Compositions and methods for treating pulmonary hypertension

Abstract

In some aspects, the invention teaches pharmaceutical compositions comprising a TGF-beta ligand trapping agent, and methods of using the TGF-beta ligand trapping agent to treat, prevent, or reduce the rate of progression of pulmonary hypertension (PH). The invention also provides methods of using the TGF-beta ligand trapping agents to treat, prevent, or reduce the rate of progression of a variety of disorders, the conditions include, but are not limited to, pulmonary vascular remodeling, pulmonary fibrosis, right ventricular hypertrophy, diseases associated with excessive TGF-beta signaling, diseases associated with excessive GDF15 signaling, and diseases associated with excessive PAI-1 signaling. The invention further provides methods of using the TGF-beta ligand trapping agent to reduce right ventricular systolic pressure in a subject.

Description

[0001] This application is a divisional application of patent application No. 201480073820.1, filed on November 21, 2014, entitled "Composition and Method for Treating Pulmonary Hypertension".

[0002] Cross-references to related applications

[0003] Pursuant to 35U.SC119(e), this application claims the benefit of U.S. Provisional Patent Application Serial No. 61 / 907,260, filed November 21, 2013, the contents of which are incorporated herein by reference in their entirety.

[0004] Statement Regarding Federally Sponsored Research

[0005] This invention was made with government support under NIH grant number 5R01AR057374. The government holds certain rights to this invention. Technical Field

[0006] This invention generally relates to the medical field, as well as the fields of cardiovascular and pulmonary diseases. Background Technology

[0007] All publications herein are incorporated by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful for understanding the invention. This is not an admission that any information provided herein is prior art or related to the invention currently claimed, or that any publications explicitly or implicitly referenced are prior art.

[0008] Numerous pathological and undesirable biological processes arise from ligand binding to cell surface receptors and excessive / abnormally active signal transduction. Therefore, compositions and methods designed to reduce or advantageously modulate such binding and signal transduction can be useful.

[0009] The TGF-β superfamily includes many biologically significant ligands. TGF-β and activin play important pathogenic roles in many diseases, including uncontrolled fibrosis and the progression of cancers such as kidney, lung, and liver fibrosis. Myostatin / GDF8 is another important ligand associated with activin, sharing binding to the same type II receptor (activin RIIb). Myostatin is a potent inhibitor of skeletal muscle growth and a proven therapeutic target for muscular atrophy diseases such as muscular dystrophy. Additional ligands in the TGF-β family include bone morphogenetic proteins (BMPs) involved in cardiovascular disease. For example, high levels of both BMP2 and BMP4 have been found in calcified atherosclerotic plaques and pathological aortic valves.

[0010] Methods have been developed to reduce ligand binding by capturing the ligand and preventing its interaction with cell surface receptors. The main agents targeting such ligands are ligand capture agents / antagonists that sequester the ligand. Two examples are: (1) anti-ligand antibodies; and (2) soluble receptor extracellular domains.

[0011] The use of anti-ligand antibodies that directly capture and neutralize ligands to inhibit some ligands has been reported. Soluble forms of the receptor extracellular domain directly antagonize ligands by binding to them and preventing the ligands from interacting with cell surface receptors. In the case of TGF-β, expression of the extracellular domain (ED) of TGF-β receptor type II (TβRII) partially restored host immunity and promoted tumor clearance in animal models, indicating that receptor extracellular domain-mediated neutralization of TGF-β inhibits tumor progression. Unfortunately, it has been shown that monovalent TβRII-ED is less effective than optimal in antagonizing TGF-β. Attempts to overcome this problem have led to the production of a divalent artificial dimerized form of TβRII-ED, which dimers via fusion with a coil-coil domain or the Fc domain of IgG. This dimerization improves the antagonistic effect. It has been shown that nonvalent dimerization of TβRII-ED (e.g., via fusion to heterodimerization of the helical chain (coil-coil TβRII-ED)) greatly enhances the antagonistic potency of TβRII-ED (De Crescenzo et al., 2004, J. Biol. Chem. 279: 26013). A significant drawback of the coil-coil fusion dimer is that the nonvalent nature of the dimerizing domain limits its potency; that is, the dimer dissociates at low concentrations, thus allowing most of the extracellular domain of the helical fusion receptor to function as a monomer rather than a dimer. The use of the Fc domain of IgG provides covalent interactions, but at the cost of large size.

[0012] Importantly, one of the obstacles to the clinical deployment of TGFβRI inhibitors developed to date for the treatment of PH is toxicity, including hemorrhagic valvular necrosis.

[0013] Given the shortcomings of the treatments attempted to date, there is a clear need in the art for receptor-based trapping / neutralizing agents that can antagonize ligand activity and have the potential to serve as therapeutic or diagnostic (imaging or non-imaging) agents for diseases / disorders caused by the overproduction / activity of the target ligands described herein. Summary of the Invention

[0014] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods, and are intended to be exemplary and illustrative, and not to limit the scope.

[0015] Several embodiments of the present invention describe pharmaceutical compositions comprising a TGF-β ligand scavenger. In some embodiments, the TGF-β ligand scavenger is a soluble recombinant TGF-β type II receptor Fc-fusion protein (TGFBRII-Fc). In some embodiments, the TGFBRII-Fc fusion protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1; or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0016] Several embodiments of the present invention describe methods for treating, preventing, or reducing the progression rate of pulmonary hypertension (PH) in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject to treat, prevent, or reduce the progression rate of PH in the subject.

[0017] Several embodiments of the present invention describe methods for treating, preventing, or slowing the progression of pulmonary vascular remodeling in subjects. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to a subject, thereby treating, preventing, or slowing the progression of pulmonary vascular remodeling in the subject.

[0018] Several embodiments of the present invention describe methods for treating, preventing, or slowing the progression of pulmonary fibrosis in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or slowing the progression of pulmonary fibrosis in the subject.

[0019] Several embodiments of the present invention describe methods for treating, preventing, or slowing the progression of right ventricular hypertrophy in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or slowing the progression of right ventricular hypertrophy in the subject.

[0020] Several embodiments of the present invention describe methods for treating, preventing, or slowing the progression of diseases associated with excessive TGF-β signaling in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or slowing the progression of the disease in the subject.

[0021] Several embodiments of the present invention describe methods for treating, preventing, or slowing the progression of diseases associated with excessive GDF15 signaling in subjects. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to a subject, thereby treating, preventing, or slowing the progression of the disease in the subject.

[0022] Several embodiments of the present invention describe methods for treating, preventing, or slowing the progression of diseases associated with excessive PAI-1 signaling in subjects. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to a subject to treat, prevent, or slow the progression of the disease in the subject.

[0023] Several embodiments of the present invention describe methods for reducing right ventricular systolic pressure in subjects. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby reducing right ventricular systolic pressure in the subject.

[0024] Several embodiments of the present invention describe a method for imaging / detecting TGF-β ligands in a subject, the method comprising administering to the subject a quantity of a TGF-β ligand trapping agent linked to an imaging molecule. Attached Figure Description

[0025] Exemplary embodiments are illustrated in the accompanying drawings. The embodiments and drawings disclosed herein are intended to be illustrative and not restrictive.

[0026] Figures 1A to 1E This is a graph showing the association between pyruvic thalassemia (MCT)-induced pulmonary hypertension in rats and increased PAI-1 and decreased Id1 transcriptional activity according to embodiments of the present invention. After treatment of Sprague-Dawley rats with MCT (40 mg / kg SC), right ventricular systolic pressure (RVSP) was periodically measured. Figure 1A ) and right ventricular hypertrophy (RVH, Figure 1BChanges in the right ventricular (RV) free wall were measured. RVSP was measured by right ventricular catheterization, and RVH was determined by the ratio of the weight of the right ventricular (RV) free wall to the sum of the left ventricular and septal (LV+S) free walls (n=3 at each time point). Quantitative RT-PCR of the lungs of MCT-treated rats showed elevated PAI-1 transcription (reflecting increased TGF-β signaling). Figure 1C The level of PAI-1 transcription is directly correlated with the pH level based on RVSP. Figure 1D and Figure 1E Conversely, decreased expression of Bmpr2 and its transcriptional target Id1 was observed, both of which were negatively correlated with RVSP levels (n = 5–6, *p < 0.05 and **p < 0.01 compared to control rats).

[0027] Figure 2 A to Figure 2 F shows the immunoblot and graph. Figure 2 A- Figure 2 C indicates that, according to an embodiment of the present invention, TGFBRII-Fc selectively inhibits the signaling of TGFβ1, TGFβ3, and GDF15 in human pulmonary artery smooth muscle cells (PASMCs). Cultured PASMCs were deserted of serum and incubated for 30 minutes with multiple concentrations of BMP4, TGFβ1, TGFβ2, TGFβ3, and GDF15 ligands. Western blotting and qPCR were performed to evaluate the ability of TGFBRII-Fc to modulate signal transduction activity in vitro. Figure 2 D- Figure 2 F indicates that, according to an embodiment of the present invention, TGFBRII-Fc selectively inhibits TGFβ1 and GDF15 signaling in vascular smooth muscle cells. Figure 2 D) Human aortic smooth muscle cells were deserled and incubated overnight, then incubated for 30 minutes with indicated concentrations of BMP4, TGFβ1, TGFβ2, or GDF15. Phosphorylation of Smad1, Smad2, Smad3, and Smad5 was analyzed by Western blotting as shown. TGFβ1, TGFβ2, and GDF15 induced activation of Smad2 and Smad3 in a dose-dependent manner and to a lesser extent of activation of Smad1 and Smad5, while BMP4 activated only Smad1 and Smad5. Figure 2 E- Figure 2F) HASMCs were deserumed and pretreated with TGFBRII-Fc (2000 ng / ml) or excipients, followed by incubation with TGFβ1 (1 ng / ml), TGFβ2 (1 ng / ml), or GDF15 (30 ng / ml) for 2 hours. Gene expression analysis by qRT-PCR showed that GDF15 and TGFβ1-induced PAI-1 and Id1 mRNA expression were effectively inhibited, but TGFβ2-induced PAI-1 and Id1 mRNA expression was not effectively inhibited (n = 3-5 samples each, *p < 0.05, **p < 0.01 compared to excipients).

