 21, 39, 53, 68, 81, 95, 109, 123, 137, 151, 165, 179, 193, 208 or 221. A protein that binds to BMP1, TLL1 and / or TLL2 is provided, comprising the above.

[0012] In one embodiment, the following six CDRs: LCDR1 of RASQSVSSYLA (sequence number 1); LCDR2 of DASNRAT (sequence number 2); LCDR3 of QQSDSWPPT (sequence number 3); HCDR1 of GYYMS (sequence number 4); HCDR2 of WINPLSGETNYAQKFQG (Sequence ID 5); and DTGELDGMNWYFDL (SEQ ID NO: 6) HCDR3 A protein that binds to BMP1, TLL1 and / or TLL2 is provided, comprising the above.

[0013] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 is provided, comprising a VH region that is 100% identical to SEQ ID NO: 7 and a VL region that is 100% identical to SEQ ID NO: 8.

[0014] In one embodiment, there is provided a protein that binds to BMP1, TLL1, and / or TLL2, comprising a light chain that is 100% identical to SEQ ID NO: 9 and a heavy chain that is 100% identical to SEQ ID NO: 10.

[0015] According to a further aspect of the present invention, there is provided a polynucleotide sequence encoding a protein that binds to BMP1, TLL1, and / or TLL2 as described herein.

[0016] According to a further aspect of the present invention, there is provided an expression vector comprising the polynucleotide sequence described herein.

[0017] According to a further aspect of the present invention, there is provided a recombinant host cell comprising the polynucleotide sequence described herein or the expression vector described herein.

[0018] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a protein that binds to BMP1, TLL1, and / or TLL2 as described herein and a pharmaceutically acceptable diluent or carrier.

[0019] According to a further aspect of the present invention, there is provided a method for treating a fibrosis-related disease or disorder in a subject that needs it, comprising administering to the subject a therapeutically effective amount of a protein that binds to BMP1, TLL1, and / or TLL2 as described herein or the pharmaceutical composition described herein.

[0020] According to a further aspect of the present invention, there is provided a protein that binds to BMP1, TLL1, and / or TLL2 as described herein or the pharmaceutical composition described herein for use in therapy.

[0021] According to a further aspect of the present invention, there is provided a protein that binds to BMP1, TLL1 and / or TLL2 described herein or a pharmaceutical composition described herein for use in the treatment of fibrosis-related diseases or disorders.

[0022] According to a further aspect of the present invention, there is provided the use of a protein that binds to BMP1, TLL1 and / or TLL2 described herein or a pharmaceutical composition described herein in the manufacture of a medicament for use in the treatment of fibrosis-related diseases or disorders.

[0023] According to a further aspect of the present invention, there is provided a method for promoting muscle growth and / or improving muscle function in a subject that needs it, the method comprising administering to the subject a therapeutically effective amount of a protein that binds to BMP1, TLL1 and / or TLL2 described herein or a pharmaceutical composition described herein.

[0024] According to a further aspect of the present invention, there is provided a protein that binds to BMP1, TLL1 and / or TLL2 described herein or a pharmaceutical composition described herein for use in promoting muscle growth and / or improving muscle function.

[0025] According to a further aspect of the present invention, there is provided the use of a protein that binds to BMP1, TLL1 and / or TLL2 described herein or a pharmaceutical composition described herein in the manufacture of a medicament for use in promoting muscle growth and / or improving muscle function. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] [Figure 1]Figure 1 shows the results of a FRET assay measuring the inhibition of 62.5 pM human BMP-1 activity by an anti-BMP1 / TLL antibody molecule. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The antibody was tested in 11 3-fold dilution systems starting from a maximum concentration of 75 nM. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 2] Figure 2 shows the FRET assay for measuring the inhibition of 50 pM mouse BMP-1 activity by anti-BMP1 / TLL antibody molecules. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The molecule was tested in 11 3-fold dilution systems starting from a maximum concentration of 75 nM. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 3] Figure 3 shows the FRET assay for measuring the inhibition of 250 pM biotinylated human TLL-1 activity by anti-BMP1 / TLL antibody molecules. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The molecules were tested in a 3-fold dilution system with 11 points, starting from a maximum concentration of 75 nM. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 4] Figure 4 shows the FRET assay for measuring the inhibition of 500 pM human TLL-2 activity by an anti-BMP1 / TLL antibody molecule. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The molecule was tested in 11 3-fold dilution systems starting from a maximum concentration of 75 nM. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 5]Figure 5 shows the FRET assay for measuring the inhibition of 800 pM biotinylated mouse TLL-1 activity by an anti-BMP1 / TLL antibody molecule. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The molecule was tested in a 3-fold dilution system with 11 points, starting from a maximum concentration of 75 nM. The figure shows the mean of the two replicate data points and the standard deviation as error bars. [Figure 6] Figure 6 shows the results of a FRET assay measuring the inhibition of 2.5 nM rat TLL-1 activity by an anti-BMP1 / TLL antibody molecule. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The molecule was tested in 11 3-fold dilution systems starting from a maximum concentration of 75 nM. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 7] Figure 7 shows the results of a FRET assay measuring the inhibition of 2.5 nM rat TLL-2 activity by an anti-BMP1 / TLL antibody molecule. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The molecule was tested in 11 3-fold dilution systems starting from a maximum concentration of 75 nM. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 8] Figure 8 shows the results of a FRET assay measuring the inhibition of 750 pM biotinylated cynomolgus monkey (cyno) TLL-1 activity by an anti-BMP1 / TLL antibody molecule. Dose-response curves were plotted for 13Y039-4B06-4334, 13Y039-3E07-2944, and 13Y039-8F02-2949. The molecule was tested in 11 3-fold dilution systems starting from a maximum concentration of 75 nM. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 9]Figure 9 shows the FRET assay for measuring the inhibition of 8 nM biotinylated cynomolgus monkey TLL-2 activity by an anti-BMP1 / TLL antibody molecule. Dose-response curves for 13Y039-4B06-4334 tested at 22 3-fold dilutions starting from a maximum concentration of 600 nM are plotted. The figure shows the mean of two replicate data points, with the standard deviation shown as error bars. [Figure 10] Figure 10 shows the binding of 13Y039-4B06-4334 (antibody expressed by HEK and antibody expressed by CHO) to human C1q as measured by ELISA. [Figure 11] Figure 11 shows the MSD assay for measuring latent complex cleavage inhibition and released myostatin by the anti-BMP-1 / TLL antibody 13Y039-4B06-4334. [Figure 12] Figure 12 shows the plasma BMP1 activity from animals examined in the mouse AngII / PE model. For reference, plasma BMP1 levels were determined in naive mice that did not receive osmotic pumps or IP injections (white bar on the far right). Compounds A-D are antibodies cloned as reverse chimeric mAbs possessing the human variable region and mouse κ on mouse IgG2a LAGA Fc (referred to herein as compound A for 4B06- and compound B for 3E07-2944) and the human variable region and rat κ on rat IgG2b LAGA Fc (referred to herein as compound C for 4B06-4334 and compound D for 3E07-2944) (AngII / PE and physiological saline compared by unpaired t-test, *p<0.05; AngII / PE and mAb group compared by one-way ANOVA, #p<0.05, ####p<0.0001; RSV control and corresponding compound A or E07, ####p<0.0001). [Figure 13]Figure 13 shows plasma PICP measurements from selected groups of the mouse AngII / PE model. PICP levels were measured using multiple immunoblots. Each gel contained all AngII / PE+ saline samples and one of the other test groups. For each gel, all band intensities were normalized to the mean of each AngII / PE+ saline group. The data were then combined into a single graph for visualization. (AngII / PE+ saline and other groups were compared using an unpaired t-test, ****p<0.0001). [Figure 14] Figure 14 shows the effect of compound A on skeletal muscle mass in the AngII / PE model. Percentages in black above the treatment group indicate an increase in normalized gastrocnemius muscle weight exceeding that of the AngII / PE group, while percentages in red indicate an increase exceeding the model window (the difference between the saline-saline group and the AngII / PE-saline group) (AngII / PE and saline compared by unpaired t-test, **p<0.01; AngII / PE and mAb group compared by one-way ANOVA, #p<0.05, ###p<0.001, ####p<0.0001). [Figure 15] Figure 15 shows the effect of compound A on total plasma myostatin (MSTN) levels in the AngII / PE model. Total plasma myostatin was measured by ELISA. The magnification change is expressed relative to the AngII / PE control (AngII / PE and physiological saline compared by unpaired t-test, **p<0.01; AngII / PE and mAb group compared by one-way ANOVA, ##p<0.01, ####p<0.0001; RSV control and corresponding compound A or compound B, ##p<0.01, ###p<0.001, ####p<0.0001). [Figure 16]Figure 16 shows the left ventricular hydroxyproline (HDXP) content in the mouse AngII / PE test. The percentage change relative to the AngII / PE group is calculated (AngII / PE and physiological saline compared by unpaired t-test, ****p<0.0001; AngII / PE and mAb group (one-sided) compared by one-way ANOVA, #p<0.05, ##p<0.01, ###p<0.001; RSV control and corresponding compound A (one-sided) compared by one-way ANOVA, #p<0.05). [Figure 17] Figure 17 shows the effects of compound C on fibrosis (Figure 17A) and skeletal muscle mass (Figure 17B) in the rat Dahl S model. Figure 17A: Left ventricular fibrosis measured by quantitative evaluation of Masson trichrome histopathological staining in the rat Dahl S study. (PBS + 0.3% NaCl and PBS + 8% NaCl compared by unpaired t-test, **p<0.01; one-way ANOVA compared PBS + 8% NaCl and compound C + 8% NaCl, #p<0.05). Figure 17B: The compound C treatment group showed a 9% increase in skeletal muscle mass compared to PBS + 8% NaCl and a 10% increase compared to the anti-RSV treatment group (PBS + 8% NaCl and mAb group compared by one-way ANOVA, **p<0.01). [Figure 18] Figure 18 shows lean body mass measurements during the recovery phase of aging mice two weeks after hind limb fixation. The data are shown as the time course of absolute lean body mass (left) and the percentage change relative to the measurement after splinting (right) (percentage change in lean body mass compared by one-way ANOVA, compound A and anti-RSV mAb: *p<0.05, **p<0.01; anti-myostatin mAb and anti-RSV mAb: #p<0.05). [Figure 19]Figure 19 shows the wet weight of the gastrocnemius (left) and soleus (right) muscles measured at the end of the hindlimb immobilization test in aging mice. The blank squares represent the weight of the left hindlimb without a splint, and the filled squares represent the weight of the right hindlimb with a splint (muscle weight compared by two-way ANOVA. Compound A or anti-myostatin mAb and anti-RSV mAb for limbs without splints: *p<0.05, ****p<0.0001; Compound A or anti-myostatin mAb and anti-RSV mAb for limbs with splints: *p<0.05, ***p<0.001, ****p<0.0001; limbs with and without splints: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 20] Figure 20 shows in vivo muscle contraction force measurements measured longitudinally in aging mice using a hindlimb fixation test. Muscle contraction force values ​​are shown as absolute tetanic contraction force (left panel), relative tetanic contraction force relative to the value after splinting (center panel), and normalized to gastrocnemius muscle wet weight (right panel) (relative tetanic contraction force compared by one-way ANOVA: compound A and anti-RSV mAb, *p<0.05; anti-myostatin mAb and anti-RSV mAb, #p<0.05. Normalized force compared by one-way ANOVA, **p<0.01). [Modes for carrying out the invention]

[0027] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in which this invention pertains. As used herein, the following terms have the following meanings:

[0028] The term "proteins that bind to BMP1, TLL1, and / or TLL2" refers, as used herein, to antibodies and other protein constructs, such as domains, that can bind to BMP1 (bone morphogenetic factor 1), TLL1 (toroidal 1), and / or TLL2 (toroidal 2). The terms "proteins that bind to BMP1, TLL1, and / or TLL2" and "antigen-binding proteins" are used interchangeably herein. This does not include native homologous ligands or receptors.

[0029] The term “antibody” is used herein in its broadest sense to refer to molecules having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies (including bispecific antibodies), and heteroconjugate antibodies; single variable domains (e.g., domain antibodies (DABs)), antigen-binding antibody fragments, Fab, F(ab')2, Fv, disulfide-linked Fv, single-stranded Fv, disulfide-linked scFv, diabodies, TANDAB, etc., and any of the above variations (for an overview of other “antibody” forms, see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126–1136).

[0030] As used herein with respect to antibodies, the terms “full,” “whole,” and “intact” are used interchangeably herein and refer to heterotetrameric glycoproteins having a molecular weight of approximately 150,000 daltons. Intact antibodies consist of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains comprising two antigen-binding fragments known as “Fab” fragments and an “Fc” crystalline fragment. The Fab fragment consists of a variable heavy chain (VH) or variable light chain (VL) as a variable domain at the amino terminus, and CH1 (heavy chain) and CL (light chain) as constant domains at the carboxyl terminus. The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. Fc can evoke effector function by binding to receptors on immune cells or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by different heavy-chain amino acid sequences called μ, α, γ, ε, and δ, respectively, with each heavy chain potentially paired with a κ or λ light chain. The majority of antibodies in serum belong to the IgG class, and human IgG has four isotypes (IgG1, IgG2, IgG3, and IgG4), whose sequences differ primarily in the hinge region.

[0031] Fully human antibodies can be obtained using various methods, such as using a yeast-based library or transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies. Yeast that displays human antibodies that bind to the target antigen on its surface can be selected by methods based on FACS (fluorescence-activated cell sorting) or by capture onto beads using labeled antigens. Transgenic animals modified to express human immunoglobulin genes can be immunized with the target antigen, and antigen-specific human antibodies can be isolated using B-cell sorting techniques. Human antibodies produced using such techniques can then be characterized for desirable properties such as affinity, developability, and selectivity.

