TGF-beta receptor type II variant and its use

Abstract

To provide compositions and methods for modulating TGFβ superfamily signaling.SOLUTION: The disclosure provides TβRII polypeptides and the use of such TβRII polypeptides as selective antagonists for GDF15, TGFβ1 or TGFβ3. As described herein, polypeptides comprising part or all of the TβRII extracellular domain (ECD), with or without additional mutations, bind to and / or inhibit GDF15, TGFβ1 or TGFβ3 with varying affinities. Thus, in certain aspects, the disclosure provides TβRII polypeptides for use in selectively inhibiting TGFβ superfamily associated disorders.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 61 / 868,713, filed on 22 August 2013; U.S. Provisional Patent Application No. 61 / 906,270, filed on 19 November 2013; and U.S. Provisional Patent Application No. 61 / 906,849, filed on 20 November 2013. The disclosures of the basic applications are incorporated herein by reference in their entirety. [Background technology]

[0002] The transforming growth factor-beta (TGFβ) superfamily members are multifaceted cytokines involved in important cellular functions such as proliferation, differentiation, apoptosis, motility, extracellular matrix production, tissue remodeling, angiogenesis, immune responses, and cell adhesion, and play crucial roles in the pathophysiology of disease states as diverse as chronic inflammatory conditions and cancer. The TGFβ superfamily members have been classified into major family groupings, including TGFβ, bone morphogenetic proteins (BMPs), osteogenic proteins (OPs), growth and differentiation factors (GDFs), inhibin / activins, Müllerian duct inhibitors (MISs), and glial-derived neurotrophic factor (GDNF).

[0003] TGFβ superfamily members transmit their signals across the plasma membrane by inducing the formation of heteromeric complexes of specific type I and type II serine / threonine kinase receptors, which in turn activate a specific subset of SMAD proteins (some inhibitory and some excitatory). These SMAD molecular compounds relay signals into the nucleus, where they interact with other proteins to direct transcriptional responses.

[0004] Dysfunctional TGFβ superfamily signaling has been associated with several clinical disorders, including cancer, fibrosis, bone disease, diabetic nephropathy, and chronic vascular diseases such as atherosclerosis. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, the object of this disclosure is to provide compositions and methods for modulating TGFβ superfamily signaling. [Means for solving the problem]

[0006] In part, this disclosure provides TβRII polypeptides and the use of such TβRII polypeptides as selective antagonists to GDF15, TGFβ1, or TGFβ3. Polypeptides comprising some or all of the TβRII extracellular domain (ECD), with or without further mutations, as described herein, bind to and / or inhibit GDF15, TGFβ1, or TGFβ3 with varying affinity. Thus, in certain embodiments, this disclosure provides TβRII polypeptides for use in selectively inhibiting TGFβ superfamily-related disorders.

[0007] In certain embodiments, the Disclosure provides a polypeptide comprising mutations and / or truncations in the extracellular domain of TβRII. In certain embodiments, the Disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence derived from the extracellular domain of TβRII and a heterologous amino acid sequence, wherein the first amino acid sequence comprises, or comprises, an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or at least 99% identical to, a) a sequence beginning at any position 23–35 of SEQ ID NO: 5 and ending at any position 153–159 of SEQ ID NO: 5, or b) a sequence beginning at any position 23–60 of SEQ ID NO: 6 and ending at any position 178–184 of SEQ ID NO: 6.