[0028] Figure 3 A to Figure 3 D is a graph showing the trend of low-dose TGFBRII-Fc treatment, according to embodiments of the present invention, to reduce right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling. After 3 weeks of MCT treatment with or without TGFBRII-Fc (5 mg / kg, twice weekly), rats were analyzed in a blinded manner via catheterization and endotracheal intubation under pentobarbital anesthesia to determine RVSP. Figure 3 A) Systemic arterial pressure (not shown), and the rats were euthanized. The degree of RVH was assessed in a blinded manner based on the measurement of Fulton's ratio (RV / (LV+S)). Figure 3 B). Values ​​are expressed as mean ± SEM, n = 6–8, *p < 0.05 and **p < 0.01 (compared to control rats). Lung tissue sections were stained with α-smooth muscle actin and von Willebrand factor to identify vascular smooth muscle vessels and endothelium, respectively. Muscletization of distal acinar vessels (10 μm–50 μm in diameter) was quantified, and the percentages of non-muscletized, partially muscletized, and fully (circumferentially) muscletized vessels were calculated. Figure 3 C). All fully muscularized intraacini vessels (diameter 10μm-50μm, Figure 3 The medial wall thickness of D) was calculated. The wall thickness index was calculated as: index = (outer diameter - inner diameter) / outer diameter × 100. TGFBRII-Fc treatment (5 mg / kg, twice weekly) tended to reduce the percentage of fully muscularized vessels and significantly reduce the medial wall thickness index. Values ​​are expressed as mean ± SEM, n = 100-150 vessels / treatment group (each from 6-8 rats), and p-values ​​are shown in the figure.

[0029] Figure 4 A to Figure 4Figure D shows a graph demonstrating that high-dose TGFBRII-Fc treatment according to embodiments of the present invention reduced right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and prevented pulmonary vascular remodeling. Rats were analyzed in a blinded manner after 3 weeks of MCT treatment with or without TGFBRII-Fc (15 mg / kg, twice weekly) to determine RVSP ( Figure 4 A). The degree of RVH was assessed using a blinded method based on Fulton's proportion measurement. Figure 4 B). Values ​​are expressed as mean ± SEM, n = 6–8. Quantification of muscularization of distal acinar vessels (10 μm–50 μm in diameter) was performed. Figure 4 C). All fully muscularized intraacini vessels (diameter 10μm-50μm, Figure 4 The medial wall thickness of D) was calculated. TGFBRII-Fc treatment (15 mg / kg, twice weekly) significantly reduced the percentage of fully muscularized vessels and the medial wall thickness index. Values ​​are expressed as mean ± SEM, n = 89–127 vessels / treatment group (each from 6–8 rats), *p < 0.05 and ***p < 0.001 compared to control rats.

[0030] Figure 5 A to Figure 5 Figure D shows a graph demonstrating that TGFBRII-Fc reduced echocardiographic RV hypertrophy according to an embodiment of the present invention. Rats were treated with either excipients or TGFBRII-Fc (15 mg / kg, twice weekly) starting 24 hours after MCT (40 mg / kg SC). Two weeks after MCT, rats were analyzed by small animal ultrasound under 1.5% isoflurane anesthesia to measure right ventricular thickness and diastolic diameter ( ). Figure 5 A and Figure 5 B) Pulmonary blood flow acceleration time (PAT) Figure 5 C) and lung ejection time (PET, Figure 5 D). Values ​​are expressed as mean ± SEM, n = 6–8, *p < 0.05 and ***p < 0.001 compared to control rats.

[0031] Figure 6 A to Figure 6 F is a graph illustrating the inhibition of TGFβ-mediated transcription by TGFBRII-Fc in lung tissue according to an embodiment of the present invention. MCT-induced PH is associated with a moderate increase in TGFβ1 and a significant decrease in TGFβ2 mRNA expression. Figure 6 A- Figure 6C). Following MCT treatment, the inhibition of Bmpr2 and Id1 expression was not affected by TGFBRII-Fc (15 mg / kg twice weekly). Figure 6 D- Figure 6 E), while treatment with TGFBRII-Fc significantly reduced TGFβ1 and its transcriptional target PAI-1 (E). Figure 6 F). Values ​​are expressed as mean ± SEM, n = 3-5, *p < 0.05 and **p < 0.01 compared to the control.

[0032] Figure 7 A to Figure 7 C is a graph showing the relationship between treatment with TGFBRII-FC after pH establishment and mortality and partial recovery of pH according to various embodiments of the present invention. Rats were treated with MCT (40 mg / kg SC) in a delayed manner starting on day 17 after pH establishment with TGFBRII-FC (15 mg / kg three times weekly). Kaplan-Meier analysis compared to rats treated with excipients (n = 12 per group, p = 0.10) showed... Figure 7 A) showed an improved survival trend in the TGFBRII-Fc-treated group. Among the surviving animals at day 35, a significant reduction in RVSP (Recovery Risk Per Spectrum) was observed in the TGFBRII-Fc-treated group. Figure 7 B). However, no significant differences were found in RVH among the surviving animals. Figure 7 C). The values ​​shown are mean ± SEM, n = 8-11 per group, **p < 0.01 compared to control.

[0033] Figure 8A and Figure 8B The hinges of human IgG1, IgG2, IgG3 and IgG4 are shown. Figure 8A ) and Fc( Figure 8B The amino acid sequences of the IgG1 hinge domains are as follows: IgG1 hinge domain (SEQ ID NO: 65); IgG2 hinge domain (SEQ ID NO: 66); IgG3 hinge domain (SEQ ID NO: 67); IgG4 hinge domain (SEQ ID NO: 68). Figure 8B With Figure 8AThe same sequence is shown: IgG1 Fc domain (SEQ ID NO: 69), i.e., the first line of the amino acid sequence; IgG2 Fc domain (SEQ ID NO: 70), i.e., the second line of the amino acid sequence; IgG3 Fc domain (SEQ ID NO: 71), i.e., the third line of the amino acid sequence; IgG4 Fc domain (SEQ ID NO: 72), i.e., the fourth line of the amino acid sequence. This is based on the Kabat EU numbering system. Figure 8A and Figure 8B The amino acid residues shown are numbered. The isotype sequence is compared with the IgG1 sequence by placing the first and last cysteine ​​residues (which form S-S bonds between heavy chains) in the same position in their respective hinge regions. Figure 8B In this context, residues in the CH2 domain are indicated by a plus sign (+), while residues in the CH3 domain are indicated by a wavy line. Any Fc domain can be used in the method of this invention, as illustrated in Figure 8, which provides guidance for various Fc domains, allowing for alignment of the entire antibody sequence.

[0034] Figures 9A to 9B Tissue sections showing no mitral valve remodeling, degenerative changes, or abnormalities in response to TGFBRII-Fc treatment are shown. Figure 9A For comparison. Figure 9B It has been treated with TGFBRII-Fc-. Detailed Implementation

[0035] All references cited herein are incorporated herein by reference in their entirety and are fully described herein. Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd edition, J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 5th edition, J. Wiley & Sons (New York, NY 2001); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide general guidance to those skilled in the art for many of the terms used in this application.

[0036] For references on how to prepare antibodies, see, for example, D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor NY, 1988); Kohler and Milstein, (1976) Eur. J. Immunol. 6:511; Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., Nature 332:323 (1988). Unless otherwise stated, the practice of this invention will be carried out using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology within the scope of the art.Such techniques are well explained in the literature, for example: Current Protocols in Immunology (edited by J. E. Olgan et al., 1999, including the 2011 supplement); Current Protocols in Molecular Biology (edited by F. M. Usubel et al., 1987, including the 2011 supplement); Short Protocols in Molecular Biology, edited by F. M. Usubel et al., 5th edition 2002, including the 2011 supplement; Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook and Russell, 2001); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); The Immunoassay Handbook (edited by D. Wild, Stockton Press NY, 1994); Bioconjugate Techniques (edited by Greg T. Hermanson, Academic Press, 1996); Methods of Immunological Analysis (R. Masseyeff, W.H. Albert and NAStaines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999; and Beaucage et al., eds., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, New York, 2000).

[0037] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used in the practice of this invention. Other features and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings, which illustrate various features of embodiments of the invention by way of example. In fact, the invention is not limited to the methods and materials described. For the purposes of this invention, some terms are defined below.

[0038] "Beneficial outcomes" may include, but are not in any way limited to, reducing or alleviating the severity of disease symptoms, preventing disease flare-ups, treating disease symptoms, preventing disease progression, reducing the patient's chance of developing disease symptoms, and extending the patient's lifespan or life expectancy. In several embodiments, the disease symptoms are pulmonary hypertension, pulmonary vascular remodeling, pulmonary fibrosis, right ventricular hypertrophy, diseases associated with excessive TGF-β signaling, diseases associated with excessive GDF15 signaling, and diseases associated with excessive PAI-1 signaling.