[0032] Alternative antibody forms include alternative scaffolds on which one or more CDRs of the antigen-binding protein can be positioned on a suitable non-immunoglobulin protein scaffold or scaffold, such as an affibody, SpA scaffold, LDL receptor class A domain, avimer (see, for example, U.S. Patent Applications Publications 2005 / 0053973, 2005 / 0089932, and 2005 / 0164301) or EGF domain.

[0033] The term "neutralize," as used herein, means that the biological activity of BMP1, TLL1, and / or TLL2 is reduced in vitro or in vivo in the presence of an antigen-binding protein, such as those described herein, compared to the activity of BMP1, TLL1, and / or TLL2 in the absence of the antigen-binding protein. Neutralization may result from one or more of the following: inhibiting BMP1, TLL1, and / or TLL2 from binding to their target substrate, and preventing BMP1, TLL1, and / or TLL2 from cleaving their target substrate. For example, fluorescence resonance energy transfer (FRET) based assays described in the examples may be used to evaluate the neutralizing ability of proteins that bind to BMP1, TLL1, and / or TLL2.

[0034] "CDR" is defined as the complementarity-determining region amino acid sequence of an antigen-binding protein. These are the hypervariable regions of the immunoglobulin heavy and light chains. The variable region of an immunoglobulin contains three heavy-chain CDRs and three light-chain CDRs (or CDR regions). Therefore, as used herein, "CDR" refers to all three heavy-chain CDRs, all three light-chain CDRs, all heavy-chain and light-chain CDRs, or at least two CDRs.

[0035] Throughout this specification, amino acid residues in variable domain sequences and variable domain regions within full-length antigen-binding sequences, such as antibody heavy chain sequences or antibody light chain sequences, are numbered according to Kabat numbering rules. Similarly, the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3”, “LCDR1”, “LCDR2”, “LCDR3”, “HCDR1”, “HCDR2”, and “HCDR3” used in the examples and shown in the sequence listings conform to Kabat numbering rules. For further details, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987).

[0036] It is obvious to those skilled in the art that there are other numbering rules for the amino acid residues in variable domain sequences and full-length antibody sequences. Similarly, there are other numbering rules for CDR sequences, such as those shown in Chothia et al. (1989) Nature 342: 877-883. The structure and folding of antigen-binding proteins may mean that other residues are considered part of the CDR sequence, and this will be understood by those skilled in the art.

[0037] Other numbering rules for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) method and the "contact" (University College London) method.

[0038] Table 1-1 below shows one definition using a numbering rule for each CDR or binding unit. Table 1 uses the Kabat numbering scheme for the variable domain amino acid sequences. Note that some CDR definitions may differ depending on the individual publication used.

[0039] [Table 1]

[0040] CDRs can be modified by at least one amino acid substitution, deletion, or addition, and the variant antigen-binding protein substantially retains the biological characteristics of the unmodified protein.

[0041] It is understood that each of CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3 can be modified individually or in any permutation or combination with any other CDR. In one embodiment, a CDR is modified by substitution, deletion, or addition of up to three amino acids, e.g., one or two amino acids, e.g., one amino acid. Generally, the modification is a substitution, particularly a conservative substitution, such as those shown in Table 1-2 below.

[0042] [Table 2]

[0043] For example, in variant CDRs, flanking residues that include a CDR as part of another definition, such as Kabat or Chothia, may be substituted with conserved amino acid residues.

[0044] Such antigen-binding proteins comprising the variant CDR described above may be referred to herein as "functional CDR variants."

[0045] The "antigen-binding site" refers to a region on an antigen-binding protein that can specifically bind to an antigen. This may be a single variable domain or a paired VH / VL domain, as can be seen in standard antibodies. Single-chain Fv(ScFv) domains can also provide antigen-binding sites.

[0046] In some embodiments, the protein that binds to BMP1, TLL1, and / or TLL2 may be an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof.

[0047] An antibody fragment (which may also be called an “antibody fragment,” “immunoglobulin fragment,” “antigen-binding fragment,” or “antigen-binding polypeptide”) refers, as used herein, to a portion of an antibody (or a construct containing such portion) that specifically binds to a target, i.e., BMP1, TLL1, and / or TLL2. Examples of binding fragments encompassed by the term antibody fragment include: (i) Fab fragment (a monovalent fragment consisting of VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragment (a divalent fragment consisting of two Fab fragments linked by disulfide bonds in the hinge region); (iii) Fd fragment (consisting of VH and CH1 domains); (iv) Fv fragment (consisting of the VL and VH domains of a single arm of the antibody); (v) Single-stranded variable fragment, scFv (consisting of a VL domain and a VH domain linked by a synthetic linker using recombination, which enables the creation of a single protein chain in which the VL and VH domains pair up to form a monovalent molecule); (vi) VH (variable domain of immunoglobulin chain consisting of VH domains); (vii) VL (variable domain of immunoglobulin chain consisting of VL domains); (viii) Domain antibody (consisting of either a dAb, VH domain, or VL domain); (ix) minibody (consisting of a pair of scFv fragments linked via CH3 domains); and (x) Diabody (consisting of a non-covalent dimer of scFv fragments, in which a VH domain derived from one antibody is linked to a VL domain derived from another antibody by a small peptide linker) These are some examples.

[0048] "Human antibodies" refer to antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in CDRs, particularly CDR3 (e.g., mutations introduced by random or site-directed mutagenesis or somatic mutations). However, this term is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mice, have been grafted onto human framework sequences. Human antibodies prepared, expressed, produced, or isolated by recombinant means such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, antibodies isolated from animals transgenic to human immunoglobulin genes (e.g., mice), or antibodies prepared, expressed, produced, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences, may also be called "recombinant human antibodies."

[0049] Replacing at least one amino acid residue in the framework region of a non-human immunoglobulin variable domain with a corresponding residue in a human variable domain is called "humanization." Humanization of variable domains may reduce immunogenicity in humans.

[0050] In one embodiment, the antigen-binding protein of this disclosure exhibits cross-reactivity between human BMP1, TLL1, and / or TLL2 and BMP1, TLL1, and / or TLL2 of another species, such as mouse, rat, and / or cynomolgus monkey BMP1, TLL1, and / or TLL. In one embodiment, the antigen-binding protein of the present invention specifically binds to human and mouse BMP1, TLL1, and / or TLL2. This is particularly useful in drug development, where it is generally necessary to test lead drug candidates in mouse systems before testing the drug in humans. By providing a drug that can bind to human and mouse species, results can be determined in these systems and data using the same drug can be compared side by side. This avoids the complexity of having to search for a drug that acts against mouse BMP1, TLL1, and / or TLL2 and another drug that acts against human BMP1, TLL1, and / or TLL2, and also avoids the need to compare results in humans and mice using non-identical drugs. Cross-reactivity between other species used in disease models, such as dogs or monkeys (e.g., cynomolgus macaques), is also anticipated.

[0051] "Specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or fragment can bind. Antibody specificity is the ability of an antibody to recognize a particular antigen as a unique molecular entity and distinguish it from other antigens. An antibody that "specifically binds" to an antigen or epitope is a well-understood term in the art. A molecule is said to exhibit "specific binding" when it reacts with a particular target antigen or epitope more frequently, more rapidly, more persistently, and / or with higher affinity compared to other targets. An antibody "specifically binds" to a target antigen or epitope when it binds with higher affinity, avidity, ease, and / or persistence than it would to other substances.

[0052] Affinity, expressed as the equilibrium constant (KD) for the dissociation of an antigen and an antigen-binding polypeptide, is an indicator of the binding strength between the antigenic determinant and the antigen-binding site of the antibody (or its fragment). A smaller KD value indicates a stronger binding affinity between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity can be expressed as an affinity constant (KA) of 1 / KD. Affinity can be determined by known methods such as equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or dynamics (e.g., BIACORE analysis), depending on the specific antigen being studied. For example, the BIACORE method described in the examples can be used to measure binding affinity.

[0053] Avidity, also known as functional affinity, is the cumulative strength of binding at multiple interaction sites. For example, it is the sum of the strengths of binding between two molecules (or more molecules in the case of bispecific or multispecific molecules) at multiple sites, taking into account, for example, the valency of the interaction.

[0054] The term "epitope," as used herein, refers to a portion of an antigen that contacts a specific binding domain of an antigen-binding protein, and is also called a paratope. Epitopes may be linear or conformal / discontinuous. Conformal or discontinuous epitopes consist of amino acid residues separated by other sequences, i.e., amino acid residues that are not in a continuous sequence in the primary sequence of the antigen assembled by the three-dimensional folding of the polypeptide chain. The residues may come from different regions of the polypeptide chain but are in close proximity in the three-dimensional structure of the antigen. In the case of multimeric antigens, conformal or discontinuous epitopes may consist of residues from different peptide chains. The specific residues contained in an epitope can be determined by the three-dimensional structure obtained by computer modeling programs or by methods known in the art, such as X-ray crystallography. Epitope mapping can be performed using various techniques known to those skilled in the art, as described in publications such as Methods in Molecular Biology 'Epitope Mapping Protocols', Mike Schutkowski and Ulrich Reineke (volume 524, 2009) and Johan Rockberg and Johan Nilvebrant (volume 1785, 2018). Exemplary methods include peptide-based approaches such as pepscan, in which a series of overlapping peptides are screened for binding using techniques such as ELISA or in vitro display of a large library of peptide or protein variants, for example, by phages. Detailed epitope information can be determined by structural techniques including X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). Mutagenesis, such as alanine scanning, is an effective approach for epitope mapping, where binding loss analysis is used. Alternatively, a combination of hydrogen / deuterium exchange (HDX), proteolysis, and liquid chromatography-mass spectrometry (LC-MS) can be used to characterize discontinuities or conformational epitopes.

[0055] Competition between the BMP1 / TLL1 / TLL2-binding protein of the present invention and a reference BMP1 / TLL1 / TLL2-binding protein, such as a reference antibody, can be determined by one or more techniques known to those skilled in the art, such as ELISA, FMAT, surface plasmon resonance (SPR), or FORTEBIO OCTET biolayer interferometry (BLI). Such techniques are sometimes called epitope binning. There are several possible reasons for this competition, including that the two proteins may bind to the same or overlapping epitopes, there may be steric inhibition of binding, or the binding of the first protein may induce conformational changes of the antigen that block or reduce the binding of the second protein.

[0056] The reduction or inhibition of biological activity may be partial or complete. The neutralizing antigen-binding protein may neutralize the activity of BMP1, TLL1 and / or TLL2 by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the activity of BMP1, TLL1 and / or TLL2 in the absence of the antigen-binding protein.

[0057] In some embodiments, the antigen-binding protein (i.e., polypeptide) of the present invention is isolated. “Isolated” antigen-binding protein is one that has been removed from its original environment. The term “isolated” may also be used to refer to an antigen-binding protein that substantially does not contain other antigen-binding proteins having different antigen specificities (e.g., an isolated antigen-binding protein that specifically binds to BMP1, TLL1 and / or TLL2, or fragments thereof, substantially does not contain antigen-binding proteins that specifically bind to antigens other than BMP1, TLL1 and / or TLL2). The term “isolated” may also be used to refer to a preparation in which the isolated antigen-binding protein is pure enough to be therapeutically administered when formulated as an active ingredient in a pharmaceutical composition, or has a purity of at least 70–80% (w / w), more preferably at least 80–90% (w / w), even more preferably 90–95% (w / w), and most preferably at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w).

[0058] In some embodiments, the polynucleotides used in the present invention are isolated. An "isolated" polynucleotide is one that has been removed from its original environment. For example, a naturally occurring polynucleotide is isolated if it has been separated from some or all of the substances that coexist in the natural system. For example, a polynucleotide is considered isolated if it has been cloned into a vector that is not part of its natural environment or if it is composed within cDNA.

[0059] For the purpose of comparing two closely related polypeptide sequences, the "sequence identity %" between the first polypeptide sequence and the second polypeptide sequence can be calculated using NCBI BLAST v2.0 with standard polypeptide sequence settings (BLASTP). For the purpose of comparing two closely related polynucleotide sequences, the "sequence identity %" between the first nucleotide sequence and the second nucleotide sequence can be calculated using NCBI BLAST v2.0 with standard nucleotide sequence settings (BLASTN).

[0060] A polypeptide or polynucleotide sequence is said to be the same or "identical" if it has 100% sequence identity with another polypeptide or polynucleotide sequence throughout its entire length. Residues in a sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus in the case of polypeptides, and from the 5' end to the 3' end in the case of polynucleotides.

[0061] A "difference" between sequences refers to the insertion, deletion, or substitution of a single amino acid residue at the position of the second sequence compared to the first sequence. Two polypeptide sequences can contain one, two, or more such amino acid differences. An insertion, deletion, or substitution in the second sequence that is otherwise identical to the first sequence (100% sequence identity) results in a decrease in sequence identity percentage. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in 88.9% sequence identity. If the first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences have more than 66% identity (the first and second polypeptide sequences have 66.7% identity).

[0062] Alternatively, for the purpose of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can be determined. “Addition” refers to the addition of one amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). “Substitution” refers to the replacement of one amino acid residue in the sequence of the first polypeptide with one different amino acid residue. Such substitutions may be conserved or non-conservative. “Deletion” refers to the deletion of one amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0063] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another ligated nucleic acid. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated to a viral genome. Certain types of vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication, as well as episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can direct the expression of the gene to which they are operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Hereinafter, “plasmid” and “vector” may be used interchangeably, as plasmids are the most common form of vector. However, the present invention is intended to include equivalent functional viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), as well as other forms of expression vectors such as bacteriophages and phagemid systems. The term “recombinant host cell” (or simply “host cell”) is intended, as used herein, to refer to a cell into which a recombinant expression vector has been introduced. Such terminology is intended to refer not only to a specific target cell but also to the offspring of such a cell.

[0064] When referring to "control," "patient," or "individual," it means the subject being treated, particularly a mammalian subject. Mammalian subjects include humans, non-human primates, farm animals (e.g., cattle), sports animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, or mice. In some embodiments, the subject is human. In other embodiments, the subject is a non-human mammal such as a mouse.