[0008] In certain embodiments, the Disclosure provides a polypeptide comprising the wild-type or modified and / or truncated extracellular domain of TβRII fused to at least a portion of the Fc domain of human IgG2. Thus, in certain embodiments, the Disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence derived from the extracellular domain of TβRII and a heterologous amino acid sequence, wherein the first amino acid sequence comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, and at least 98% of a) a sequence beginning at any position 23–35 of SEQ ID NO: 5 and ending at any position 153–159 of SEQ ID NO: 5, or b) a sequence beginning at any position 23–60 of SEQ ID NO: 6 and ending at any position 178–184 of SEQ ID NO: 6. , providing a TβRII fusion polypeptide having an amino acid sequence that is at least 99% identical or identical to, or consisting of, such amino acid sequence, wherein the polypeptide comprises at least the constant domain of human IgG2 and optionally comprises a second polypeptide sequence that may contain or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, SEQ ID NO: 19, and a linker optionally positioned between the first polypeptide and the second polypeptide. An example of this is provided as SEQ ID NO: 50, which is encoded by the nucleic acid sequence of SEQ ID NO: 51. In certain embodiments, the Disclosure provides a polypeptide having an amino acid sequence that contains 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: 50. In certain embodiments, the Disclosure provides polypeptides comprising or encoded by nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 51.

[0009] In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 23 of SEQ ID NO: 5 and ends at position 159 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 29 of SEQ ID NO: 5 and ends at position 159 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 35 of SEQ ID NO: 5 and ends at position 159 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 23 of SEQ ID NO: 5 and ends at position 153 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 29 of SEQ ID NO: 5 and ends at position 153 of SEQ ID NO: 5. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 35 of SEQ ID NO: 5 and ends at position 153 of SEQ ID NO: 5.

[0010] In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 23 of SEQ ID NO: 6 and ends at position 184 of SEQ ID NO: 6. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 29 of SEQ ID NO: 6 and ends at position 184 of SEQ ID NO: 6. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 23 of SEQ ID NO: 6 and ends at position 178 of SEQ ID NO: 6. In some embodiments, the first amino acid sequence includes or consists of a sequence that begins at position 29 of SEQ ID NO: 6 and ends at position 178 of SEQ ID NO: 6.

[0011] In some embodiments, this first amino acid sequence includes or consists of a sequence having D at the position corresponding to position 36 of SEQ ID NO: 47 and / or K at the position corresponding to position 76 of SEQ ID NO: 47.

[0012] In certain embodiments, the Disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence or an active fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the sequence of SEQ ID NO: 7 or SEQ ID NO: 13, and a second heterologous portion, wherein the first amino acid sequence has D at the position corresponding to position 36 of SEQ ID NO: 47 and / or K at the position corresponding to position 76 of SEQ ID NO: 47.

[0013] In some embodiments, this first amino acid sequence includes an N-terminal truncation of 1 to 12 amino acids corresponding to amino acids 1 to 12 of SEQ ID NO: 7 or 1 to 37 amino acids corresponding to amino acids 1 to 37 of SEQ ID NO: 13. In some embodiments, this first amino acid sequence includes an N-terminal truncation of 6 amino acids corresponding to amino acids 1 to 6 of SEQ ID NO: 7 or SEQ ID NO: 13. In some embodiments, this first amino acid sequence includes an N-terminal truncation of 12 amino acids corresponding to amino acids 1 to 12 of SEQ ID NO: 7 or 37 amino acids corresponding to amino acids 1 to 37 of SEQ ID NO: 13. In some embodiments, this first amino acid sequence includes a C-terminal truncation of 1 to 6 amino acids corresponding to amino acids 137 to 132 of SEQ ID NO: 7 or amino acids 162 to 157 of SEQ ID NO: 13. In some embodiments, this first amino acid sequence includes a C-terminal truncation of 6 amino acids corresponding to amino acids 132 to 137 of SEQ ID NO: 7 or amino acids 157 to 162 of SEQ ID NO: 13. In some embodiments, this first amino acid sequence includes an insertion corresponding to SEQ ID NO: 18 between the residues corresponding to positions 117 and 118 of SEQ ID NO: 47.

[0014] In some embodiments, this heterologous portion comprises one or more polypeptide moieties that enhance one or more of the following: in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, protein complex formation, and / or purification. In some embodiments, this heterologous portion comprises an immunoglobulin Fc domain and a polypeptide moiety selected from serum albumin. In further embodiments, this immunoglobulin Fc domain is linked to this TβRII polypeptide by a linker.