[0039] As used in this article, "treatment / management" refers to therapeutic treatment / management and preventative or preventative measures, where the goal is to alleviate (reduce) the targeted pathological symptoms, prevent the pathological symptoms, pursue or achieve beneficial outcomes, or reduce an individual's chance of developing the condition even if the treatment is ultimately unsuccessful. Those requiring treatment / management include individuals who already have the condition, those who are susceptible to the condition, or those in whom preventative measures are being taken.

[0040] As used in this article, "pulmonary hypertension" (PH) can include elevated blood pressure in the pulmonary arteries (pulmonary arterial hypertension), pulmonary veins, or pulmonary capillaries (collectively known as the pulmonary vascular system), leading to shortness of breath, dizziness, fainting, lower extremity swelling, and other symptoms. PH can be a serious condition accompanied by significantly reduced exercise tolerance and heart failure. PH can be one of at least five different possible types, including: arterial PH, venous PH, hypoxic PH, thromboembolic PH, or mixed PH.

[0041] As used herein, “TGF-β ligand trapper” refers to a protein that can trap TGF-β ligands (even if only transiently), thereby modulating the ligand’s ability to interact with one or more additional molecules.

[0042] In some implementations, the TGF-β ligand may mean a ligand selected from TGF-β1, TGF-β2, TGF-β3, and GDF 15.

[0043] Examples of TGF-β ligand traps include, but are in no way limited to, soluble recombinant TGF-β receptor Fc-fusion proteins containing a TGF-β receptor TGF-β ligand-binding domain and an immunoglobulin Fc domain.

[0044] Therefore, in one embodiment, a method is provided for treating, preventing, or reducing the progression of pulmonary hypertension (PH) in a subject. The method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject to treat, prevent, or reduce the progression of PH in the subject, wherein the TGF-β ligand trapping agent comprises: 1) a TGF-β ligand-binding domain of a TGF-β receptor; 2) an Fc domain of an immunoglobulin; and 3) optionally a linker (immunoglobulin linker or other linker) between the ligand-binding domain and the Fc domain.

[0045] In one embodiment, the TGF-β ligand-binding domain of the TGF-β receptor comprises SEQ ID NO:63, or a portion thereof, or a variant thereof: TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCSITSICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI MKEKKKPGET FFMCSCSSDECNDNIIFSEE YNTSNPD (SEQ ID NO:63).

[0046] In one embodiment, the TGF-β ligand-binding domain of the TGF-β receptor includes SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or a portion thereof, or a variant thereof.

[0047] In one implementation, the Fc domain contains SEQ ID NO:64, or a portion / fragment of SEQ ID NO:64, or a variation thereof. SEQ ID NO:64: ECPPCPAP PVAGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVV SVLTVVHQDW LNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK (SEQ ID NO:64).

[0048] Furthermore, Figure 8BExemplary Fc domains, such as SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72, are described herein. In some embodiments, the Fc domain comprises SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72, or comprises a fragment of SEQ ID NO:69, a fragment of SEQ ID NO:70, a fragment of SEQ ID NO:71, or a fragment of SEQ ID NO:72, or a variant of SEQ ID NO:69, a variant of SEQ ID NO:70, a variant of SEQ ID NO:71, or a variant of SEQ ID NO:72.

[0049] Based on the disclosure herein, the selection of suitable binding domains is within the capabilities of those skilled in the art. In some instances, the binding domains may be selected from the extracellular domains of the TGF-β1 and TGF-βII receptors. A non-limiting example is the soluble recombinant TGF-βII receptor Fc-fusion protein (TGFBRII-Fc).

[0050] In a further example, the natural receptors for designing peptide-binding domains from this can be TβR-I-ED or TβR-II-ED.

[0051] In one embodiment, the TGF-β ligand-binding domain of the TGF-β receptor includes a sequence of, or a portion thereof, of the extracellular domain of the TGF-β type I receptor; for example, SEQ ID NO:73 or a portion thereof, GVQVETISPG DGRTFPKRGQTCVVHYTGML EDGKKFDSSR DRNKPFKFML GKQEVIRGWE EGVAQMSVGQ RAKLTISPDY AYGATGHPGIIPPHATLVFD VELLKLE (SEQ ID NO:73); or for example, SEQ ID NO:74 or a portion / fragment thereof, EDPSLDRPFI SEGTTLKDLI YDMTTSGSGS GLPLLVQRTI ARTIVLQESI GKGRFGEVWRGKWRGEEVAVKIFSSREERS WFREAEIYQT VMLRHENILGFIAADNKDNG TWTQLWLVSD YHEHGSLFDYLNRYTVTVEGMIKLALSTAS GLAHLHMEIV GTQGKPAIAH RDLKSKNILVKKNGTCCIAD LGLAVRHDSATDTIDIAPNH RVGTKRYMAPEVLDDSINMK HFESFKRADI YAMGLVFWEI ARRCSIGGIHEDYQLPYYDLVPSDPSVEEM RKVVCEQKLR PNIPNRWQSCEALRVMAKIM RECWYANGAA RLTALRIKKT LSQLSQQEGIKM(SEQ ID NO:74)(Chain A, cytoplasmic domain of unphosphorylated TGF-β type I receptor crystallized without Fkbp12, GenBank accession 1IAS_A GI:15988007).

[0052] In one embodiment, the TGF-β ligand-binding domain of the TGFβ receptor includes the sequence of TβR-III-ED, or a portion of SEQ ID NO:75; MTSHYVIAIF ALMSSCLATA GPEPGALCEL SPVSASHPVQALMESFTVLSGCASRGTTGL PQEVHVLNLR TAGQGPGQLQ REVTLHLNPISSVHIHHKSV VFLLNSPHPLVWHLKTERLA TGVSRLFLVSEGSVVQFSSA NFSLTAETEE RNFPHGNEHL LNWARKEYGAVTSFTELKIARNIYIKVGED QVFPPKCNIG KNFLSLNYLAEYLQPKAAEG CVMSSQPQNE EVHIIELITPNSNPYSAFQVDITIDIRPSQ EDLEVVKNLI LILKCKKSVN WVIKSFDVKG SLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKW ALDNGYSPITSYTMAPVANR FHLRLENNEE MGDEEVHTIPPELRILLDPGALPALQNPPI RGGEGQNGGL PFPFPDISRR VWNEEGEDGLPRPKDPVIPS IQLFPGLREPEEVQGSVDIA LSVKCDNEKMIVAVEKDSFQ ASGYSGMDVT LLDPTCKAKM NGTHFVLESPLNGCGTRPRWSALDGVVYYN SIVIQVPALG DSSGWPDGYEDLESGDNGFP GDMDEGDASL FTRPEIVVFNCSLQQVRNPSSFQEQPHGNI TFNMELYNTD LFLVPSQGVF SVPENGHVYVEVSVTKAEQE LGFAIQTCFISPYSNPDRMS HYTIIENICPKDESVKFYSP KRVHFPIPQA DMDKKRFSFV FKPVFNTSLLFLQCELTLCTKMEKHPQKLP KCVPPDEACT SLDASIIWAMMQNKKTFTKP LAVIHHEAES KEKGPSMKEP NPISPPIFHGLDTLT (SEQ ID NO:75) (also known as soluble TGF-β receptor III, for example, human recombinant soluble TGF-β receptor III is described in the following: Moren A et al.,Molecular cloning and characterization of thehuman and porcine transforming growth factor-beta type III receptors,1992,J.Biochem.Biophys.Res.Commun.189(1),356-362)。,

[0053] The recombinant soluble TGF-βR type II cDNA is described in: Melissa A. Rowland-Goldsmith et al., Soluble Type II Transforming Growth Factor-β (TGF-β) Receptor Inhibits TGF-β Signaling in COLO-357 Pancreatic Cancer Cells in Vitro and Attenuates Tumor Formationl, 2001, Clin Cancer Res, 7:2931. The complete human TβRII cDNA was used as a template for PCR amplification of the coding sequence of the extracellular domain of TβRII (nucleotides 1-477 containing the signal sequence). PCR was performed using a positive primer (5′-AAGCTTGCCGCCGCCATGGGTCG (SEQ ID NO:76)) and an antisense primer (5′-CTGGAATTCGTCAGGATTGCTGG (SEQ ID NO:77)). SEQ ID NO:78 is an example of the extracellular domain of the type II transforming growth factor-β (TGF-β) receptor: MGRGLLRGLW PLHIVLWTRI ASTIPPHVQK SVNNDMIVTDNNGAVKFPQL CKFCDVRFST CDNQKSCMSN CSITSICEKPQEVCVAVWRK NDENITLETV CHDPKLPYHDFILEDAASPKCIMKEKKKPG ETFFMCSCSSDECNDNIIFS EEYNTSNPDLLLVIFQVTGI SLLPPLGVAISVIIIFYCYR VNRQQKLSSTWETGKTRKLM EFSEHCAIIL EDDRSDISST CANNINHNTELLPIELDTLVGKGRFAEVYK AKLKQNTSEQ FETVAVKIFPYEEYASWKTE KDIFSDINLK HENILQFLTAEERKTELGKQYWLITAFHAK GNLQEYLTRH VISWEDLRKL GSSLARGIAHLHSDHTPCGR PKMPIVHRDLKSSNILVKND LTCCLCDFGLSLRLDPTLSV DDLANSGQVG TARYMAPEVL ESRMNLENVESFKQTDVYSMALVLWEMTSR CNAVGEVKDY EPPFGSK (SEQ ID NO 78).