[0065] The term "sufficient amount" means an amount sufficient to produce the desired effect. The term "therapeutic amount" is an amount that is effective in improving the symptoms of a disease or disorder. A therapeutic amount may also be a "preventive amount," as prevention can sometimes be considered a treatment.

[0066] As used herein, the term "approximately" includes values ​​from 10% greater to 10% less than a specified value, and especially from 5% greater to 5% less than a specified value. The term "between" includes values ​​within a specified boundary.

[0067] Those skilled in the art will understand that post-translational modifications can occur during the production of antigen-binding proteins, such as antibodies, in host cells. For example, these include cleavage of specific leader sequences, addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycation, deamidation, oxidation, disulfide bond scrambling and other cysteine ​​variants (e.g., free sulfhydryl, racemized disulfide, thioether, and trisulfide bonds), isomerization, C-terminal lysine clipping, and N-terminal glutamine cyclization. The present invention encompasses the use of antigen-binding proteins that have undergone or are subject to one or more post-translational modifications. Accordingly, the “antigen-binding protein” or “antibody” of the present invention includes the “antigen-binding protein” or “antibody” described herein, respectively, that have undergone post-translational modifications as described herein.

[0068] Glycation is a post-translational, non-enzymatic chemical reaction between reducing sugars such as glucose and free amine groups in proteins, and is commonly found at the ε-amine of the lysine side chain or at the N-terminus of proteins. Glycation can only occur in the presence of reducing sugars during production and storage.

[0069] Deamidation reactions that can occur during production and storage are primarily enzymatic reactions that convert asparagine (N) to isoaspartic acid and aspartic acid (D) in a ratio of approximately 3:1. Therefore, this deamidation reaction is related to the isomerization of aspartic acid (D) to isoaspartic acid. Both asparagine deamidation and aspartic acid isomerization involve the intermediate succinimide. Deamidation can also occur, albeit to a lesser degree, at glutamine residues. Deamidation can occur in the CDR, Fab (non-CDR region), or Fc region.

[0070] Oxidation occurs during production and storage (i.e., in the presence of oxidative conditions) and results in covalent modifications of proteins, directly induced by reactive oxygen species or indirectly induced by reactions with secondary byproducts of oxidative stress. Oxidation primarily occurs at methionine residues, but can also occur at tryptophan and free cysteine ​​residues. Oxidation can occur in CDR, Fab (non-CDR), or Fc regions.

[0071] Disulfide bond scrambling can occur during production and under basic storage conditions. Under certain circumstances, disulfide bonds may be cleaved or improperly formed, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryl (-SH) can promote shuffling.

[0072] Thioether formation and racemization of disulfide bonds can occur under basic conditions during production or storage by beta-quenching of disulfide bridges returning to cysteine ​​residues via dehydroalanine and persulfate intermediates. Subsequently, thioether bonds may be formed by cross-linking of dehydroalanine and cysteine, or free cysteine ​​residues may form disulfide bonds with a mixture of D-cysteine ​​and L-cysteine.

[0073] Trisulfides are formed when a sulfur atom is inserted into a disulfide bond (Cys-SS-Cys) and are created due to the presence of hydrogen sulfide in the cell culture.

[0074] The N-terminal glutamine (Q) and glutamic acid (E) of the heavy and / or light chains are thought to form pyroglutamic acid (pGlu) through cyclization. While much of pGlu formation occurs within the production bioreactor, it can also occur non-enzymatically due to pH and temperature conditions during processing and storage. Cyclization of the N-terminal Q or E is commonly observed in naturally occurring human antibodies.

[0075] C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in recombinant and native human antibodies. Variants of this process include the removal of lysine from one or both heavy chains by cellular enzymes from recombinant host cells. When administered to human subjects / patients, the remaining C-terminal lysine may be removed.

[0076] Fc engineering can be applied to modulate the functional or pharmacokinetic properties of antibodies. Effector function can be altered by introducing mutations in the Fc region that increase or decrease binding to C1q or Fcγ receptors, thereby altering CDC or ADCC activity, respectively. Effector function can also be altered by changing the glycosylation pattern of the antibody. The in vivo half-life of an antibody can be altered by introducing mutations that affect the binding of Fc to the neonatal Fc receptor (FcRn).

[0077] As used herein, the term "effector function" refers to one or more of the following: antibody-mediated action, including antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-mediated complement activation, including complement-dependent cell-mediated cytotoxicity (CDC); complement-dependent cell-mediated phagocytosis (CDCP); antibody-dependent complement-mediated cytolysis (ADCML); and Fc-mediated phagocytosis or antibody-dependent cell-mediated phagocytosis (ADCP).

[0078] The interaction between the Fc region of antigen-binding proteins or antibodies and various Fc receptors (FcRs), including FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), FcRn, C1q, and type II Fc receptors, is thought to mediate the effector function of antigen-binding proteins or antibodies. Important biological effects may result from this effector function. Typically, the ability to mediate effector function requires the binding of the antigen-binding protein or antibody to an antigen, and not all antigen-binding proteins or antibodies mediate all effector functions.

[0079] Effector function can be evaluated in many ways, including, for example, evaluating the ADCC effector function of antibodies coating target cells mediated by natural killer (NK) cells via FcγRIII or by monocytes / macrophages via FcγRI, or evaluating the CDC effector function of antibodies coating target cells mediated by the C1q complement cascade. For example, the antigen-binding protein of the present invention can be evaluated for ADCC effector function in a natural killer cell assay. Examples of such assays can be found in Shields et al, 2001, The Journal of Biological Chemistry, Vol. 276, p. 6591-6604; Chappel et al, 1993, The Journal of Biological Chemistry, Vol 268, p. 25124-25131; Lazar et al, 2006, PNAS, 103; 4005-4010. An example of an assay for determining CDC function is described in J Imm Meth, 1995, 184: 29-38.

[0080] The effects of mutations on effector functions (e.g., FcRn binding, FcγR and C1q binding, CDC, ADCML, ADCC, ADCP) can be evaluated, for example, as described in Grevys et al., J Immunol. 2015 Jun 1; 194(11): 5497-5508.

[0081] In this specification, amino acid residues in the Fc region of an antibody sequence or full-length antigen-binding protein sequence are numbered according to the EU index numbering rules.

[0082] Several isotypes of the human constant region, particularly the IgG4 and IgG2 isotypes, essentially lack a) classical complement activation and b) ADCC function. The heavy chain constant region of antigen-binding proteins may undergo various modifications to alter effector function, depending on the desired effector characteristics. IgG1 constant regions containing specific mutations that reduce binding to the Fc receptor and diminish effector functions such as ADCC and CDC have been described (Duncan et al. Nature 1988, 332; 563-564; Lund et al. J. Immunol. 1991, 147; 2657-2662; Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. Immunol. 1992, 51;1-84; Morgan et al., Immunology 1995, 86; 319-324; Hezareh et al., J. Virol. 2001, 75 (24); 12161-12168).

[0083] In one embodiment, BMP1, TLL1 and / or TLL2 binding proteins are provided, wherein the antigen-binding protein comprises a constant region such that effector function is reduced, such as a decrease in ADCC and / or CDC. In such an embodiment, the heavy chain constant region may comprise a naturally impaired constant region of an IgG2 or IgG4 isotype, or a mutant IgG1 constant region. Examples of preferred modifications are described in EP0307434. One example comprises a substitution with alanine at 235 and 237 (EU index numbering), i.e., L235A and G237A (commonly referred to as "LAGA" mutations). Another example comprises a substitution with alanine at 234 and 235 (EU index numbering), i.e., L234A and L235A (commonly referred to as "LALA" mutations). Further examples described in EP2691417 and US8969526 include P329G or P329R (EU index numbering) in combination with LALA mutations for IgG1 Fcs, and P329G or P329R (EU index numbering) in combination with S228P and L235E for IgG4 Fcs.

[0084] Further modifications and mutations to reduce effector function include the following (referring to IgG1 unless otherwise noted): aglycosylated N297A or N297Q or N297G; L235E; IgG4: F234A / L235A; and chimeric IgG2 / IgG4. IgG2: H268Q / V309L / A330S / P331S, and IgG2: V234A / G237A / P238S / H268A / V309L / A330S / P331S can reduce FcγR and C1q binding (Wang et al. 2018 and US8961967).

[0085] Other mutations that reduce effector function include: L234F / L235E / P331S; chimeric antibodies created using the CH1 and hinge regions from human IgG2 and the CH2 and CH3 regions from human IgG4; IgG2m4 based on an IgG2 isotype with changes in four key amino acid residues from IgG4 (H268Q, V309L, A330S, and P331S); IgG2σ containing V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions to remove affinity for the Fcγ receptor and C1q complement protein; IgG2m4 (H268Q / V309L / A330S / P331S, changed to IgG4); IgG4 (S228P / L234A / L235A); huIgG1 L234A / L235A(AA); huIgG4 Examples include S228P / L234A / L235A;IgG1σ(L234A / L235A / G237A / P238S / H268A / A330S / P331S);IgG4σ1(S228P / F234A / L235A / G237A / P238S); and IgG4σ2(S228P / F234A / L235A / ΔG236 / G237A / P238S, where Δ represents deletion) (Tam et al., Antibodies 2017, 6(3)).

[0086] Antigen-binding protein The present invention provides antigen-binding proteins that can specifically bind to BMP1, TLL1, and / or TLL2. In some embodiments, such antigen-binding proteins are anti-BMP1, TLL1, and / or TLL2 antibodies or fragments thereof.

[0087] The antigen-binding proteins described herein neutralize the activity of BMP1, TLL1, and / or TLL2 by binding to their catalytic domains. While not bound by any particular theory, the antigen-binding proteins of the present invention are thought to limit fibrosis and delay organ dysfunction by neutralizing BMP1, TLL1, and / or TLL2. For example, BMP1 / TLL converts soluble procollagen I to insoluble collagen fibers, leading to fibrosis, and the antigen-binding proteins described herein can neutralize the cleavage of procollagen I. Fibrosis is thought to occur in response to tissue damage in many, but not all, organ systems. While early fibrosis is beneficial for maintaining tissue integrity, excessive fibrosis leads to scarring and impaired normal organ function. Therefore, mitigating this pathological fibrosis may slow disease progression. Furthermore, inhibition of BMP1 has also been shown to promote muscle growth and enhance muscle function, which may consequently alleviate weakness.

[0088] In one embodiment, the antigen-binding protein is an antibody or a fragment thereof, and this antibody or fragment is an scFv, Fab, Fab', F(ab')2, Fv, variable domain (e.g., VH or VL), diabody, minibody, or monoclonal antibody. In a further embodiment, the antibody or fragment thereof is a monoclonal antibody.

[0089] The antibody of the present invention may be of any class, such as IgG, IgA, IgM, IgE, IgD, or any isotype thereof, and may contain a κ or λ light chain. In one embodiment, the antibody is an IgG antibody, for example, at least one of the isotypes IgG1, IgG2, IgG3, or IgG4. In a further embodiment, the antibody is in a form such as an IgG form modified to confer desired properties, such as having a mutated Fc for reduced effector function, extended half-life, altered ADCC, or improved hinge stability. Such modifications are described above.

[0090] In one embodiment, the antibody or its fragment is human. Therefore, the antibody or its fragment may be derived from a human immunoglobulin (Ig) sequence. The CDR, framework, and / or constant region of the antibody (or its fragment) may be derived from a human Ig sequence, particularly a human IgG sequence. The CDR, framework, and / or constant region may be substantially identical to a human Ig sequence, particularly a human IgG sequence. The advantage of using human antibodies is that they have low or no immunogenicity in humans.

[0091] The antibody or its fragment may also be a chimera, for example, a mouse-human antibody chimera.

[0092] Alternatively, the antibody or its fragment may be derived from a non-human species, such as a mouse. Such non-human antibodies can be modified to increase their similarity to naturally occurring antibody variants in humans, and thus the antibody or its fragment can be partially or completely humanized. Therefore, in one embodiment, the antibody or its fragment is humanized.

[0093] In one embodiment, the protein that binds to BMP1, TLL1, and / or TLL2 is an IgG antibody.

[0094] In some embodiments, the protein that binds to BMP1, TLL1, and / or TLL2 is an IgG antibody comprising at least one mutation to reduce Fc-mediated effector function, such as reduction of ADCC.

[0095] In some embodiments, the protein that binds to BMP1, TLL1, and / or TLL2 is an IgG antibody comprising mutants L235A and G237A (also known as "LAGA" mutants) to reduce Fc-mediated effector functions such as reduction of ADCC.

[0096] In some embodiments, the protein that binds to BMP1, TLL1, and / or TLL2 is a fully human monoclonal antibody. In some embodiments, the protein that binds to BMP1, TLL1, and / or TLL2 is a fully human monoclonal IgG1 antibody comprising mutants L235A and G237A to reduce Fc-mediated effector functions such as reduction of ADCC.

[0097] Sequence of antigen-binding proteins The proteins that bind to the BMP1, TLL1, and / or TLL2 antigens of the present invention, such as anti-BMP1, TLL1, and / or TLL2 antibodies or fragments, can be described with reference to their CDR sequences.

[0098] According to a first aspect of the present invention, (a)(i) Any one or combination of CDRH1, CDRH2, CDRH3 in SEQ ID NOs. 7, 22, 40, 54, 67, 82, 96, 110, 124, 138, 152, 166, 180, 194, 207 and 222 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NOs. 8, 21, 39, 53, 68, 81, 95, 109, 123, 137, 151, 165, 179, 193, 208 and 221 of the CDR; or (ii) A variant of the CDR of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 7, 22, 40, 54, 67, 82, 96, 110, 124, 138, 152, 166, 180, 194, 207 or 222, and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 8, 21, 39, 53, 68, 81, 95, 109, 123, 137, 151, 165, 179, 193, 208 or 221. A protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, is provided.

[0099] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 7 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 8; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 7 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 8. It consists of one or more of the following.

[0100] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 22 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 21; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 22 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 21. It consists of one or more of the following.

[0101] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 40 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 39; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 40 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 39. It consists of one or more of the following.

[0102] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 54 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 53; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 54 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 53. It consists of one or more of the following.

[0103] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 67 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 68; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 67 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 68. It consists of one or more of the following.

[0104] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 82 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 81; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) comprising one or more VH regions comprising a sequence that is at least 80% identical to the sequence of sequence number 82 and / or VL regions comprising a sequence that is at least 80% identical to the sequence of sequence number 81.

[0105] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 96 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 95; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 96 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 95. It consists of one or more of the following.

[0106] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 110 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 109; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 110 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 109. It consists of one or more of the following.

[0107] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 124 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 123; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 124 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 123. It consists of one or more of the following.

[0108] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 138 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 137; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 138 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 137. It consists of one or more of the following.

[0109] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 152 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 151; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 152 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 151. It consists of one or more of the following.

[0110] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 166 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 165; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 166 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 165. It consists of one or more of the following.

[0111] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 180 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 179; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 180 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 179. It consists of one or more of the following.

[0112] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 194 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 193; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 194 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 193. It consists of one or more of the following.

[0113] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 207 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 208; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 207 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 208. It consists of one or more of the following.

[0114] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, (a)(i) any one or combination of CDRs selected from CDRH1, CDRH2, CDRH3 in SEQ ID NO: 222 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NO: 221; or (ii) a CDR variant of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 222 and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 221. It consists of one or more of the following.

[0115] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, for example, an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, LCDR1 comprising a sequence having at least 80% sequence identity with RASQSVSSYLA (SEQ ID NO: 1); and / or LCDR2 comprising a sequence having at least 80% sequence identity with DASNRAT (SEQ ID NO: 2); and / or LCDR3 comprising a sequence having at least 80% sequence identity with QQSDSWPPT (SEQ ID NO: 3); and / or HCDR1 comprising a sequence having at least 80% sequence identity with GYYMS (SEQ ID NO: 4); and / or HCDR2 comprising a sequence having at least 80% sequence identity with WINPLSGETNYAQKFQG (Sequence ID 5); and / or HCDR3 comprising a sequence having at least 80% sequence identity with DTGELDGMNWYFDL (SEQ ID NO: 6) It consists of one or more of the following.

[0116] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VH region comprising a CDR1 having at least 80% sequence identity with GYYMS (SEQ ID NO: 4).

[0117] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VH region comprising a CDR2 having at least 80% sequence identity with WINPLSGETNYAQKFQG (SEQ ID NO: 5).

[0118] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VH region comprising a CDR3 having at least 80% sequence identity with DTGELDGMNWYFDL (SEQ ID NO: 6).

[0119] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VH region comprising a CDR1 containing the sequence GYYMS (SEQ ID NO: 4), a CDR2 containing the sequence WINPLSGETNYAQKFQG (SEQ ID NO: 5), and a CDR3 containing the sequence DTGELDGMNWYFDL (SEQ ID NO: 6).

[0120] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VL region comprising a CDR1 having at least 80% sequence identity with RASQSVSSYLA (SEQ ID NO: 1).

[0121] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VL region comprising a CDR2 having at least 80% sequence identity with DASNRAT (SEQ ID NO: 2).

[0122] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VL region comprising a CDR3 having at least 80% sequence identity with QQSDSWPPT (SEQ ID NO: 3).

[0123] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VL region comprising CDR1 comprising the sequence RASQSVSSYLA (SEQ ID NO: 1), CDR2 comprising the sequence DASNRAT (SEQ ID NO: 2), and CDR3 comprising the sequence QQSDSWPPT (SEQ ID NO: 3).

[0124] In one embodiment, a protein that binds to BMP1, TLL1 and / or TLL2 antigens, such as an anti-BMP1, TLL1 and / or TLL2 antibody or a fragment thereof, comprises the following six CDRs: LCDR1 of RASQSVSSYLA (SEQ ID NO: 1); LCDR2 of DASNRAT (SEQ ID NO: 2); LCDR3 of QQSDSWPPT (SEQ ID NO: 3); HCDR1 of GYYMS (SEQ ID NO: 4); HCDR2 of WINPLSGETNYAQKFQG (SEQ ID NO: 5); and HCDR3 of DTGELDGMNWYFDL (SEQ ID NO: 6).

[0125] A further aspect of the present invention provides a protein that binds to BMP1, TLL1 and / or TLL2 antigens, for example, an anti-BMP1, TLL1 and / or TLL2 antibody or a fragment thereof, comprising a VH region comprising the CDR1, CDR2 and CDR3 sequences described herein and a VL region comprising the CDR1, CDR2 and CDR3 sequences described herein.

[0126] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VL region comprising LCDR1 of RASQSVSSYLA (SEQ ID NO: 1); LCDR2 of DASNRAT (SEQ ID NO: 2); and LCDR3 of QQSDSWPPT (SEQ ID NO: 3); and a VH region comprising HCDR1 of GYYMS (SEQ ID NO: 4); HCDR2 of WINPLSGETNYAQKFQG (SEQ ID NO: 5); and HCDR3 of DTGELDGMNWYFDL (SEQ ID NO: 6).

[0127] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VH region having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7.

[0128] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VL region having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8.

[0129] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VH region having an amino acid sequence having 100% sequence identity with SEQ ID NO: 7.

[0130] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VL region having an amino acid sequence having 100% sequence identity with SEQ ID NO: 8.

[0131] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a VH region comprising the amino acid sequence of SEQ ID NO: 7 and a VL region comprising the amino acid sequence of SEQ ID NO: 8.

[0132] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a light chain having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9.

[0133] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a heavy chain having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10.

[0134] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a light chain having an amino acid sequence having 100% sequence identity with SEQ ID NO: 9.

[0135] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a heavy chain having an amino acid sequence having 100% sequence identity with SEQ ID NO: 10.

[0136] In one embodiment, a protein that binds to BMP1, TLL1, and / or TLL2 antigens, such as an anti-BMP1, TLL1, and / or TLL2 antibody or a fragment thereof, comprises a light chain comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 10.

[0137] In one aspect of the present invention, the protein that binds to BMP1, TLL1 and / or TLL2 is selected from the following and is a fully human Fc-impaired monoclonal antibody that binds to BMP-1, TLL1 and / or TLL2, defined according to the CDR, VH / VL and / or HC / LC sequences: 13Y039-4B06-4334, as shown in sequence numbers 1-10; 13Y039-3E07-2944, as shown in sequence numbers 15-24; 13Y039-8F02-2949, as shown in sequence numbers 33-42; 13Y039-4B06-4376, as shown in sequence numbers 47-56; 13Y039-4B06-4373, as shown in sequence numbers 61-70; 13Y039-4B06-4364, as shown in sequence numbers 75-84; 13Y039-4B06-4351, as shown in sequence numbers 89-98; 13Y039-4B06-4348, as shown in sequence numbers 103-112; 13Y039-4B06-4328, as shown in sequence numbers 117-126; 13Y039-4B06-4327, as shown in sequence numbers 131-140; 13Y039-4B06-4325, as shown in sequence numbers 145-154; 13Y039-4B06-4324, as shown in sequence numbers 159-168; 13Y039-127G03-2890, as shown in sequence numbers 173-182; 13Y039-152B02-2948, as shown in sequence numbers 187-196; 13Y039-152B02-2940, as shown in sequence numbers 201-210; and 13Y039-152B02-2935, as shown in sequence numbers 215-224.

[0138] In further embodiments, anti-BMP-1, TLL1, and / or TLL2 antibodies are selected from the following and defined according to the CDR, VH / VL, and / or HC / LC sequences: 13Y039-4B06-4334, as shown in sequence numbers 1-10; 13Y039-3E07-2944, as shown in sequence numbers 15-24; and 13Y039-8F02-2949, as shown in sequence numbers 33-42.

[0139] In further embodiments, anti-BMP-1, TLL1, and / or TLL2 antibodies are selected from the following and defined according to the CDR, VH / VL, and / or HC / LC sequences: 13Y039-4B06-4334, as shown in sequence numbers 1-10; and 13Y039-3E07-2944, as shown in sequence numbers 15-24.

[0140] In further embodiments, anti-BMP-1, TLL1 and / or TLL2 antibodies: 13Y039-4B06-4334, as shown in sequence numbers 1-10.

[0141] Embodiments indicated herein as “at least 80%” or “80% or more” are understood to include all values ​​equal to or greater than 80%, for example, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity. In one embodiment, an antigen-binding protein such as an antibody or fragment of the present invention comprises at least 85%, for example, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with a particular sequence.

[0142] Binding to target antigen The antigen-binding protein of the present invention can bind to the catalytic domain of human BMP1 with a binding affinity (KD) of less than 100 nM, as measured by SPR. In further embodiments, the KD is 50 nM or less, for example, 10 nM or less. In even further embodiments, the KD is 5 nM or less, for example, 2 nM or less. For example, according to one embodiment, a human anti-BMP1 antibody is provided that binds to the catalytic domain of BMP1 with a binding affinity (KD) of less than 2 nM, as measured by SPR.

[0143] The antigen-binding protein of the present invention can bind to the catalytic domain of human TLL1 with a binding affinity (KD) of less than 100 nM, as measured by SPR. In further embodiments, the KD is 50 nM or less, for example, 10 nM or less. In even further embodiments, the KD is 5 nM or less, for example, 2 nM or less. For example, according to one embodiment, a human anti-TLL1 antibody is provided that binds to the catalytic domain of TLL1 with a binding affinity (KD) of less than 2 nM, as measured by SPR.

[0144] The antigen-binding protein of the present invention can bind to the catalytic domain of human TLL2 with a binding affinity (KD) of less than 100 nM, as measured by SPR. In further embodiments, the KD is 50 nM or less, for example, 10 nM or less. In yet another embodiment, the KD is 5 nM or less, for example, 4 nM or less. For example, according to one embodiment, a human anti-TLL2 antibody is provided that binds to the catalytic domain of TLL2 with a binding affinity (KD) of less than 4 nM, as measured by SPR.

[0145] For example, according to one embodiment, human anti-BMP1 antibody, human anti-TLL1 antibody, and human anti-TLL2 antibody are provided, which bind to the catalytic domain of BMP1 with a binding affinity (KD) of less than 2 nM as measured by SPR, which binds to the catalytic domain of TLL2 with a binding affinity (KD) of less than 2 nM as measured by SPR, or which bind to the catalytic domain of TLL2 with a binding affinity (KD) of less than 4 nM as measured by SPR.

[0146] This specification also describes other assays that may be used to define the function of antigen-binding proteins.

[0147] Polynucleotides and expression vectors In one aspect of the present invention, a polynucleotide encoding a protein that binds to the BMP1, TLL1, and / or TLL2 antigens described herein is provided.

[0148] In one embodiment, the polynucleotide sequence encoding a protein that binds to BMP1, TLL1, and / or TLL2 antigens comprises a VL that has at least 70% sequence identity with SEQ ID NO: 11.

[0149] In one embodiment, the polynucleotide sequence encoding a protein that binds to BMP1, TLL1, and / or TLL2 antigens comprises a VH chain having at least 70% sequence identity with SEQ ID NO: 12.

[0150] In one embodiment, the polynucleotide sequence encoding a protein that binds to BMP1, TLL1, and / or TLL2 antigens comprises a heavy chain having a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 13.

[0151] In one embodiment, the polynucleotide sequence encoding a protein that binds to BMP1, TLL1, and / or TLL2 antigens comprises a light chain having at least 70% sequence identity with SEQ ID NO: 14.

[0152] In one embodiment, the polynucleotide sequence encoding the protein that binds to BMP1, TLL1, and / or TLL2 antigens consists of the sequence of SEQ ID NOs. 13 and / or 14.

[0153] To express antigen-binding proteins such as antibodies or fragments thereof, polynucleotides encoding partial or full-length light and heavy chains, as described herein, are inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. Accordingly, one aspect of the present invention provides an expression vector comprising the polynucleotide sequences described herein.

[0154] In one embodiment, the expression vector comprises the heavy chain of SEQ ID NO: 13.

[0155] In one embodiment, the expression vector comprises the light chain of SEQ ID NO: 14.

[0156] In one embodiment, the expression vector comprises the heavy chain of SEQ ID NO: 13 and the light chain of SEQ ID NO: 14.

[0157] The nucleotide sequences described herein include additional sequences encoding amino acid residues to aid in translation, purification, and detection, but it will be understood that other sequences may be used depending on the expression system used. These optional sequences may be deleted, modified, or substituted if a different design, translation, purification, or detection strategy is employed.

[0158] While the amino acid sequence of polypeptides remains silent, mutations can be used to create DNA or cDNA encoding polypeptides that provide preferred codons for translation in specific hosts. Preferred codons for nucleic acid translation are known, for example, in Escherichia coli (E. coli), S. cerevisiae, and mammals, particularly humans.

[0159] Polypeptide mutations can be achieved, for example, by substitution, addition, or deletion of the nucleic acid encoding the polypeptide. Substitutions, additions, or deletions of polypeptide-encoding nucleic acids can be introduced by many methods, including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recurrent ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, gene reassembly, artificial gene synthesis, gene site saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or combinations thereof. Furthermore, modifications, additions, or deletions to nucleic acids can also be introduced by methods including recombination, recurrent sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped double-strand mutagenesis, point mismatch repair mutagenesis, repair-deficient host-strain mutagenesis, chemical mutagenesis, radiation mutagenesis, deletion mutagenesis, restriction selection mutagenesis, restriction purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimerization, or combinations thereof.