[0054] The complete extracellular portion of the TGF-β receptor typically includes unstructured portions located on either side of its folded ligand-binding domain. These unstructured extracellular portions are readily apparent from: experimentally determined 3D structures available from the PDB database (Berman et al., 2000, Nucl. Acid Res. 28:235) (e.g., extracellular domains of type I TGF-β receptors (Groppe et al., 2008, Mol. Cell 29:157) or crystal structures of extracellular domains of type II TGF-β receptors (Hart et al., 2002 Nat. Struct. Biol. 9:203; Boesen et al., 2002, Structure 10:913; Grope et al., 2008, Mol. Cell 29:157)), or NMR structures of extracellular domains of type II TGF-β receptors (Deep et al., 2003, Biochemistry 42:10126). Those skilled in the art are adept at identifying the ligand-binding domains of the TGF-β receptor. With regard to TGF-β ligand trappers, ligand binding can be confirmed using standard ligand binding assays known to those skilled in the art (e.g., radioligand binding assays) (see, for example, Sittampalam, GS; Kahl, SD; Janzen, WP, High-throughput screening: Advances in assay technologies, 1997, Current Opinion in Chemical Biology 1(3):384-391; and De Jong, LAA et al., Receptor-ligand binding assays: Technologies and Applications, 2005, Journal of Chromatography B 829(1-2):1-25).

[0055] As used herein, "TGFBRII-Fc" refers to a fusion protein comprising: a TGF-β ligand-binding domain or a variant thereof of the TGF-β type II receptor or its biologically active portion thereof, and an Fc domain of an immunoglobulin. In several embodiments, a linker may be included between the TGF-β ligand-binding domain and the Fc domain. Also according to the invention, the fusion protein may comprise the entire extracellular portion of the TGF-β type II receptor or a variant thereof, and an Fc domain of an immunoglobulin. In some embodiments, the fusion protein may comprise a portion of: the extracellular portion of the TGF-β type II receptor or a variant thereof, and an Fc domain of an immunoglobulin. Examples of variants may include, but are not limited to, variants comprising conventional amino acid mutations, SNP variants, and splice variants. A non-limiting example is a IIb splice variant of the TGF-β type II receptor. In several embodiments, the TGF-β ligand-binding domain and / or the Fc domain may be modified, for example, to facilitate purification, provided that such modifications do not reduce the function of these domains to an unacceptable level.

[0056] The basic techniques for Fc-fusion have been generally described in this field, for example in Czajkowsky et al., Fc-fusion proteins: new developments and future perspectives, EMBO Mol Med. 2012 Oct, 4(10):1015-28, which is incorporated herein by reference in its entirety. TGF-β type II receptors can be derived from mammals. In some instances, the receptor is derived from humans, monkeys, apes, dogs, cats, cattle, horses, goats, sheep, pigs, rabbits, mice, or rats. Immunoglobulins can be derived from mammals. By way of example only, immunoglobulins can be derived from humans, monkeys, apes, dogs, cats, cattle, horses, goats, sheep, pigs, rabbits, mice, or rats.

[0057] When referring to antibody domains, amino acids are assigned to each domain according to Kabat's definition (see, Elvin A. Kabat, Tai Te Wu, Kay S. Gottesman, Carl Foeller, "Sequences of Proteins of Immunological Interest," 5th edition, Publisher 913242, National Institutes of Health, Bethesda, Md., 1991, and earlier). Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are indicated by their position within the chain. Kabat describes a number of amino acid sequences for antibodies, identifies common amino acid sequences for each subgroup, and assigns a residue number to each amino acid. Kabat's numbering scheme can be extended to antibodies not included in his generalization by comparing a questionable antibody to one of the common sequences in Kabat, referring to a conserved amino acid list. This method of assigning residue numbers has become standard in the art and conveniently identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of the human antibody light chain occupies the equivalent position of the amino acid at position 50 of the mouse antibody light chain.

[0058] As used herein, the terms “Fc region”, “Fc domain”, or similar terms are used to define the CH2 / CH3 C-terminal region of the IgG heavy chain. Figure 8B An example of an amino acid sequence containing human IgG1 is shown. As per the Kabat system numbering, although the boundaries may vary slightly, the Fc domain extends from amino acid 231 to amino acid 447 (according to the Kabat system). Figure 8B The amino acid residues in the sequence are numbered: see Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th ed., Public Health Service, NIH, MD (1991), which is incorporated herein by reference in its entirety. Figure 8B Examples of amino acid sequences of the Fc region of IgG isotypes IgG1, IgG2, IgG3 and IgG4 are also provided.

[0059] The Fc region of IgG contains two constant domains: CH2 and CH3. According to the Kabat numbering system, the CH2 domain of the human IgG Fc region typically extends from amino acid 231 to amino acid 341. Figure 8B According to the Kabat numbering system, the CH3 domain of the human IgG Fc region typically extends from amino acid 342 to amino acid 447. Figure 8BThe CH2 domain (also known as the "Cγ2" domain) of the human IgG Fc region is unique because it does not pair tightly with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of intact native IgG.

[0060] Examples of TGFBRII-Fc include, but are not limited to, proteins having the sequence listed in SEQ ID NO:1 or a variant thereof. In one embodiment, a variant of SEQ ID NO:1 comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:1. TGG G VECPPCPAP PVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEV QFNWYVDGVEVHNAKTKPRE EQFNSTFRVV SVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQP REPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK (SEQ ID NO: 1).

[0061] In SEQ ID NO:1, amino acids 1-137 are the TGF-β ligand-binding domain, amino acids 138-141 are the linker, and amino acids 142-364 are the Fc domain. This exemplary TGFBRII-Fc can be represented by a nucleic acid containing the nucleotide sequence listed in SEQ ID NO:2 or a degenerate variant thereof. As used herein, a “degenerate variant” refers to a variant with a mutated nucleotide sequence that still encodes the same polypeptide due to redundancy in the genetic code.

[0062]

[0063]

[0064]

[0065] Using Kabat numbering, the “hinge region” or “hinge domain” of heavy chain IgG is typically defined as the Glu216 extension of human IgG1 to Pro230. Figure 8A An example of the amino acid sequence of the hinge region of human IgG1 is shown (based on the Kabat system). Figure 8A (The amino acid residues in the residues are numbered). For example... Figure 8A As shown, by placing the first and last cysteine ​​residues forming the S-S bond between heavy chains in the same position, the hinge region of other IgG isotypes can be aligned with the IgG1 sequence. In some embodiments, the linker between the ligand-binding domain and the Fc domain includes a hinge region, such as any one of SEQ ID NO:65 to SEQ ID NO:68 (see [link to documentation]). Figure 8A In one embodiment, the connector comprises TGG G (SEQ ID NO: 79). In some embodiments, the connector comprises any one of SEQ ID NO: 6 to SEQ ID NO: 48 (see Example 3).

[0066] It will be readily understood by those skilled in the art that peptides substantially identical to those specifically described herein are considered and may contain one or more conventional amino acid mutations. It is known in the art that making one or more conventional amino acid mutations to a reference peptide can produce a mutant peptide that is not substantially different from the reference peptide in physiological, chemical, or functional properties; in such cases, the reference peptide and the mutant peptide may be considered “substantially identical” polypeptides.

[0067] Traditional amino acid mutations can include the addition, deletion, or substitution of amino acids. In this document, a traditional amino acid substitution is defined as replacing an amino acid residue with another amino acid residue having similar chemical properties (e.g., size, charge, or polarity). In non-limiting examples, a traditional mutation can be an amino acid substitution. Such a traditional amino acid substitution may replace a basic, neutral, hydrophobic, or acidic amino acid with another amino acid having the same functional group.

[0068] As used herein, “basic amino acids” include hydrophilic amino acids with a side-chain pKa value greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). As used herein, “neutral amino acids” (also known as “polar amino acids”) means hydrophilic amino acids with side chains that are uncharged at physiological pH, but which contain at least one bond in which a pair of electrons shared by two atoms is closer to one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). According to Eisenberg’s standardized general hydrophobicity scale (1984), the term “hydrophobic amino acids” (also known as “nonpolar amino acids”) means amino acids that exhibit hydrophobicity greater than zero. Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acids" refer to hydrophilic amino acids with a side chain pKa value less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0069] Sequence identity is used to evaluate the similarity between two sequences; when comparing the maximum correspondence between residue positions of two sequences, sequence identity is determined by calculating the percentage of identical residues. Any known method can be used to calculate sequence identity; for example, computer software can be used to calculate sequence identity. As a non-limiting example, sequence identity can be calculated using software such as BLAST-P, Blast-N, or FASTA-N, or any other suitable software known in the art. The substantially identical sequences of the present invention are at least 80% identical. In other examples, substantially identical sequences are at least 80%, 85%, 90%, 95%, or 100% identical at the amino acid level to the sequences described herein.

[0070] As described above, in several embodiments, a linker may be present between the TGF-β ligand-binding domain and the Fc domain. Sequences of such linkers are provided herein. In one embodiment, the linker is an unstructured and flexible polypeptide sequence. The linker region provides a segment distinct from the structured ligand-binding and Fc domains, and thus can be used to conjugate to auxiliary molecules (e.g., molecules for improving the stability of PEGylation moieties) or load molecules (e.g., contrast agents and toxins for imaging) without chemical modification of the ligand-binding and Fc domains. Conjugation methods vary, but are generally implemented using commercially available kits capable of conjugation via common reactive groups such as primary amines, succinimide (NHS) esters, and thiol reactive groups. Some non-limiting examples are the Alexa Fluor 488 Protein Labeling Kit (molecular probe, Invitrogen detection technologies) and the PEGylation Kit (Pierce Biotechnology Inc.).