[0160] In particular, artificial gene synthesis can be used. The gene encoding the polypeptide of the present invention can be produced synthetically, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo without the need for precursor template DNA. To obtain the desired oligonucleotide, the constituent blocks are sequentially attached to the growing oligonucleotide chain in the order required by the product sequence. Once the chain assembly is complete, the product is released from the solid phase into solution, deprotected, and recovered. The product can be separated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide with high purity.

[0161] Expression vectors include, for example, plasmids, retroviruses, cosmids, yeast artificial chromosomes (YACs), and Epstein-Barr virus (EBV)-derived episomes. Polynucleotides are ligated into the vector so that transcription and translation control sequences within the vector perform the intended function of controlling the transcription and translation of the polynucleotides. Expression and / or control sequences may include promoters, enhancers, transcription terminators, the 5' start codon (i.e., ATG) of the coding sequence, intron splicing signals, and stop codons. Expression vectors and expression control sequences are selected to be compatible with the expression host cells used. Sequence IDs 11-12 comprise nucleotide sequences encoding the single-stranded variable fragment of the present invention, comprising a VH region and a VL region. It will be understood that the polynucleotides or expression vectors of the present invention may encode the VH region, the VL region, or both; or they may encode the heavy chain, the light chain, or both. Therefore, polynucleotides encoding the VH and VL regions (or heavy and light chains) can be inserted into separate vectors, or sequences encoding both regions or chains can be inserted into the same expression vector. Polynucleotides are inserted into the expression vector by standard methods (e.g., ligation of the polynucleotide with a complementary restriction site on the vector, or blunt-end ligation if no restriction site exists).

[0162] Convenient vectors encode functionally complete human CH or CL immunoglobulin sequences, with appropriate restriction sites manipulated to allow for the easy insertion and expression of any VH or VL sequence, as described herein. Expression vectors can also encode signal peptides that facilitate the secretion of antigen-binding proteins, such as antibodies (or fragments thereof), from host cells. Polynucleotides can be cloned into vectors such that the signal peptide is in-frame linked to the amino terminus of the antigen-binding protein. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0163] In one aspect of the present invention, a cell (e.g., a host cell or recombinant host cell) comprising a polynucleotide or expression vector described herein is provided. It will be understood that the cell may comprise a first vector encoding the light chain of an antibody or fragment thereof, and a second vector encoding the heavy chain of an antibody or fragment thereof. Alternatively, both the heavy and light chains encoded in the same expression vector may be introduced into the cell.

[0164] In one embodiment, a polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to an antibody or fragment thereof, and the antibody or fragment thereof is presented on the extracellular surface of a cell.

[0165] Transformation can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides into liposomes, microparticle gun injection, and direct microinjection of DNA into the nucleus. Furthermore, nucleic acid molecules can also be introduced into mammalian cells by viral vectors.

[0166] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and numerous other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly advantageous cell lines are selected by determining which cell lines have high expression levels. Other usable cell lines include insect cell lines (e.g., Sf9 cells), amphibian cells, bacterial cells, plant cells, and fungal cells. Antigen-binding fragments of antibodies, e.g., scFv and Fv fragments, can be isolated using methods known in the art and expressed in Escherichia coli.

[0167] Antigen-binding proteins are produced by culturing host cells for a period sufficient to allow expression of the antigen-binding protein in the host cells, or more preferably, secretion of the antigen-binding protein into the culture medium in which the host cells are growing. Antigen-binding proteins can be recovered from the culture medium using standard protein purification methods.

[0168] The antibody (or fragment) of the present invention can be obtained and manipulated using, for example, the techniques disclosed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012) 4th Edition, Cold Spring Harbour Laboratory Press.

[0169] Monoclonal antibodies, in particular, can be produced using hybridoma technology by fusing specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for their ability to proliferate in tissue culture and their inability to synthesize antibody chains.

[0170] Monoclonal antibodies against the determined antigen are, for example, a) Immortalizing lymphocytes obtained from the peripheral blood of animals pre-immunized with the determined antigen in order to form hybridomas, preferably with immortalized cells, myeloma cells. b) Culture the formed immortalized cells (hybridomas) and collect cells that produce antibodies with the desired specificity. It can be obtained by doing so.

[0171] Alternatively, the use of hybridoma cells is not necessary. Antigen-binding proteins capable of binding to target antigens as described herein can be isolated from suitable antibody libraries by conventional practices, such as using phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Therefore, monoclonal antibodies are particularly, for example, a) A step of cloning a DNA or cDNA sequence obtained from animal lymphocytes (preferably pre-immunized with a determined antigen), particularly peripheral blood lymphocytes, into a vector, in particular a phage, more specifically a filamentous bacteriophage. b) A step of transforming prokaryotic cells with the above vector under conditions that enable antibody production, c) A step of selecting an antibody by performing antigen affinity selection, and d) A step of recovering antibodies having the desired specificity. It can be obtained by a method that includes [a certain element].

[0172] Pharmaceutical composition In a further embodiment, a composition is provided comprising proteins that bind to BMP1, TLL1, and / or TLL2 as described herein. In such embodiments, the composition may comprise the antigen-binding protein in combination with other excipients as optional. A composition is also provided comprising one or more additional active ingredients (e.g., active ingredients suitable for treating the diseases described herein).

[0173] In a further embodiment, a pharmaceutical composition is provided comprising a protein that binds to BMP1, TLL1, and / or TLL2 as described herein, together with a pharmaceutically acceptable diluent or carrier. The antigen-binding protein described herein can be incorporated into a pharmaceutical composition suitable for administration to a subject. Generally, the pharmaceutical composition comprises the antigen-binding protein described herein and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic agent and absorption retarder, etc. Examples of pharmaceutically acceptable carriers include one or more of water, physiological saline, salt, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Often, it is preferable to include an isotonic agent in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride. Furthermore, the pharmaceutical composition may include pharmaceutically acceptable substances such as humectants, emulsifiers, preservatives, or buffers, or other trace amounts of auxiliary substances that enhance the shelf life or efficacy of the antibody or its fragments.

[0174] The compositions described herein may be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and injectable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. A typical preferred composition is in the form of an injectable or injectable solution administered by injection or serial infusion (e.g., but not limited to intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and portal vein infusions).

[0175] The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antigen-binding protein is administered by intravenous infusion or injection. In another preferred embodiment, the antigen-binding protein is administered by intramuscular or subcutaneous injection. In yet another preferred embodiment, the antigen-binding protein is administered by subcutaneous injection, generally once a month.

[0176] Therapeutic compositions generally must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations.

[0177] It is within the scope of the present invention to use the pharmaceutical compositions of the present invention as an adjunct to, or in combination with, other established therapies commonly used for the treatment of diseases such as those described herein.

[0178] In a further embodiment of the present invention, an antigen-binding protein, composition, or pharmaceutical composition is administered sequentially, simultaneously, or separately with at least one active ingredient.

[0179] The pharmaceutical composition may be included in a kit containing an antigen-binding protein together with other pharmaceuticals, and / or with instructions for use. For convenience, the kit may also include a predetermined amount of reagents and instructions for use. The kit may also include a device used for administering the pharmaceutical composition.

[0180] The antigen-binding proteins described herein may also be used in therapeutic methods. Those skilled in the art will understand that, as used herein, "therapeutic" refers to the treatment of an established condition. However, under certain conditions, the compounds of the present invention may also be useful for the prevention of certain diseases. The antigen-binding proteins described herein are used in effective amounts for therapeutic, prophylactic, or preventative measures. A therapeutically effective amount of the antigen-binding proteins described herein is an amount effective for improving or reducing one or more symptoms of a disease, or for preventing or curing a disease.

[0181] Treatment method A further aspect of the present invention provides proteins that bind to BMP1, TLL1 and / or TLL2 as described herein, or pharmaceutical compositions as described herein, for use as pharmaceuticals or in therapy.

[0182] The antigen-binding proteins of the present invention neutralize the activity of BMP1, TLL1, and / or TLL2, and may be particularly useful in the treatment of diseases related to BMP1, TLL1, and / or TLL2 activity, including, for example, the treatment of diseases in which inhibition of BMP1, TLL1, and / or TLL2 is therapeutically beneficial. For example, the antigen-binding proteins of the present invention may be particularly useful in the treatment of diseases in which inhibition of tissue ECM (extracellular matrix) production and / or maturation is beneficial, or inhibition of myostatin activity is beneficial, or inhibition of fibrosis is beneficial.

[0183] In some embodiments, diseases associated with BMP1, TLL1, and / or TLL2 activity are selected from fibrosis-related diseases or disorders in organs or tissues of the body, e.g., pathological fibrotic conditions or diseases associated with fibrosis (e.g., prevention and regression of fibrosis), such as the following conditions: Cardiac conditions (e.g., myocardial infarction ("MI"), prevention of post-MI heart failure, heart failure (e.g., reduced ejection fraction heart failure (HFrEF), maintained ejection fraction heart failure), cardiac arrhythmias (e.g., atrial fibrillation), cardiac fibrosis (e.g., hypertrophic cardiomyopathy), acute decompression heart failure, atrial fibrillation); Lungs (e.g., chronic obstructive pulmonary disease ("COPD"), lung / pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis ("IPF"), pulmonary arterial hypertension (PAH)); Kidney (e.g., diabetic nephropathy, post-acute kidney injury, chronic kidney disease ("CKD"), delayed renal function development after transplantation, renal fibrosis, peritoneal fibrosis, and prevention of peritoneal fibrosis in peritoneal dialysis patients (e.g., delaying the transition to hemodialysis in end-stage renal disease patients), focal segmental glomerulosclerosis (FSGS)); The liver (e.g., cirrhosis, non-alcoholic steatohepatitis ("NASH"), hepatic fibrosis (e.g., post-HCV hepatic fibrosis)); Eyes (e.g., glaucoma, corneal scarring); Skeletal muscle (e.g., muscular dystrophy (including Duchenne, Becker limb, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreyfus types), recurrent muscle injury); Skin (e.g., keloids, wound healing, adhesions, hypertrophic scars and other scars (e.g., scars associated with burns, surgery or other trauma), Dupuytren's contracture, lymphedema, scleroderma); Vascular system (e.g., stroke and collagen disease vascular diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis and scleroderma)); and The nervous system (e.g., spinal cord injury, multiple sclerosis).

[0184] In some embodiments, diseases associated with BMP1, TLL1, and / or TLL2 activity are selected from cancer and cancer cell metastasis.

[0185] In some embodiments, diseases associated with BMP1, TLL1, and / or TLL2 activity are Idiopathic pulmonary fibrosis; Hypertrophic cardiomyopathy; and Prevention of peritoneal fibrosis in peritoneal dialysis patients Selected from.

[0186] In a particular embodiment, the disease associated with BMP1, TLL1, and / or TLL2 activity is NASH (non-alcoholic steatohepatitis). NASH is a subtype of non-alcoholic fatty liver disease characterized by liver inflammation and a substantial risk of progression to cirrhosis, with more advanced stages of these diseases being characterized by inflammation.

[0187] In some embodiments, diseases associated with BMP1, TLL1 and / or TLL2 activity are selected from muscle diseases characterized by decreased muscle function and / or muscle mass, such as muscular dystrophy (e.g., Duchenne, Becker limb, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreyfus types), sarcopenia, and cachexia (e.g., heart failure, CKD, COPD, cancer, or age-related cachexia).

[0188] Accordingly, the following are provided: proteins that bind to BMP1, TLL1 and / or TLL2 as described herein, or pharmaceutical compositions as described herein, for use in the treatment of fibrosis-related diseases or disorders as described herein.

[0189] In a further embodiment, a method is provided for treating a fibrosis-related disease or disorder described herein in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of a protein or pharmaceutical composition described herein that binds to the BMP1, TLL1 and / or TLL2 antigens.

[0190] In a further embodiment, the use of proteins that bind to BMP1, TLL1 and / or TLL2 as described herein, or the pharmaceutical compositions described herein, in the manufacture of a pharmaceutical for use in the treatment of fibrosis-related diseases or disorders described herein is provided.

[0191] The proteins that bind to BMP1, TLL1, and / or TLL2 described herein may also be used to promote muscle growth and / or improve muscle function, for example, to alleviate weakness (e.g., muscle wasting) in cachexia patient populations.

[0192] Therefore, according to a further embodiment, a method is provided for promoting muscle growth and / or improving muscle function in a subject requiring such action, comprising administering to the subject a therapeutically effective amount of a protein or pharmaceutical composition described herein that binds to BMP1, TLL1 and / or TLL2.

[0193] In a further embodiment, proteins that bind to BMP1, TLL1, and / or TLL2 as described herein, or pharmaceutical compositions as described herein, are provided for use in promoting muscle growth and / or improving muscle function.

[0194] A further aspect of the present invention provides the use of proteins that bind to BMP1, TLL1 and / or TLL2 as described herein, or pharmaceutical compositions as described herein, in the manufacture of a pharmaceutical for promoting muscle growth and / or improving muscle function.

[0195] Other features and advantages of the present invention will become apparent from the description provided herein. However, the description and specific examples illustrating preferred embodiments of the present invention should be understood to be for illustrative purposes only, as various changes and modifications will become apparent to those skilled in the art. The present invention will be described below using the following non-limiting examples. [Examples]

[0196] Antibody generation and characterization Using an in vitro antibody discovery platform, we identified and performed affinity maturation of fully human antibodies specific to human BMP1 / TLL.

[0197] The clones obtained from the selection process were screened in a series of experiments to understand their binding rate, potency, and biophysical properties. Following this, a lead panel of 16 monoclonal antibodies with desirable functional characteristics was selected. These 16 antibody lead panels were expressed and purified from HEK293-6E cells for further functionalization and characterization studies. Functional characterization was performed against recombinant human and homologous species, as well as human serum and rat plasma (endogenous expression).

[0198] Since effector function is not required for the desired mechanism of action, residues L235 and G237 in the CH2 domain of the heavy chain constant region were mutated to alanine residues (LAGA mutation). These mutations have been shown to eliminate the ability of the IgG1 antibody to lyse target cells via ADCC or CDC [Bartholomew et al. (1995). Immunology 85, 41-48; Bret et al. (1997) Immunology 91, 346-353].

[0199] 13Y039-4B06-4334 and 13Y039-3E07-2944 were selected for in vivo characterization. These antibodies were cloned as reverse chimera mAbs having human variable regions in mouse IgG2a LAGA Fc and mouse κ (referred to herein as compound A for 4B06-4334 and compound B for 3E07-2944) and human variable regions in rat IgG2b LAGA Fc and rat κ (referred to herein as compound C for 4B06-4334 and compound D for 3E07-2944). These reverse chimeras were tested in the AngII / PE efficacy test described below.

[0200] Characterization of binding of anti-BMP1 / TLL antibodies expressed in mammals. Characterization of the binding of anti-BMP1 / TLL antibodies expressed in mammals by surface plasmon resonance (SPR).

[0201] Binding of the entire read panel expressed in HEK to human BMP1 CD+CUB1 via surface plasmon resonance (SPR). The binding of 16 lead panel antibodies expressed in HEK cells to terminally cleaved human BMPs (human BMP1 CD+CUB1 terminally cleaved antigens) containing catalytic and terminal cleavage domains was evaluated by SPR using BIACORE T200. Experiments were performed at 25°C using HBS-EP+ buffer. Protein A was immobilized on a CM5 sensor chip by amine coupling. The lead panel antibodies were captured on the surface of protein A. Next, human BMP1 CD+CUB1 was passed over the captured antibodies at various concentrations. The results are shown in Table 1 below.

[0202] [Table 3]

[0203] The results in Table 1 show that all 16 antibodies bound to human BMP1 CD+CUB1 and exhibited affinities in the range of 0.3 to 4.3 nM, with an affinity of a single order of magnitude nM. 13Y039-4B06-4334 and 13Y039-3E07-2944 were further evaluated.

[0204] SPR-mediated binding of HEK-expressed antibodies 13Y039-4B06-4334 and 13Y039-3E07-2944 and reverse chimeras to human TLL2 CD+CUB1 and mouse BMP1 CD+CUB1. Both antibodies, 13Y039-4B06-4334 and 13Y039-3E07-2944, along with the reverse chimeric antibody, were evaluated for binding to human TLL2 CD+CUB1 and mouse BMP1 CD+CUB1 cleaved-end antigens by SPR using a BIACORE T200. Protein A / G was immobilized on a CM5 sensor chip via amine coupling, and the antibodies were captured on the surface of the protein A / G. Antigens were passed over the captured antibodies at various concentrations. The measured affinity for human TLL2 CD+CUB1 is shown in Table 2, and the measured affinity for mouse BMP1 CD+CUB1 is shown in Table 3.

[0205] [Table 4]

[0206] The results in Table 2 show that 13Y039-4B06-4334 in HBS-EP+ buffer has an affinity of 3.08 nM for human TLL2 CD+CUB1, and 13Y039-3E07-2944 is not a binder for human TLL2 CD+CUB1.

[0207] [Table 5]

[0208] The results in Table 3 show that the affinity of both antibodies against mouse BMP1 CD+CUB1 is within a single-digit nM range (1.7 to 6.1 nM).

[0209] Reverse chimeric antibodies possessing the human variable region and mouse constant κ region on mouse IgG2a LAGA Fc were also evaluated for binding to all CD+CUB1 terminal cleavages. These experiments included negative antibody controls of anti-RSV mouse IgG2a LAGA Fc and anti-MOPC21 mouse IgG2a, both possessing the human variable region and mouse constant κ region. Neither negative control was a binder for any antigen (see Tables 4, 5, and 6).

[0210] Table 4 shows the affinity of the reverse chimera for human BMP1 CD+CUB1. When performed in HBS-EP+, the affinity for both antibodies was in the single order of nM (2.33–5.31 nM). When performed in enzyme dilution buffer, the affinity was stronger, in the double order of pM (40–70 pM). These results are comparable to the affinity of the human antibody reported in Table 1.

[0211] Table 5 shows the affinity of reverse chimeras to human TLL2 CD+CUB1. The affinity measured for 13Y039-4B06-4334 was 3.54 nM. 13Y039-3E07-2944 did not bind to human TLL2 CD+CUB1. These results are comparable to the affinity of the human antibodies reported in Table 2.

[0212] Table 6 shows the affinity of reverse chimeras for mouse BMP1 CD+CUB1. The affinity ranges in the single-digit nM range (0.82 to 6.2 nM). These results are comparable to the affinity of human antibodies reported in Table 3.

[0213] Both antibody 13Y039-4B06-4334 and its reverse skimer exhibited similar affinity for human BMP1 CD-CUB1, mouse BMP1 CD-CUB1, and human TLL2 CD-CUB1, further supporting the use of the reverse skimer as an alternative to 13Y039-4B06-4334 in mouse preclinical efficacy studies.

[0214] [Table 6]

[0215] [Table 7]

[0216] [Table 8]

[0217] Table 7 summarizes all kinetic and affinity data for antibody 13Y039-4B06-4334, expressed in HEK cells and binding to all CD+CUB1 antigens. This shows that this antibody has an affinity of one order of magnitude nM for all truncated CD+CUB1 targets when tested with HBS-EP+, and its affinity for human BMP1 CD+CUB1 is stronger (40 pM) in enzyme dilution buffer.

[0218]

Table 9

[0219] Binding of anti-BMP1 / TLL antibody 13Y039-4B06-4334 to full-length human and homologous BMP1 / TLL antigens by surface plasmon resonance (SPR). 13Y039-4B06-4334 was expressed in CHO cells and tested in humans and all homologous species (cynomolgus monkey, rat, and mouse) for binding to all full-length BMP1 antigen and TLL antigen. The proteins used in this assay were recombinant forms of naturally occurring proteins as a comparison to the truncated antigens used in the above selection assay. The results were obtained in two buffers: HBS-EP+ and HBS-N+ 5 mM CaCl2 and 1 μM ZnCl2. Protein A was immobilized on a CM5 sensor chip by amine coupling, and antibodies were captured on the Protein A surface. The antigens were diluted in both buffers and passed over the captured antibodies at various concentrations.

[0220] 13Y039-4B06-4334 bound to recombinant human BMP1 with an affinity of 23.6 pM, to TLL-1 with an affinity of 880 pM, and to TLL-2 with an affinity of 4270 pM. The high-affinity binding was dependent on the addition of Zn 2+ and Ca 2+ .

[0221] The results are shown in Table 8.

[0222]

Table 10

[0223] In vitro target association efficacy Screening of a mammalian expression read panel of 16 anti-BMP-1 / TLL monoclonal antibodies (mAbs) for activity against recombinant human BMP-1-1, human TLL-1, mouse BMP-1-3 (707-terminus truncated), mouse TLL-1, rat TLL-1, rat TLL-2, and cynomolgus monkey TLL-1 enzymes using a FRET-based assay. The activity of the BMP-1 / TLL enzyme was detected using a fluorescence resonance energy transfer (FRET) assay. Peptide substrates across the BMP1 / TLL cleavage site of the physiological protein substrate prolysyl oxidase were labeled with two fluorophores, specifically donor and quencher (acceptor) molecules (hereinafter referred to as "prolysyl oxidase FRET peptide"). When the donor fluorophore is excited by light of its excitation wavelength, it typically produces transient, higher-wavelength emission as the molecule returns to its ground state. However, in the case of FRET peptides, the proximity of the acceptor fluorophore transfers this energy to the emission of the fluorescence signal, effectively quenching the fluorescence. When the peptide substrate is cleaved by the enzyme (in this case, the relevant species BMP-1, TLL-1, or TLL-2), the fluorophores are separated, allowing emission of fluorescence from the donor fluorophore. Antibody inhibition of BMP1 / TLL enzyme-mediated peptide degradation was determined by measuring the donor emission.

[0224] Serial dilutions of antibody samples were prepared in enzyme dilution buffer. The antibody samples were pre-incubated with the enzyme (enzyme dilution buffer) while gently shaking. After this pre-incubation, prolysyl oxidase FRET peptide was added and incubated. After incubation, the enzymatic reaction was stopped with EDTA. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0225] Screening was performed using a lead panel of 16 antibodies produced in HEK cells. 50 The values ​​were calculated and compared (Table 9). In this initial screening, the 4B06 family had ICs of 0.01 nM to 0.09 nM. 50 The value showed effective inhibition of human BMP-1, which was generally lower than other families tested. Early IC of 13Y039-4B06-4334 50 The values ​​showed effective inhibitory activity for all enzymes: huBMP-1 (IC) 50 ), 0.03nM;huTLL-1, 0.02nM;huTLL-2, 0.03nM;mTLL-1, 0.06nM;cynoTLL1, 0.05nM.

[0226] [Table 11]

[0227] The characteristics of three antibodies—13Y039-3E07-2944, 13Y039-8F02-2949, and 13Y039-4B06-4334—were further determined. They were tested to investigate the inhibition of huBMP-1, mBMP-1, huTLL-1, huTLL-2, mTLL-1, ratTLL-1, ratTLL-2, cynoTLL-1, and cynoTLL-2 activity. Total dose-response curves were plotted for each enzyme, and representative graphs for each enzyme are shown in Figures 1-9 (Figure 1, huBMP-1; Figure 2, mBMP-1; Figure 3, huTLL-1; Figure 4, huTLL-2; Figure 5, mTLL-1; Figure 6, ratTLL-1; Figure 7, ratTLL-2; Figure 8, cynoTLL-1; Figure 9, cynoTLL-2).

[0228] Antibody 13Y039-4B06-4334 inhibited all enzymes tested. IC for all tests 50 The values, along with the calculated mean and standard deviation, are shown in Table 10. The mean IC for each enzyme. 50 The values ​​were huBMP-1, 0.04nM; huTLL-1, 0.05nM; huTLL-2, 0.05nM; mBMP-1, 0.02nM; mTLL-1, 0.23nM; ratTLL-1, 0.50nM; ratTLL-2, 0.67nM; cynoTLL-1, 0.10nM; cynoTLL-2, 0.31nM.

[0229] [Table 12]

[0230] 13Y039-4B06-4334 was expressed in HEK cells and CHO cells, and the inhibition of recombinant human BMP1, human TLL1, and human TLL2 activities by the protein batches expressed in HEK and CHO was measured by FRET as generally described above. Profiling against recombinant human BMP1, human TLL1, human TLL2, and human serum showed that the potency was equivalent between the protein batches expressed by HEK293 and the protein batches expressed by polyclonal CHO (Table 11).

[0231]

Table 13

[0232] To test the biological efficacy of 13Y039-4B06-4334 in preclinical rodents, reverse chimeras were generated by fusing the variable domains of 13Y039-4B06-4334 to mouse or rat IgG2a Fc mutants. These constructs were profiled for activity against recombinant human BMP1 and mouse BMP1, as well as human serum, mouse plasma, and rat plasma, using the prollyl oxidase FRET peptide as described above.

[0233] The reverse chimeric antibodies and antibody 13Y039-4B06-4334 showed similar pKi,app values in both the recombinant human BMP1 assay and the mouse BMP1 assay, supporting their use in preclinical efficacy testing as an alternative to 13Y039-4B06-4334. Activity in human serum and mouse or rat plasma was confirmed for 13Y039-4B06-4334 and the reverse chimeric antibodies (Table 12).

[0234]

Table 14

[0235] Binding to Fcγ receptor, FcRN, and C1q The binding of an anti-BMP1 / TLL antibody containing 13Y039-4B06-4334 on the hIgG1 LAGA skeleton to soluble recombinant Fcγ receptor, FcRn, and C1q was determined by surface plasmon resonance (SPR).

[0236] The antibodies 13Y039-4B06-4334 and 13Y039-3E07-2944, expressed by HEK, and the antibody 13Y039-4B06-4334, expressed by CHO, were evaluated for their binding to recombinant soluble human Fcγ receptor (FcγR). The antibodies were analyzed against a positive control antibody (Fix Fc+) containing the wild-type human IgG1 Fc region and a negative control antibody (Fix Fc-) containing two point mutations (L235A / G237A) in the Fc region that reduce interaction with the Fcγ receptor. The binding of 13Y039-4B06-4334 expressed by HEK, 13Y039-4B06-4334 expressed by CHO, and 13Y039-3E07-2944 expressed by HEK to human Fcγ receptors, mouse Fcy receptors, and cynomolgus monkey Fcγ receptors was evaluated by surface-to-surface regeneration (SPR) using Fcγ receptors captured on the surface, and the test antibody was then flowed onto the receptors at the desired concentration.

[0237] As expected in cases of Fc-damaging mutations, all antibodies possessing the hIgG1 LAGA scaffold did not bind to human Fcγ receptors (FcγRI, FcγRIIaH, FcγRIIaR, FcγRIIb, FcγRIIIaV, and FcγRIIIaF), mouse Fcγ receptors (FcγRI, FcγRIIb, FcγRIIIa / b, and FcγRIV), or cynomolgus monkey Fcγ receptors (FcγRIIa, FcγRIIb, and FcγRIII), thus reducing their likelihood of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).

[0238] The binding of anti-BMP1 / TLL antibodies to human, cynomolgus monkey, and mouse neonatal type Fc receptors (FcRn) was evaluated by SPR. 13Y039-4B06-4334, 13Y039-3E07-2944, and human IgG1 WT control Fix Fc+ were evaluated for binding to recombinant soluble human and cynomolgus monkey FcRn. Compounds A (13Y039-4B06-4334, mIgG2a LAGA), B (13Y039-3E07-2944, mIgG2a LAGA), anti-RSV mouse IgG2a LAGA, anti-RSV rat IgG2b LAGA, compound C (13Y039-4B06-4334 rat IgG2b LAGA), and compound D (13Y039-3E07-2944 rat IgG2b) were evaluated for binding to recombinant soluble mouse FcRn. This included Fix Fc+, rat IgG2b wild-type control, and mouse IgG2a control (anti-MOPC). Human and cynomolgus monkey FcRn were tested with hIgG1, and mouse FcRn was tested with rat IgG2b and mIgG2a.