[0071] The linker may comprise an unstructured amino acid sequence that is identical to, or derived from, a naturally occurring unstructured region in the extracellular portion of another receptor in the TGF-β superfamily or a ligand of interest, or from conventional modifications of that naturally occurring unstructured region. In other instances, such linkers may be entirely artificial in composition and origin, but will contain selected amino acids to provide an unstructured, flexible linker that is less likely to encounter electrostatic or steric hindrance disturbances when brought close to the ligand of interest.

[0072] The length of a linker is considered to be the number of amino acids between two factors: (a) the C-terminal carbon atom of the binding domain at the N-terminus of the linker; and (b) the N-terminal nitrogen atom of the binding domain at the C-terminus of the linker. A linker length is considered acceptable when it allows the binding domains to bind their native binding sites to their native ligands. Examples of natural and artificial linker sequences of different lengths are given in Table 2. For example, without wishing to be restricted in any way, linker lengths can be approximately 18–80 amino acids, 25–60 amino acids, 35–45 amino acids, or any other suitable length.

[0073] In some instances, it may be desirable to optimize the peptide-based linker design of the ligand-binding reagents disclosed herein to accommodate properties desired for a specific application. For example, to improve binding affinity, specificity, immunogenicity, and stability, the linker can be modified in length and composition based on atomic-level simulations and knowledge-based design. This is applicable to a wide range of molecular systems exhibiting homopolymeric, heteropolymeric, dimeric, and polypolymeric ligand-receptor structural properties. Additional, distinct binding domains can be incorporated to generate multivalent traps with even higher binding valences.

[0074] The linker can be designed to facilitate the purification of the linker and / or ligand binding trap. The chosen precise purification protocol will determine what modifications are needed, for example, and undesirably, the addition of purification "tags" (such as His tags) is under consideration; in other instances, the linker may contain regions that facilitate the addition of loading or auxiliary molecules. When such additions affect the unstructured properties of the linker or introduce potential electrostatic or steric interference, the linker length will be appropriately increased to ensure that the binding domains can bind their sites to the ligands. Such determinations can be routinely made by those skilled in the art in accordance with the methods and teachings herein.

[0075] In embodiments of the invention, the ligand-binding domain and the linker primarily comprise natural sequences, which are generally not expected to be highly immunogenic or toxic in typical patients.

[0076] The peptides of the present invention can be used as therapeutic agents to neutralize the effects of disease-associated covalently stable dimerizing ligands (such as growth factors). They also have commercial potential as diagnostic reagents to detect the presence of disease-associated covalently stable dimerizing ligands (such as growth factors) in both imaging and non-imaging diagnostic applications.

[0077] This invention also covers nucleotide sequences encoding the polypeptides of the invention. These nucleotide sequences can be cloned and inserted into any suitable vector (including expression vectors), and therefore these nucleotide sequences are well-suited for producing the polypeptides of the invention.

[0078] As used herein, the term "vector" refers to a vector nucleic acid molecule into which a nucleic acid sequence can be inserted for the purpose of introducing the nucleic acid sequence into a cell in which it can replicate. The nucleic acid sequence can be "exogenous," meaning that the nucleic acid sequence is foreign to the cell in which the vector is introduced, or that the sequence is homologous to a sequence in the cell but located in a position within the host cell's nucleic acid where the sequence is not normally found. Vectors include plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well prepared to construct vectors using standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference). Furthermore, the techniques illustrated in the referenced figures and described herein are also illustrative for the efficient construction of vectors.

[0079] The term "expression vector" refers to any type of genetic construct that contains nucleic acids encoding RNA that can be transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. In others, such as in the production of antisense molecules or ribozymes, these sequences are not translated. Expression vectors may contain a variety of "control sequences," which are nucleic acid sequences essential in a particular host cell for the possible translation and transcription of the operatively linked coding sequences. In addition to the control sequences governing transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions, as described below.

[0080] As used herein, the terms “peptide” or “protein” refer to a polymer of amino acids linked in a specific sequence by peptide bonds. As used herein, unless otherwise specifically specified, the term “amino acid” refers to the D or L stereoisomer of an amino acid.

[0081] As used herein, the “biologically active” part of a molecule refers to a portion of a larger molecule capable of performing a function similar to that of a larger molecule. By way of non-limiting example only, the biologically active part of a protein is any portion of the protein that retains (even only slightly) the ability to perform one or more biological functions of the full-length protein (e.g., binding to another molecule, phosphorylation, etc.). By way of non-limiting example, the ligand-binding domain is the biological part of the TGFβ receptor.

[0082] As used herein, the term "therapeutic effective dose" means the amount of TGF-β ligand trapping agent that attenuates or inhibits excessive TGF-β signaling and thus treats, prevents, or slows the progression of the disease described herein. The effective dose will vary depending on the condition or pathology to be treated, depending on the patient and his or her condition, and other factors known to those skilled in the art. The effective dose can be readily determined by those skilled in the art. In some embodiments, the therapeutic dose is administered via subcutaneous, intrathecal, convection-enhancing, intravenous, or intraarterial routes at intervals from daily to monthly, in doses ranging from 0.05 mg / kg body weight to 50 mg / kg body weight, and optionally 1.0 mg / kg body weight to 10 mg / kg body weight, or 0.3 mg / kg body weight to 3.0 mg / kg body weight. In many embodiments, the TGF-β ligand trapping agent is administered to the subject 1-7 times per week, or once per week, or once every two weeks, once every three weeks, or once every four weeks. In several implementations, subjects are given a TGF-β ligand trap for 1–5 days, 1–5 weeks, 1–5 months, or 1–5 years.

[0083] While some exemplary administration routes are provided according to the present invention, any suitable administration route for TGF-β ligand trapping agents can be modified, and therefore the administration routes described herein are not intended to be limiting. Administration routes may include, but are not limited to, intravenous, oral, buccal, intranasal, inhalation, topical application to mucous membranes, or injection (including intradermal, intrathecal, intracisional, intralesional, or any other type of injection). Administration may be continuous or intermittent and may vary depending on the subject and the condition to be treated. It will be readily understood by those skilled in the art that the various administration routes described herein will enable the delivery of TGF-β ligand trapping agents or compositions at, near, or at the site of lung disease or target cells. It will also be readily understood by those skilled in the art that the various administration routes described herein will enable the delivery of the TGF-β ligand trapping agents or compositions described herein to regions adjacent to the diseased tissue, organ, or single cell to be treated. "Proximity" may include any tissue or fluid in the subject that is sufficiently close to or in full communication with the diseased tissue, organ, or single cell, such that at least a portion of the TGF-β ligand trap or composition administered to the subject reaches its intended target and exerts its therapeutic effect.

[0084] Pharmaceutical Composition

[0085] In several embodiments, the present invention provides pharmaceutical compositions comprising the TGF-β ligand trapping agents described herein. In several embodiments, the pharmaceutical compositions are formulated for improved release, sustained release, controlled release, or combinations thereof. In several embodiments, the pharmaceutical compositions are formulated for oral, inhalation, nasal, sublingual, buccal, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, or parenteral administration.

[0086] In several embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. Examples of excipients include, but are not limited to, starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, wetting agents, emulsions, colorants, release agents, coating agents, sweeteners, flavoring agents, aroma agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardening agents, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.

[0087] In several embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known in the art and include aqueous solutions, such as physiologically buffered saline, other solvents, or excipients (such as ethylene glycol, glycerol, vegetable oils (e.g., olive oil), or injectable organic esters). Pharmaceutically acceptable carriers can be used to administer the compositions of the invention to cells in vitro or to a subject. Pharmaceutically acceptable carriers may contain physiologically acceptable compounds, for example, compounds that have a stabilizing effect on the composition or increase the absorption of reagents. Physiologically acceptable compounds may include, for example, carbohydrates (such as glucose, sucrose, or dextrose), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives, with preservatives particularly used to prevent microbial activity or growth. A variety of preservatives are known and include, for example, phenol and ascorbic acid. Those skilled in the art will recognize that the choice of pharmaceutically acceptable carriers (including physiologically acceptable compounds) depends on, for example, the route of administration of the peptide. For instance, physiologically acceptable compounds (such as aluminum monostearate or gelatin) are specifically used as delaying agents to prolong the rate of absorption of a pharmaceutical composition administered to a subject. Further examples of carriers, stabilizers, or adjuvants can be found in Martin, Remington's Pharm. Sci., 15th edition, (Mack Publ. Co., Easton, 1975), which is incorporated herein by reference. Other examples of carriers include, but are not limited to, nanoparticle-based carriers (e.g., polymer N-(2-hydroxypropyl)methacrylamide (HPMA), glutamate, PEG, dextrose) and nanocarriers (e.g., nanoshells, liposomes, nanoliposomes).

[0088] Treatment

[0089] In several embodiments, the present invention provides a method for treating, preventing, or reducing the progression of pulmonary hypertension (PH) in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject to treat, prevent, or reduce the progression of PH in the subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand trapping agent before administering the therapeutically effective amount to the subject. Pulmonary hypertension is a type of pulmonary hypertension that is particularly suitable for treatment with a TGF-β ligand trapping agent. Therefore, in some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject to treat, prevent, or reduce the progression of pulmonary hypertension (including any of the following subclasses of pulmonary hypertension) in the subject. Pulmonary hypertension may be secondary to other conditions or arise as primary or idiopathic pulmonary hypertension. Of particular interest are the fact that some types of familial pulmonary hypertension are associated with reduced expression or function of bone morphogenetic protein receptor type II (BMPRII), which is thought to lead to excessive signaling of TGF-β.

[0090] Pulmonary hypertension can have five main types, therefore a series of tests are performed to differentiate pulmonary arterial hypertension from venous pulmonary hypertension, hypoxic pulmonary hypertension, thromboembolic pulmonary hypertension, or mixed pulmonary hypertension. These tests generally include: pulmonary function tests; blood tests to rule out HIV, autoimmune diseases, and liver disease; electrocardiogram (ECG); arterial blood gas measurements; chest X-ray (followed by a high-resolution CT scan if interstitial lung disease is suspected); and ventilation-perfusion or V / Q scans to rule out chronic thromboembolic pulmonary hypertension. The diagnosis of PAH requires the presence of pulmonary hypertension. Although pulmonary artery pressure can be estimated based on echocardiography, pressure measurements using a Swan-Ganz catheter inserted through the right side of the heart provide the most accurate assessment for diagnosis.

[0091] Those skilled in the art are adept at monitoring improvements in pulmonary hypertension, for example, by measuring clinical improvement typically through the "six-minute walk test" (i.e., the distance a patient can walk in six minutes). Stability and improvement in this measurement are associated with better survival. Blood BNP levels are now also used to track progression in patients with pulmonary hypertension. Symptom improvement can also be monitored by analyzing arterial pressure. For example, normal resting pulmonary artery pressure in people living at sea level has a mean of 8 mmHg–20 mmHg (1066 Pa–2666 Pa). Pulmonary hypertension is present when the resting mean pulmonary artery pressure exceeds 25 mmHg (3300 Pa). Mean pulmonary artery pressure (mPAP) should not be confused with pulmonary systolic pressure (sPAP), which is typically reported in echocardiographic reports. A systolic pressure of 40 mmHg generally implies a mean pressure exceeding 25 mmHg. Roughly, mPAP = 0.61·sPAP + 2.

[0092] In several embodiments, the present invention provides a method for treating, preventing, or slowing the progression of pulmonary vascular remodeling in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or slowing the progression of pulmonary vascular remodeling in the subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand trapping agent prior to administering the therapeutically effective amount of the TGF-β ligand trapping agent to the subject.

[0093] In several embodiments, the present invention provides a method for treating, preventing, or slowing the progression of vascular remodeling in the heart of a subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with a TGF-β ligand trapping agent before administering a therapeutically effective amount of the TGF-β ligand trapping agent to the subject. In some embodiments, the method of the present invention reduces mitral valve degeneration, or, for example, mitral valve prolapse. The beneficial effects can be monitored by echocardiography.

[0094] In several embodiments, the present invention provides a method for treating, preventing, or slowing the progression of pulmonary fibrosis in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or slowing the progression of pulmonary fibrosis in the subject. In several embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand trapping agent prior to administering the therapeutically effective amount of the TGF-β ligand trapping agent to the subject.

[0095] In several embodiments, the present invention provides a method for treating, preventing, or slowing the progression of right ventricular hypertrophy in a subject. In several embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or slowing the progression of right ventricular hypertrophy in the subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand trapping agent before administering the therapeutically effective amount to the subject.

[0096] In several embodiments, the present invention provides a method for treating, preventing, or slowing the progression of lung disease associated with excessive TGF-β signaling in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand scavenger to the subject, thereby treating, preventing, or slowing the progression of the disease in the subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand scavenger before administering the therapeutically effective amount to the subject.

[0097] In several implementations, TGF-β may be TGF-β1, TGF-β3, or a combination thereof.

[0098] In several embodiments, the present invention provides a method for treating, preventing, or slowing the progression of lung disease associated with excessive GDF15 signaling in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand scavenger to the subject, thereby treating, preventing, or slowing the progression of the disease in the subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand scavenger before administering the therapeutically effective amount to the subject.

[0099] In several embodiments, the present invention provides a method for treating, preventing, or slowing the progression of lung disease associated with excessive PAI-1 signaling in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand scavenger to the subject, thereby treating, preventing, or slowing the progression of the disease in the subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand scavenger before administering the therapeutically effective amount to the subject.

[0100] In several embodiments, the present invention provides a method for reducing right ventricular systolic pressure in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby reducing right ventricular systolic pressure in the subject. In some embodiments, the method may further include mixing a pharmaceutically acceptable carrier with the TGF-β ligand trapping agent prior to administering the therapeutically effective amount of the TGF-β ligand trapping agent to the subject.

[0101] In several embodiments, the subjects in the above examples are mammals. In some embodiments, the subjects are humans, monkeys, apes, dogs, cats, cattle, horses, goats, sheep, pigs, rabbits, mice, or rats. In several embodiments, TGF-β is TGF-β1, TGF-β3, or a combination thereof.

[0102] In several embodiments, the amount of TGF-β ligand scavenger administered to the subject is 0.05 mg / kg body weight to 50 mg / kg body weight, optionally 1.0 mg / kg body weight to 10 mg / kg body weight, or 0.3 mg / kg body weight to 3.0 mg / kg body weight. In several embodiments, the TGF-β ligand scavenger is administered to the subject 1-7 times per week, once per week, once every two weeks, once every three weeks, or once every four weeks. In several embodiments, the TGF-β ligand scavenger is administered to the subject for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years. The TGF-β ligand scavenger can be administered via any route of administration for protein therapy, including but not limited to subcutaneous, intravenous, or intramuscular administration.

[0103] As described above, in several embodiments, the TGF-β ligand scavenger is administered to the subject orally, via inhalation, nasal administration, sublingual administration, buccal administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, intraperitoneal administration, or parenteral administration. In several embodiments, the TGF-β ligand scavenger is administered before, during, or after the development of a disease condition in the subject, including but not limited to: pulmonary hypertension, pulmonary vascular remodeling, pulmonary fibrosis, right ventricular hypertrophy, lung disease associated with excessive TGF-β signaling, lung disease associated with excessive GDF15 signaling, and lung disease associated with excessive PAI-1 signaling.

[0104] In several embodiments, the TGF-β ligand trap is part of the pharmaceutical composition. In several embodiments, the pharmaceutical composition is formulated for improved release, sustained release, controlled release, or a combination thereof. In several embodiments, the pharmaceutical composition is formulated for oral, inhalation, nasal, sublingual, buccal, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, or parenteral administration.

[0105] In several embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. Examples of excipients include, but are not limited to, starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, wetting agents, emulsions, colorants, release agents, coating agents, sweeteners, flavoring agents, aroma agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, coagulants, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.

[0106] Some embodiments of the present invention may be defined by any of the following numbered paragraphs:

[0107] Paragraph 1. A method for treating, preventing, or reducing the progression of pulmonary hypertension (PH) in a subject, the method comprising: administering to the subject a therapeutically effective amount of a TGF-β ligand trapping agent, thereby treating, preventing, or reducing the progression of PH in the subject.

[0108] Paragraph 2. The method as described in paragraph 1, wherein the PH is mediated by excessive TGF-β signaling.

[0109] Paragraph 3. The method described in any of paragraphs 1-2, wherein the subject is a human being.

[0110] Paragraph 4. The method as described in any of paragraphs 1-3, wherein the TGF-β ligand trapping agent comprises: 1) a TGF-β ligand binding domain of a TGF receptor; and 2) an Fc domain of an immunoglobulin.

[0111] Paragraph 5. The method as described in paragraph 4, wherein the TGF-β ligand trapping agent further comprises a linker between the TGF-β ligand binding domain and the Fc domain of the TGF receptor.

[0112] Paragraph 6. The method as described in any of paragraphs 1-5, wherein the TGF-β ligand scavenger is a soluble recombinant TGF-β type II receptor Fc-fusion protein (TGFBRII-Fc).

[0113] Paragraph 7. The method as described in any of paragraphs 1-6, wherein the TGFBRII-Fc consists of a sequence listed in SEQ ID NO:1 or a variant thereof.

[0114] Paragraph 8. The method as described in any of paragraphs 1-6, wherein the TGFBRII-Fc comprises the sequence listed in SEQ ID NO:1 or a variant thereof.

[0115] Paragraph 9. The method as described in any of paragraphs 1-6, wherein the TGFBRII-Fc comprises one or more bioactive portions of the sequence listed in SEQ ID NO:1.

[0116] Paragraph 10. The method as described in any of paragraphs 1-6, wherein the TGFBRII-Fc is encoded by a nucleic acid comprising the nucleotide sequence listed in SEQ ID NO:2 or a degenerate variant thereof.

[0117] Paragraph 11. The method as described in any of paragraphs 1-10, wherein the amount of TGF-β ligand capture agent administered to the subject is 0.1 mg / kg body weight to 10 mg / kg body weight.

[0118] Paragraph 12. The method as described in any of paragraphs 1-11, wherein the TGF-β ligand trapping agent is administered to the subject 1-7 times per month.

[0119] Paragraph 13. The method as described in any of paragraphs 1-12, wherein the subject is given the TGF-β ligand trap for 1-5 days, 1-5 weeks, 1-5 months, or 1-5 years.

[0120] Paragraph 14. The method as described in any of paragraphs 1-13, wherein the TGF-β ligand capture agent is administered to the subject orally, via inhalation, nasal, sublingual, buccal, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, or parenteral.

[0121] Paragraph 15. The method as described in any of paragraphs 1-14, wherein the TGF-β ligand trapping agent is administered before, during, or after the subject develops PH.

[0122] Paragraph 16. The method as described in any of paragraphs 1-15, wherein the method further comprises mixing a pharmaceutically acceptable carrier with the TGF-β ligand trapping agent before administering a therapeutically effective amount of the TGF-β ligand trapping agent to the subject.