[0239] Compounds 13Y039-4B06-4334 and 13Y039-3E07-2944 showed binding to human and cynomolgus monkey FcRn at pH 6 but not at pH 7.4, indicating that the LAGA mutation does not affect binding to human or cynomolgus monkey FcRn. The relative binding affinity of 13Y039-4B06-4334 expressed in CHO to human FcRn was equivalent to that of cynomolgus monkeys with IgG1(Fix Fc+) control antibody. Compounds A, B, and D showed binding to mouse FcRn at pH 6 but not at pH 7.4. Capture of compound C on the surface of protein A was extremely unstable, so there were insufficient antibodies on the surface to evaluate binding to mouse FcRn.

[0240] The binding of 13Y039-4B06-4334 to human C1q was evaluated by SPR using Fix Fc+ and Fix Fc- as controls. The antibodies were diluted and immobilized on sensor chips by amine coupling. Human C1q was diluted with HBS-N + 10 mM CaCl2 and injected onto the immobilized constructs. Fix Fc+ bound to C1q as expected (KD = 36.3 nM), while Fix Fc- did not bind to C1q as expected for an Fc-damaging antibody. 13Y039-4B06-4334 showed comparable binding to the Fix Fc+ control antibody (KD = 36.3 nM).

[0241] The binding of 13Y039-4B06-4334 to human C1q was also evaluated by ELISA. Human C1q protein was added to the antibody, and binding was detected using an anti-C1q biotin detection reagent and streptavidin-HRP. The colorimetric quantitative signal was detected using SureBlue TMB, and after developing the colorimetric quantitative reaction, it was measured at 450 nm. The positive control mAb (anti-RSV IgG1) showed a dose-response effect as expected, but the binding of the negative control mAb (anti-RSV LAGA) to C1q was minimal, which was also as expected. ELISA confirmed that 13Y039-4B06-4334 expressed by CHO bound to C1q with affinity equivalent to that of the hIgG1 WT control.

[0242] 13Y039-4B06-4334, expressed in HEK and CHO cells, binds to human C1q at higher levels than the positive binding control antibody, anti-RSV IgG1 with wild-type Fc. Each data point in Figure 10 is representative of an experiment with n=3, except for 13Y039-4B06-4334 expressed in CHO cells with only one repeat. The data demonstrates that this molecule behaved similarly to other CHO materials and to anti-RSV WT, which represents n=2 due to its known binding activity.

[0243] These results, taken together, demonstrate that while 13Y039-4B06-4334 still binds to human C1q, it is an impaired Fc gene with impaired binding to human and cynomolgus monkey Fcγ receptors. The Fc-impaired mutation did not affect binding to human FcRn.

[0244] in vitro cell activity 13Y039-4B06-4334 also inhibits the cleavage of its intrinsic substrate procollagen I, catalyzed by BMP1, in a fibroblast-based collagen formation assay ("scar-in-a-jar," SIJ). In this assay, stimulation of human primary cardiac fibroblasts with Ficol induces procollagen formation and cleavage by endogenous BMP1, as measured by the release of type I procollagen C-terminal peptide (PICP). 13Y039-4B06-4334 dose-dependently inhibits PICP formation, indicating that this antibody blocks the cleavage of this endogenous protein substrate, attenuating a key component of the fibrotic mechanism in disease-associated cell types. The mean pIC50 value of 13Y039-4B06-4334 in normal human cardiac fibroblasts (NHCF) across multiple studies was 9.6 (±0.2, n=3).

[0245] The mouse reverse chimera of 13Y039-4B06-4334 (compound A) showed activity in the SIJ assay with a pIC50 of 9.8 (±0.6, n=2), which was comparable to that observed with 13Y039-4B06-4334.

[0246] Myostatin latent complex cleavage assay Anti-BMP1 / TLL antibodies were profiled for their inhibition of human BMP-1 in the cleavage of myostatin latent complexes. Recombinant human BMP-1 was pretreated with anti-BMP1 / TLL antibodies in a certain concentration range and then added to recombinant human myostatin latent complexes. BMP-1 alone cleaved the myostatin latent complex, releasing active myostatin, which was measured to indicate BMP-1 activity. When BMP-1 was pre-incubated with anti-BMP1 / TLL antibodies, its total enzyme activity decreased with increasing antibody concentration. The level of myostatin released from the complexes was measured using a mesoscale discovery (MSD) assay with anti-myostatin antibodies for capture and detection of myostatin homodimers. The inhibition rate % of anti-BMP1 / TLL antibodies was calculated using the MSD measurements of myostatin levels.

[0247] The data in Figure 11 shows that the anti-BMP1 / TLL antibody 13Y039-4B06-4334 inhibited BMP1 cleavage of the myostatin latent complex in a dose-response manner. These results confirm that 13Y039-4B06-4334 inhibits the enzymatic activity of BMP1 by preventing BMP-1 from cleaving the myostatin latent complex and releasing myostatin.

[0248] In vivo target association, pharmacodynamic markers, antifibrotic and anabolic activity Pharmacodynamic markers in the mouse AngII / PE model Mice administered angiotensin-II (AngII) and phenylephrine (PE) via subcutaneous osmotic pumps developed cardiac fibrosis within a two-week treatment period (hereinafter referred to as the AngII / PE model). Using this AngII / PE model, the effects of compound A, a reverse chimeric construct combining the variable region of 13Y039-4B06-4334 and the mouse IgG2a LAGA Fc domain, on BMP1 inhibition and pharmacodynamic markers of cardiac fibrosis were evaluated. Treatment was initiated at the time of osmotic pump implantation and administered to mice once a week for two weeks. Anti-RSV (mouse IgG2a LAGA / mouse cK) antibody and MOPC-21 (mouse variable region and constant region mouse IgG2a / mouse cK) antibody were used as controls. 13Y039-152B02-1 ("B02") is a mouse reverse chimera (human variable region on mouse IgG2a / mouse cK) tool anti-BMP1 / TLL mAb, and compound B is a mouse reverse chimera (mouse IgG2a LAGA / mouse cK) of another antibody, 13Y039-3E07-2944. In this study, both compound A / B02 and compound B dose-dependently inhibited ex vivo BMP1 activity from collected plasma (Figure 12).

[0249] A significant decrease in circulating type I procollagen C-terminal peptide (PICP) levels was also detected in AngII / PE mice treated with 5 mg / kg of compound A using a Western blot assay (Figure 13).

[0250] A further effect of compound A in the mouse AngII / PE model is a significant increase in skeletal muscle mass at high levels of BMP1 inhibition. AngII / PE infusion significantly reduced left gastrocnemius muscle weight normalized to total body weight (6.24±0.10 mg / g vs. 5.89±0.08 mg / g, p<0.01, unpaired t-test). Treatment with both 0.5 mg / kg and 5 mg / kg of compound A restored muscle mass to levels greater than the osmotic pump control in physiological saline (Figure 14). Concurrent with these findings, accumulation of total myostatin was observed in the plasma of these animals, as determined by a commercially available ELISA assay detecting total (i.e., free and bound) myostatin species (Figure 15).

[0251] Circulating myostatin has been shown to be largely bound to the latent complex MSTN-LC by an inhibitory prodomain fragment (over 70% in mice, the remainder to other inhibitory complexes; Hill, 2002). When the prodomain is cleaved by BMP1 / TLL, active myostatin is released, which signals as a negative growth factor. Therefore, the increase in muscle mass and total plasma myostatin levels upon administration of compound A may be due to reduced prodomain degradation by BMP1 / TLL and the subsequent easing of negative growth regulation that would otherwise occur with the release of mature myostatin in local tissue sites of skeletal muscle.

[0252] Anti-fibrotic effect of mouse reverse chimera (compound A) in the mouse AngII / PE model Injecting mice with AngII / PE for two weeks results in a significant increase in collagen production in cardiac tissue, as measured by left ventricular hydroxyproline (HDXP) content (comparing the first two bars in Figure 16) as measured by liquid chromatography / mass spectrometry (LC / MS).

[0253] In this model, treatment with compound A resulted in a statistically significant reduction in HDXP levels (47% at 0.5 mg / kg and 58% at 5 mg / kg) compared to either the AngII / PE control or the corresponding anti-RSV mAb control. However, despite this reduction in fibrosis biomarkers, no significant changes in fibrosis were observed by histopathological analysis.

[0254] Effect of rat river skimer (compound C) in a Dahl salt-sensitive rat model Dahl salt-sensitive (Dahl S) rat strains rapidly develop hypertension when fed a high-salt diet (8% NaCl), accompanied by complications such as renal failure, hyperlipidemia, and insulin resistance. Previous studies have shown that this strain also develops cardiac and renal fibrosis.

[0255] To evaluate the cardiac antifibrotic effect of BMP1 inhibition in this model, Dahl S rats were fed a 0.3% NaCl diet (normal salinity) until 4–5 weeks of age, then increased to a 1% NaCl diet on day 0, and subsequently to an 8% NaCl diet on day 7. 5 mg / kg of anti-RSV mAb control or 5 mg / kg of compound C (rat reverse chimera 13Y039-4B06-4334, n=12 in each group) was administered subcutaneously weekly from day 0 to day 28, and the rats were euthanized on day 35. This study also included a control group (n=12) that received a 0.3% diet and vehicle injections throughout. For both compound C and the anti-RSV control, both peak and trough exposures were within the expected target range between 1,180 and 11,800 ng / mL, as measured by the IC90 and IC95 values ​​of the rat plasma assay (Table 12).

[0256] At the end of the study, left ventricular sections were stained with Masson's trichrome to identify fibrotic areas, which were then quantified by image analysis. Vehicle-injected rats fed an 8% NaCl diet showed a significant increase in LV fibrosis (~24%) compared to normal salt controls, demonstrating a model effect. Treatment with compound C resulted in a significant decrease in LV fibrosis (~88%) compared to vehicle-treated controls, but this was not the case with the anti-RSV control mAb (Figure 17A).

[0257] To evaluate the muscle effects of BMP1 inhibition in this model, gastrocnemius muscles were isolated and weighed at the end of the study. As observed in the AngII / PE model (Figure 14), treatment with anti-BMP1 antibodies resulted in a significant increase in skeletal muscle mass (e.g., 9% compared to PBS + 8% NaCl) compared to vehicle or control animals (Figure 17B).

[0258] Assimilation effect of mouse reverse chimera (compound A) in a mouse hindlimb fixation model To evaluate the effects of BMP1 inhibition on skeletal muscle growth and function, reverse chimeric compound A was tested in a mouse hindlimb immobilization model. In the initial test to examine muscle recovery after immobilization, aged male mice (22 months old) had their right hindlimb immobilized for two weeks with a splint. After splint removal, the mice were placed in one of three treatment groups: (i) anti-RSV control mAb, 5 mg / kg / week, sc; (ii) compound A, 5 mg / kg / week, sc; or (iii) anti-myostatin mAb (positive control), 30 mg / kg, administered three times over two weeks, sc (n-10 in each group), for two weeks. Body composition, skeletal muscle function, and muscle wet weight were measured by quantitative NMR (qNMR) after this two-week recovery period. Pharmacodynamic markers associated with BMP1 inhibition all showed significant effects in the compound A treatment group, including a 92% decrease in plasma BMP1 activity, a 77% decrease in plasma PICP, and a 7.9-fold increase in total myostatin levels. Two weeks of treatment with compound A resulted in an increase of approximately 5% in lean body mass, which was significantly greater than that seen with the control anti-RSV mAb (Figure 18).

[0259] Furthermore, treatment with compound A resulted in increased wet weight of the gastrocnemius and soleus muscles in both the control limb and the splinted limb, as measured at the end of a two-week recovery period. This increase was significantly greater than that observed in control mice (Figure 19).

[0260] Furthermore, in addition to the previously observed increase in muscle mass in the AngII / PE model, treatment with compound A resulted in a significant improvement in muscle function. The increase in muscle mass was not proportional to the increase in force, and the force-to-muscle weight ratio decreased (right panel of Figure 20), a phenomenon previously observed in myostatin knockout mouse lines (Amthor, 2007).

[0261] The results of this study demonstrate that inhibition of BMP1 / TLL by compound A not only promotes skeletal muscle growth in recovery from disuse atrophy in aging mice but also improves muscle function. These results suggest that inhibition of BMP1 / TLL by 13Y039-4B06-4334 in a clinical setting is beneficial in patient populations where fibrosis progression is accompanied by frailty and skeletal muscle loss.

[0262] Pharmacokinetics of 13Y039-4B06-4334 The pharmacokinetics of 13Y039-4B06-4334 were determined after both single and repeated administration to Wistar Han rats.

[0263] Pharmacokinetics of single-dose rats In Wistar Han rats (n=3), the pharmacokinetics of 13Y039-4B06-4334 were determined after a single intravenous (bolus) or subcutaneous administration at a nominal dose of 1 mg / kg.

[0264] Table 13 shows the individual and mean pharmacokinetic parameters after a single intravenous or subcutaneous administration of 1 mg / kg. Except for animal 1, which showed remarkably rapid clearance, 13Y039-4B06-4334 was slowly cleared with a mean terminal half-life of approximately 5 days. The mean partition volume was 94 mL / kg, close to the blood volume, suggesting that this antibody primarily remains in the systemic circulation.

[0265] [Table 15]

[0266] Pharmacokinetics of repeated doses in rats After subcutaneous administration of 1 mg / kg weekly for 4 weeks, 2 out of 3 animals maintained their expected exposure levels (Cmax and AUC6-168). The accumulation level (2.9-fold increase in AUC6-168) was as expected for a monoclonal antibody with a half-life of 5 days (Table 14). The decrease in exposure observed in the third animal may be due to an ADA response, but this could not be confirmed.