[0123] Paragraph 17. The method as described in any of paragraphs 1-16, wherein the TGF-β ligand scavenger is part of the pharmaceutical composition.

[0124] Paragraph 18. The method as described in paragraph 17, wherein the pharmaceutical composition is formulated for improved release, sustained release, or controlled release, or a combination thereof.

[0125] Paragraph 19. The method as described in paragraph 17, wherein the pharmaceutical composition is formulated for oral, inhalation, nasal, sublingual, buccal, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, or parenteral administration.

[0126] Paragraph 20. The method as described in paragraph 17, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0127] Paragraph 21. The method as described in paragraph 17, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

[0128] Paragraph 22. A method for treating, preventing, or slowing the progression of pulmonary vascular remodeling in a subject, the method comprising: administering to the subject a therapeutically effective amount of a TGF-β ligand trapping agent, thereby treating, preventing, or slowing the progression of pulmonary vascular remodeling in the subject.

[0129] Paragraph 23. A method for treating, preventing, or reducing the progression of pulmonary fibrosis in a subject, the method comprising: administering to the subject a therapeutically effective amount of a TGF-β ligand trapping agent, thereby treating, preventing, or reducing the progression of pulmonary fibrosis in the subject.

[0130] Paragraph 24. The method as described in paragraph 23, wherein the method uses the TGF-β ligand trapping agent described in any of paragraphs 4-10.

[0131] Paragraph 25. A method for treating or preventing right ventricular hypertrophy in a subject or for slowing the progression of right ventricular hypertrophy in a subject, the method comprising: administering to the subject a therapeutically effective amount of a TGF-β ligand trapping agent, thereby treating or preventing right ventricular hypertrophy in the subject or slowing the progression of right ventricular hypertrophy in the subject.

[0132] Paragraph 26. The method as described in paragraph 25, wherein the method uses the TGF-β ligand trapping agent described in any of paragraphs 4-10.

[0133] Paragraph 27. A method for treating, preventing, or reducing the progression of lung disease associated with excessive TGF-β signaling in a subject, the method comprising: administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or reducing the progression of the disease in the subject.

[0134] Paragraph 28. The method as described in paragraph 27, wherein the method uses the TGF-β ligand trapping agent described in any of paragraphs 4-10.

[0135] Paragraph 29. The method as described in any of paragraphs 1-28, wherein the TGF-β is TGF-β1, TGF-β3, or a combination thereof.

[0136] Paragraph 30. A method for treating, preventing, or reducing the progression of lung disease associated with excessive GDF15 signaling in a subject, the method comprising: administering to the subject a therapeutically effective amount of a TGF-β ligand trapping agent, thereby treating, preventing, or reducing the progression of the disease in the subject.

[0137] Paragraph 31. The method as described in paragraph 30, wherein the method uses the TGF-β ligand trapping agent described in any of paragraphs 4-10.

[0138] Paragraph 32. A method for treating, preventing, or reducing the progression of a lung disease associated with excessive PAI-1 signaling in a subject, the method comprising: administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby treating, preventing, or reducing the progression of the disease in the subject.

[0139] Paragraph 33. The method as described in paragraph 32, wherein the method uses the TGF-β ligand trapping agent described in any of paragraphs 4-10.

[0140] Paragraph 34. A method for reducing right ventricular systolic pressure in a subject, the method comprising: administering a therapeutically effective amount of a TGF-β ligand trapping agent to the subject, thereby reducing right ventricular systolic pressure in the subject.

[0141] Paragraph 35. The method as described in paragraph 34, wherein the method uses the TGF-β ligand trapping agent described in any of paragraphs 4-10.

[0142] Example

[0143] The following embodiments are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Specific materials mentioned are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent methods or reactants without inventiveness and without departing from the scope of the invention.

[0144] Example 1: Brief summary of additional background and results

[0145] As described above, transforming growth factor-β (TGF-β) ligands coordinate important developmental processes and regulate fibrosis and tissue remodeling in disease. Excessive TGF-β signaling has been involved in arterial remodeling in pulmonary hypertension (PH), partly based on the ability of TGFβ type I receptor (ALK5) kinase inhibitors to improve experimental PH in animal models. However, the clinical deployment of ALK5 inhibitors has been limited by cardiovascular toxicity. The experiments and results disclosed in this paper demonstrate that soluble recombinant TGFβ type II receptor Fc-fusion protein (TGFBRII-Fc) inhibits TGFβ signaling in limonene (MCT)-induced PH rats. When administered prophylactically after MCT, TGFBRII-Fc treatment reduced right ventricular systolic pressure, right ventricular hypertrophy, and attenuated pulmonary vascular remodeling. Consistent with the attenuation of TGFβ signaling, TGFBRII-Fc corrected for elevated mRNA levels of PAI-1, a TGFβ transcriptional target, in the lungs of MCT rats. When administered 2.5 weeks after MCT, TGFBRII-Fc partially revived established PH at week 5 and showed an improved survival trend. Notably, no cardiac structural or valvular abnormalities were found associated with treatment with any dose of TGFBRII-Fc. Overall, the data presented in this paper support the conclusion that TGFβ ligand traps can be an effective and acceptable safe strategy for correcting TGFβ-mediated pulmonary vascular remodeling and PH.

[0146] Example 2

[0147] Table 1. Non-limiting exemplary TGFβ ligand-binding domains

[0148]

[0149] Example 3

[0150] Table 2. Non-limiting exemplary connectors

[0151]

[0152]

[0153]

[0154] Also under consideration are nucleic acid sequences encoding the aforementioned adapters and binding domains.

[0155] Example 4

[0156] Materials and methods

[0157] Rat model of PAH

[0158] Male Sprague-Dawley rats (6–8 weeks old, weighing 150–170 g) were purchased from the Charles River Laboratories. All protocols and surgeries were approved by the local animal protection committee. Animals were housed at 24°C under a 12-hour light-dark cycle. Food and water were provided freely. Rats were given a single subcutaneous injection of limonene (MCT, 40 mg / kg) to induce PAH. Table 3 summarizes the total number of rats included in this study and the number of deaths.

[0159] Table 3

[0160]

[0161] Drug treatment

[0162] Prevention protocol – 24 hours after PAH induction, rats were randomly assigned to either the TGFBRII-Fc group (5 mg / kg or 15 mg / kg, twice weekly) or the adjuvant group. Rats were treated for 21 days. On day 14, ventricular function and RV remodeling were assessed by echocardiography. On day 21, hemodynamic and right ventricular hypertrophy measurements were performed on the rats.

[0163] Resuscitation Protocol – In another cohort, the ability of TGFBRII-Fc to reverse PAH progression was examined. On day 18, rats were injected with MCT and randomly administered TGFBRII-Fc (15 mg / kg, three times weekly) or excipients. Hemodynamics and right ventricular hypertrophy (RVH) were examined on day 35.

[0164] Echocardiographic assessment of LV and RV function

[0165] On day 14 following PAH induction, rats were anesthetized with 1.5% isoflurane and held in a supine position. Pulmonary blood flow acceleration, right ventricular function and hypertrophy, and left ventricular function were assessed using a VisualSonics small animal high-frequency ultrasound probe. Doppler ultrasound was applied through the mitral and tricuspid valves to determine whether TGFBRII-Fc treatment induced any significant regurgitation or damage.

[0166] Hemodynamics and RVH Measurement

[0167] At specific time points, rats were anesthetized with pentobarbital and intubated via the trachea. Rats were mechanically ventilated using a rodent ventilator, and hemodynamic assessments were performed using a fluid-filled catheter through the apex of the right ventricle (RV) as previously described (Megalou, AJ; Glava, C.; Vilaeti, AD; Oikonomidis, DL; Baltogiannis, GG; Papalois, A.; Vlahos, AP; and Kolettis, TM (2012) Pulm Circ 2, 461-469). The lungs were perfused with PBS, and a right lobe was removed and rapidly frozen for RNA and protein extraction. The lungs were further perfused into the pulmonary artery with 1% paraformaldehyde (PFA), followed by tracheal perfusion for 1 minute. The left lobe was embedded in paraffin. To assess the degree of RVH, the heart was removed, the free wall of the RV was dissected from the left ventricle plus the septal wall (LV+S), and weighed separately. The degree of RVH is determined by the RV / (LV+S) ratio.

[0168] Quantitative analysis of vascular remodeling

[0169] To determine the extent of pulmonary vascular remodeling, lung tissue sections were stained with α-smooth muscle actin and von Willebrand factor. Muscle formation in distal acinar vessels (10 μm–50 μm in diameter) was quantified, and the percentages of non-muscledized, partially muscled, and fully muscled vessels were calculated.

[0170] The medial wall thickness of all fully muscularized intraacini vessels (diameter 10μm-50μm) was calculated. The wall thickness index was calculated as: index = (outer diameter - inner diameter) / outer diameter × 100.

[0171] Expression Research

[0172] Frozen lung samples were homogenized, and total RNA was extracted using TRIZOL reagent as previously described (Long, L., Crosby, A., Yang, X., Southwood, M., Upton, PD, Kim, DK, and Morrell, NW (2009) Circulation 119, 566-576). Reverse transcription and quantitative PCR were performed as previously described (Long, L., Crosby, A., Yang, X., Southwood, M., Upton, PD, Kim, DK, and Morrell, NW (2009) Circulation 119, 566-576). The proportion of specific genes to β-actin was calculated and expressed as a fold change. Rat-specific sequences are summarized in Table 4.

[0173] Table 4

[0174]

[0175] reagents

[0176] Wild lily alkaloids were purchased from Oakwood Products. Recombinant human BMP4, TGFβ1, TGFβ2, and GDF15 were obtained from R&D Systems. Primary antibodies specific to phosphorylated-Smad 3 were purchased from Abcam, while other primary antibodies specific to phosphorylated-Smad 2, phosphorylated-Smad 1 / 5, and total Smad 3 were obtained from Cell Signaling.

[0177] Statistical analysis

[0178] All analyses of hemodynamic and RVH measurements and quantification of pulmonary vascular remodeling were performed blinded. Data are presented as mean ± SEM and compared between groups using t-tests. p < 0.05 was considered statistically significant.

[0179] Vascular remodeling of the mitral valve

[0180] Figures 9A to 9B Heart tissue sections showing no mitral valve remodeling, degenerative changes, or abnormalities in response to TGFBRII-Fc treatment are shown. Figure 9A For comparison. Figure 9B It has been treated with TGFBRII-Fc-.

[0181] The aforementioned methods and techniques provide multiple ways to implement this application. It should be understood, of course, that not all objectives and advantages described herein may be achieved according to any particular implementation thereof. Therefore, for example, those skilled in the art will recognize that the method can be implemented by achieving or optimizing one or more advantages as taught herein without necessarily achieving other objectives or advantages as taught or implied herein. Various alternatives are mentioned herein. It should be understood that some preferred embodiments specifically include one or more features, while other embodiments specifically exclude one or more features, and still others mitigate specific features by including one or more advantageous features.

[0182] Furthermore, those skilled in the art will recognize the applicability of various features from different implementations. Similarly, those skilled in the art can employ in various combinations the various elements, features, and steps disclosed above, as well as other known equivalents of such elements, features, or steps, to implement the method according to the principles described herein. Among the various elements, features, and steps, some will be specifically included in the different implementations, while others will be specifically excluded from the different implementations.

[0183] Although this application has been disclosed in the context of some implementation methods and embodiments, those skilled in the art will understand that the implementation methods of this application extend beyond the specifically disclosed implementation methods and to other alternative implementation methods and / or uses, as well as modifications and equivalents thereof.

[0184] In some embodiments, the terms “a,” “an,” “the,” and similar expressions used in the context of describing particular embodiments of this application (especially in the context of some of the claims below) may be interpreted to cover both singular and plural. The range of values ​​listed herein is intended only as a shorthand method for each individual value falling within that range when individually mentioned. Unless otherwise specified herein, each individual value is incorporated into this specification as if individually listed herein. All methods described herein may be implemented in any suitable order unless otherwise specified herein or where the context clearly conflicts. The use of any and all instances or exemplary language (e.g., “such as”) provided with respect to some embodiments herein is intended only to better clarify the application and does not limit the scope of the application unless they are claimed. The language in this specification should not be construed as indicating any unclaimed element necessary to practice this application.

[0185] This document describes preferred embodiments of this application, including the best modes known to the inventors for carrying out this application. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the above description. It is to be considered that such variations may be employed where appropriate, and that this application may be practiced in ways other than those specifically described herein. Therefore, as permitted by applicable law, many embodiments of this application include all modifications and equivalents of the subject matter listed in the appended claims. Furthermore, unless otherwise indicated herein, or unless obviously conflicting with the context, this application covers any combination of the foregoing elements in all possible variations.

[0186] Except as otherwise provided, all patents, patent applications, published texts of patent applications, and other materials (such as articles, books, specifications, publications, documents, things, and / or the like) referenced herein are hereby incorporated in their entirety by reference for all purposes: any examination document history relating to the same material, any identical material inconsistent with or conflicting with this document, or any identical material that may have a limiting effect on the broadest scope of the claims currently or subsequently relating to this document. For example, if there is any inconsistency or conflict between the description, definition, and / or use of terms relating to any incorporated material and the description, definition, and / or use of terms relating to this document, the description, definition, and / or use of terms in this document shall prevail.

[0187] It should be understood that the embodiments disclosed herein are illustrative of the principles of the embodiments of this application. Other modifications may be made within the scope of this application. Therefore, alternative constructions of the embodiments of this application may be used in accordance with the teachings herein, exemplified rather than limited. Thus, the embodiments of this application are not limited to those precisely shown and described.

[0188] Several embodiments of the invention have been described in the detailed description above. While these descriptions directly depict the embodiments above, it should be understood that modifications and / or variations will be conceived by those skilled in the art regarding the particular embodiments shown and described herein. Any such modifications or variations falling within the scope of this specification are also intended to be included herein. Unless specifically indicated, it is intended that the words and phrases in the specification and claims be given their common and conventional meanings known to those skilled in the art.

[0189] The foregoing description of various embodiments of the invention known to the applicant at the time of filing of this application is provided for illustrative and descriptive purposes. This description is not intended to be exhaustive, nor is it intended to limit the invention to the precise forms disclosed, and many modifications and variations are possible in accordance with the foregoing teachings. The described embodiments are intended to explain the principles of the invention and its practical application, and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications suitable for the particular intended use. Therefore, the invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention.

[0190] While specific embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications may be made in accordance with the teachings herein without departing from the invention and its broader aspects, and therefore, the appended claims are intended to cover all such changes and modifications falling within the true spirit and scope of the invention. Those skilled in the art will understand that, in general, the terminology used herein is intended to be “open” (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “including” should be interpreted as “comprising but not limited to,” etc.).

Claims

1. Use of TGF-β ligand scavengers in the preparation of drugs for the following purposes: (1) To treat or prevent the following diseases in the subject or to reduce the progression rate of the following diseases in the subject, wherein, The diseases mentioned include: pulmonary hypertension, pulmonary vascular remodeling, pulmonary fibrosis, right ventricular hypertrophy, lung diseases associated with excessive TGF-β signaling, lung diseases associated with excessive GDF15 signaling, and lung diseases associated with excessive PAI-1 signaling. (2) Reduce right ventricular systolic pressure in the subjects; or (3) Imaging / detecting TGF-β ligands in the subject, wherein the TGF-β ligand trapping agent is linked to the imaging molecule.

2. The use as described in claim 1, wherein, The pulmonary hypertension is mediated by excessive TGF-β signaling.

3. The use as described in any one of claims 1-2, wherein, The subjects were humans, monkeys, apes, dogs, cats, cows, horses, goats, sheep, pigs, rabbits, mice, or rats.

4. The use as described in any one of claims 1-3, wherein, The TGF-β ligand trapping agent comprises: 1) a TGF-β ligand-binding domain of the TGF receptor; and 2) an Fc domain of the immunoglobulin.

5. The use as described in claim 4, wherein, The TGF-β ligand trapping agent is further contained in a linker between the TGF-β ligand binding domain and the Fc domain of the TGF receptor.

6. The use as described in claim 5, wherein, The joint includes a hinge area.

7. The use as described in claim 5, wherein, The connector includes any one of SEQ ID NO:65-SEQ ID NO:

68.

8. The use as described in claim 5, wherein, The connector contains TGG G.

9. The use as described in claim 5, wherein, The connector includes any one of SEQ ID NO:6 to SEQ ID NO:

48.

10. The use as described in any one of claims 1-9, wherein, The TGF-β ligand scavenger is a soluble recombinant TGF-β type II receptor Fc-fusion protein (TGFBRII-Fc).

11. The use as described in any one of claims 1-10, wherein, The TGFBRII-Fc has a sequence of SEQ ID NO:1 or a variant thereof.

12. The use as described in any one of claims 1-10, wherein, The TGFBRII-Fc is encoded by the following nucleic acid, which has the sequence SEQ ID NO:2 or a degenerate variant thereof.

13. The use as described in any one of claims 1-12, wherein, The amount of TGF-β ligand scavenger administered to the subject was 0.05 mg / kg body weight to 50 mg / kg body weight, preferably 0.1 mg / kg body weight to 10 mg / kg body weight, and more preferably 0.3 mg / kg body weight to 3.0 mg / kg body weight.

14. The use as described in any one of claims 1-13, wherein, The TGF-β ligand trapping agent is administered to the subject 1-7 times per month, preferably 1-7 times per week, preferably once per week, preferably once every two weeks, preferably once every three weeks, and more preferably once every four weeks.

15. The use as described in any one of claims 1-14, wherein, The subjects were given the TGF-β ligand capture agent for 1–5 days, 1–5 weeks, 1–5 months, or 1–5 years.

16. The use as described in any one of claims 1-15, wherein, The TGF-β ligand scavenger was administered orally, via inhalation, nasal, sublingual, buccal, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, or parenteral administration to the subject.

17. The use as described in any one of claims 1-16, wherein, The TGF-β ligand trapping agent was administered before, during, or after the subject developed pulmonary hypertension.

18. The use as described in any one of claims 1-17, wherein, The drug is a pharmaceutical composition containing a TGF-β ligand trapping agent.

19. The use as described in claim 18, wherein, The pharmaceutical composition is formulated to improve release, sustain release, or control release, or a combination thereof.

20. The use as described in claim 18 or 19, wherein, The pharmaceutical composition is formulated for oral, inhalation, nasal, sublingual, buccal, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, or parenteral administration.

21. The use as described in any one of claims 18-20, wherein, The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

22. The use as described in any one of claims 18-21, wherein, The pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

23. The use as described in any one of claims 1-22, wherein, The TGF-β is TGF-β1, TGF-β3, or a combination thereof.

24. The use as described in any one of claims 1-23, wherein, The TGF-β receptor's TGF-β ligand-binding domain has a sequence selected from any of the following sequences: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:63, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:

75.

25. The use as described in any one of claims 1-24, wherein, The Fc domain has a sequence selected from any of the following sequences: SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72.

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