[0267] [Table 16]

[0268] Embodiment Embodiment 1 is, (a)(i) Any one or combination of CDRH1, CDRH2, CDRH3 in SEQ ID NOs. 7, 22, 40, 54, 67, 82, 96, 110, 124, 138, 152, 166, 180, 194, 207 and 222 and / or CDRL1, CDRL2, CDRL3 in SEQ ID NOs. 8, 21, 39, 53, 68, 81, 95, 109, 123, 137, 151, 165, 179, 193, 208 and 221 of the CDR; or (ii) A variant of the CDR of (i) having one, two or three amino acid modifications; or (b) A VH region comprising a sequence that is at least 80% identical to the sequence of sequence number 7, 22, 40, 54, 67, 82, 96, 110, 124, 138, 152, 166, 180, 194, 207 or 222, and / or a VL region comprising a sequence that is at least 80% identical to the sequence of sequence number 8, 21, 39, 53, 68, 81, 95, 109, 123, 137, 151, 165, 179, 193, 208 or 221. It is a protein that binds to BMP1, TLL1, and / or TLL2, comprising the above components.

[0269] Embodiment 2 is a protein that binds to BMP1, TLL1 and / or TLL2 of Embodiment 1, wherein the CDR of (a)(i) is CDRL1 of SEQ ID NO: 1; CDRL2 of SEQ ID NO: 2; CDRL3 of SEQ ID NO: 3; CDRH1 of SEQ ID NO: 4; CDRH2 of SEQ ID NO: 5; and / or CDRH3 of SEQ ID NO: 6.

[0270] Embodiment 3 is, LCDR1 comprising a sequence having at least 80% sequence identity with RASQSVSSYLA (SEQ ID NO: 1); and / or LCDR2 comprising a sequence having at least 80% sequence identity with DASNRAT (SEQ ID NO: 2); and / or LCDR3 comprising a sequence having at least 80% sequence identity with QQSDSWPPT (SEQ ID NO: 3); and / or HCDR1 comprising a sequence having at least 80% sequence identity with GYYMS (SEQ ID NO: 4); and / or HCDR2 comprising a sequence having at least 80% sequence identity with WINPLSGETNYAQKFQG (Sequence ID 5); and / or HCDR3 comprising a sequence having at least 80% sequence identity with DTGELDGMNWYFDL (SEQ ID NO: 6) This is a protein that binds to BMP1, TLL1 and / or TLL2 according to Embodiment 1 or 2, comprising one or more of the above.

[0271] Embodiment 4 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 3, comprising a VH region comprising CDR1, which includes a sequence having at least 80% sequence identity with GYYMS (SEQ ID NO: 4); CDR2, which includes a sequence having at least 80% sequence identity with WINPLSGETNYAQKFQG (SEQ ID NO: 5); and / or CDR3, which includes a sequence having at least 80% sequence identity with DTGELDGMNWYFDL (SEQ ID NO: 6).

[0272] Embodiment 5 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 4, comprising a VH region comprising CDR1 comprising the sequence GYYMS (SEQ ID NO: 4), CDR2 comprising the sequence WINPLSGETNYAQKFQG (SEQ ID NO: 5), and / or CDR3 comprising the sequence DTGELDGMNWYFDL (SEQ ID NO: 6).

[0273] Embodiment 6 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 5, comprising a VL region comprising CDR1 comprising a sequence having at least 80% sequence identity with RASQSVSSYLA (SEQ ID NO: 1); CDR2 comprising a sequence having at least 80% sequence identity with DASNRAT (SEQ ID NO: 2); and / or CDR3 comprising a sequence having at least 80% sequence identity with QQSDSWPPT (SEQ ID NO: 3).

[0274] Embodiment 7 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 6, comprising a VL region comprising CDR1 comprising the sequence of RASQSVSSYLA (SEQ ID NO: 1); CDR2 comprising the sequence of DASNRAT (SEQ ID NO: 2); and / or CDR3 comprising the sequence of QQSDSWPPT (SEQ ID NO: 3).

[0275] Embodiment 8 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 7, comprising LCDR1 comprising the sequence RASQSVSSYLA (SEQ ID NO: 1), LCDR2 comprising the sequence DASNRAT (SEQ ID NO: 2), LCDR3 comprising the sequence QQSDSWPPT (SEQ ID NO: 3), HCDR1 comprising the sequence GYYMS (SEQ ID NO: 4), HCDR2 comprising the sequence WINPLSGETNYAQKFQG (SEQ ID NO: 5), and / or HCDR3 comprising the sequence DTGELDGMNWYFDL (SEQ ID NO: 6).

[0276] Embodiment 9 is a BMP1, TLL1, and / or TLL2-binding protein of any of Embodiments 1 to 8, wherein all six CDRs are present in the BMP1, TLL1, and / or TLL2-binding protein.

[0277] Embodiment 10 includes the following six CDRs: LCDR1 of RASQSVSSYLA (sequence number 1); LCDR2 of DASNRAT (sequence number 2); LCDR3 of QQSDSWPPT (sequence number 3); HCDR1 of GYYMS (sequence number 4); HCDR2 of WINPLSGETNYAQKFQG (Sequence ID 5); and DTGELDGMNWYFDL (SEQ ID NO: 6) HCDR3 It is a protein that binds to BMP1, TLL1, and / or TLL2, comprising [the specified components].

[0278] Embodiment 11 is a protein that binds to BMP1, TLL1 and / or TLL2 of Embodiment 10, comprising a VH region that is 80% identical to SEQ ID NO: 7 and / or a VL region that is 80% identical to SEQ ID NO: 8.

[0279] Embodiment 12 is a protein that binds to BMP1, TLL1 and / or TLL2 of Embodiment 10 or 11, comprising a VH region that is 100% identical to SEQ ID NO: 7 and / or a VL region that is 100% identical to SEQ ID NO: 8.

[0280] Embodiment 13 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 10 to 12, comprising a heavy chain (HC) sequence that is 80% identical to SEQ ID NO: 10; and / or a light chain (LC) sequence that is 80% identical to SEQ ID NO: 9.

[0281] Embodiment 14 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 10 to 13, comprising a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 10; and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 9.

[0282] Embodiment 15 is a protein that binds to BMP1, TLL1, and / or TLL2, comprising a VH region that is 100% identical to SEQ ID NO: 7 and a VL region that is 100% identical to SEQ ID NO: 8.

[0283] Embodiment 16 is a protein that binds to BMP1, TLL1 and / or TLL2 of Embodiment 15, comprising a light chain that is 100% identical to SEQ ID NO: 9 and a heavy chain that is 100% identical to SEQ ID NO: 10.

[0284] Embodiment 17 is a polynucleotide sequence encoding a protein that binds to BMP1, TLL1, and / or TLL2 of any of Embodiments 1 to 16.

[0285] Embodiment 18 is the polynucleotide sequence of Embodiment 17, comprising SEQ ID NO: 13 encoding a heavy chain and / or SEQ ID NO: 14 encoding a light chain.

[0286] Embodiment 19 is an expression vector comprising a polynucleotide sequence as defined in Embodiment 17 or 18.

[0287] Embodiment 20 is a recombinant host cell comprising a polynucleotide sequence as defined in Embodiment 17 or 18, or an expression vector as defined in Embodiment 19.

[0288] Embodiment 21 is a method for producing proteins that bind to BMP1, TLL1, and / or TLL2, comprising culturing the recombinant host cells of Embodiment 20 under conditions suitable for the expression of the polynucleotide sequence or expression vector, thereby producing a polypeptide comprising proteins that bind to BMP1, TLL1, and / or TLL2.

[0289] Embodiment 22 is a protein that binds to BMP1, TLL1, and / or TLL2 produced by the method of Embodiment 21.

[0290] Embodiment 23 is a pharmaceutical composition comprising a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, and a pharmaceutically acceptable diluent or carrier.

[0291] Embodiment 24 is the pharmaceutical composition of Embodiment 23, comprising a protein that binds to BMP1, TLL1 and / or TLL2 of Embodiment 15 or 16.

[0292] Embodiment 25 is a method for treating a fibrosis-related disease or disorder in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, or a pharmaceutical composition of Embodiment 23 or 24.

[0293] Embodiment 26 is the method of Embodiment 25, wherein the subject is a human.

[0294] Embodiment 27 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, or a pharmaceutical composition of Embodiment 23 or 24, for use in therapy.

[0295] Embodiment 28 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, or a pharmaceutical composition of Embodiment 23 or 24, for use in the treatment of fibrosis-related diseases or disorders.

[0296] Embodiment 29 is the use of a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, or a pharmaceutical composition of Embodiment 23 or 24, in the manufacture of a pharmaceutical for use in the treatment of fibrosis-related diseases or disorders.

[0297] Embodiment 30 is any method or use of Embodiments 25, 26, and 29, wherein the fibrosis-related disease or disorder is cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, peritoneal fibrosis, or non-alcoholic steatohepatitis (NASH).

[0298] Embodiment 31 is the method or use of Embodiment 30, wherein the cardiac fibrosis is hypertrophic cardiomyopathy and the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0299] Embodiment 32 is a method for promoting muscle growth and / or improving muscle function in a subject requiring it, comprising administering to the subject a therapeutically effective amount of a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, or a pharmaceutical composition of Embodiment 23 or 24.

[0300] Embodiment 33 is the method of Embodiment 32, wherein the subject is a human.

[0301] Embodiment 34 is a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, or a pharmaceutical composition of Embodiment 23 or 24, for use in promoting muscle growth and / or improving muscle function.

[0302] Embodiment 35 involves the use of a protein that binds to BMP1, TLL1 and / or TLL2 of any of Embodiments 1 to 16 or 22, or a pharmaceutical composition of Embodiment 23 or 24, in the manufacture of a pharmaceutical for promoting muscle growth and / or improving muscle function.

[0303] Sequence List [Table 17] TIFF0007848181000018.tif239157TIFF0007848181000019.tif244157TIFF0007848181000020.tif245157TIF F0007848181000021.tif244157TIFF0007848181000022.tif245156TIFF0007848181000023.tif244157TIFF000 7848181000024.tif243157TIFF0007848181000025.tif245157TIFF0007848181000026.tif244157TIFF0007848 181000027.tif244157TIFF0007848181000028.tif240158TIFF0007848181000029.tif243157TIFF00078481810 00030.tif244157TIFF0007848181000031.tif240156TIFF0007848181000032.tif243156TIFF00078481810000 33.tif245157TIFF0007848181000034.tif244157TIFF0007848181000035.tif244157TIFF0007848181000036.t if240157TIFF0007848181000037.tif243157TIFF0007848181000038.tif245157TIFF0007848181000039.tif24 4157TIFF0007848181000040.tif244157TIFF0007848181000041.tif245157TIFF0007848181000042.tif239158

[0304]

Table 18

Claims

1. The following six CD-Rs: LCDR1 of RASQSVSSYLA (Sequence ID 1); DASNRAT (SEQ ID NO: 2) LCDR2; LCDR3 of QQSDSWPPT (Sequence ID 3); HCDR1 of GYYMS (SEQ ID NO: 4); HCDR2 of WINPLSGETNYAQKFQG (SEQ ID NO: 5); and HCDR3 of DTGELDGMNWYFDL (SEQ ID NO: 6) A protein comprising the above, which binds to BMP1, TLL1 and / or TLL2.

2. A protein that binds to BMP1, TLL1 and / or TLL2 according to claim 1, comprising a VH region that is at least 80% identical to SEQ ID NO: 7 and / or a VL region that is at least 80% identical to SEQ ID NO:

8.

3. A protein that binds to BMP1, TLL1 and / or TLL2 according to claim 1 or 2, comprising a VH region that is 100% identical to SEQ ID NO: 7 and / or a VL region that is 100% identical to SEQ ID NO:

8.

4. A protein that binds to BMP1, TLL1 and / or TLL2 according to any one of claims 1 to 3, comprising a heavy chain (HC) sequence that is at least 80% identical to SEQ ID NO: 10; and / or a light chain (LC) sequence that is at least 80% identical to SEQ ID NO:

9.

5. A protein that binds to BMP1, TLL1 and / or TLL2 according to any one of claims 1 to 4, comprising a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 10; and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO:

9.

6. A protein that binds to BMP1, TLL1, and / or TLL2, comprising a VH region that is 100% identical to SEQ ID NO: 7 and a VL region that is 100% identical to SEQ ID NO:

8.

7. A protein that binds to BMP1, TLL1 and / or TLL2 according to claim 6, comprising a light chain that is 100% identical to SEQ ID NO: 9 and a heavy chain that is 100% identical to SEQ ID NO:

10.

8. The protein that binds to BMP1, TLL1 and / or TLL2 according to any one of claims 1 to 7, wherein the protein that binds to BMP1, TLL1 and / or TLL2 is an anti-BMP1, TLL1 and / or TLL2 antibody.

9. The protein that binds to BMP1, TLL1 and / or TLL2 according to claim 8, wherein the anti-BMP1, TLL1 and / or TLL2 antibody is a humanized antibody.

10. A nucleic acid comprising a polynucleotide sequence encoding a protein that binds to BMP1, TLL1 and / or TLL2 as described in any one of claims 1 to 9.

11. The nucleic acid according to claim 10, comprising a polynucleotide sequence comprising SEQ ID NO: 13 encoding a heavy chain and SEQ ID NO: 14 encoding a light chain.

12. An expression vector comprising the nucleic acid described in claim 10 or 11.

13. Recombinant host cells comprising the nucleic acid according to claim 10 or 11, or the expression vector according to claim 12.

14. A method for producing proteins that bind to BMP1, TLL1 and / or TLL2, comprising culturing the recombinant host cells described in claim 13 under conditions suitable for the expression of the nucleic acid or expression vector, thereby producing a polypeptide comprising proteins that bind to BMP1, TLL1 and / or TLL2.

15. A pharmaceutical composition comprising a protein that binds to BMP1, TLL1 and / or TLL2 as described in any one of claims 1 to 9, and at least one pharmaceutically acceptable diluent or carrier.

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