[0015] In some embodiments, the polypeptide comprises one or more modified amino acid residues selected from glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, and amino acids conjugated to an organic derivatizer. In some embodiments, the polypeptide is glycosylated.

[0016] In certain embodiments, the Disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. In certain embodiments, the Disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 96% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. In certain embodiments, the Disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. In certain embodiments, the disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. In certain embodiments, the disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. In certain embodiments, the disclosure provides a TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion.

[0017] In certain embodiments, the Disclosure provides polypeptides comprising or consisting of amino acid sequences or portions thereof that are at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acid sequences selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43, with the leader sequences removed, for example, polypeptides comprising or consisting of amino acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acid sequences selected from SEQ ID NOs. 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. In certain embodiments, the Disclosure provides a polypeptide comprising or consisting of an amino acid sequence or portion thereof that is at least 96% identical to an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43, from which the leader sequence has been removed, for example, a polypeptide comprising or consisting of an amino acid sequence that is at least 96% identical to an amino acid sequence selected from SEQ ID NOs. 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. In certain embodiments, the Disclosure provides a polypeptide comprising or consisting of an amino acid sequence or portion thereof that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43, from which the leader sequence has been removed, for example, a polypeptide comprising or consisting of an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs. 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.In certain embodiments, the Disclosure provides a polypeptide comprising or consisting of an amino acid sequence or portion thereof that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43, from which the leader sequence has been removed, for example, a polypeptide comprising or consisting of an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs. 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. In certain embodiments, the Disclosure provides polypeptides comprising or consisting of amino acid sequences or portions thereof that are at least 99% identical to amino acid sequences selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43, with the leader sequences removed, for example, polypeptides comprising or consisting of amino acid sequences that are at least 99% identical to amino acid sequences selected from SEQ ID NOs. 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. In certain embodiments, the Disclosure provides polypeptides comprising or consisting of amino acid sequences or portions thereof that are selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43, with the leader sequences removed, for example, polypeptides comprising or consisting of amino acid sequences selected from SEQ ID NOs. 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62.

[0018] In certain embodiments, the Disclosure provides a TβRII polypeptide comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to a complement of a nucleotide sequence selected from SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44.

[0019] In each of the above, a TβRII polypeptide that does not contain full-length TβRII ECD may be selected. The TβRII polypeptide may be used as a monomeric protein or in a dimerized form. The TβRII polypeptide may also be fused to a second polypeptide moiety to provide improved properties, such as an increased half-life or greater ease of production or purification. The fusion may be direct, or a linker may be inserted between the TβRII polypeptide and any other moiety. The linker may be structured or unstructured, may consist of 1, 2, 3, 4, 5, 10, 15, 20, 30, 50 or more amino acids, and may optionally contain relatively little secondary structure.

[0020] In some embodiments, the TβRII polypeptide of this disclosure is 1 × 10 -8 Equilibrium dissociation constant (K) less than M D It binds to human GDF15.

[0021] In some embodiments, the TβRII polypeptide of this disclosure has a glycosylation pattern characteristic of the expression of this polypeptide in CHO cells.

[0022] In some embodiments, the Disclosure provides a homodimer comprising two TβRII polypeptides of the Disclosure.

[0023] In some embodiments, the Disclosure provides an isolated polynucleotide comprising the coding sequence of the TβRII polypeptide of the Disclosure. In some embodiments, the Disclosure provides a recombinant polynucleotide comprising a promoter sequence operably ligated to this isolated polynucleotide. In some embodiments, the Disclosure provides cells transformed with the isolated polynucleotide or recombinant polynucleotide of the Disclosure. In some embodiments, these cells are mammalian cells. In some embodiments, these cells are CHO cells or human cells. In some embodiments, these cells are HEK-293 cells.

[0024] In certain embodiments, the Disclosure provides pharmaceutical preparations comprising the TβRII polypeptide or homodimer of the Disclosure and pharmaceutically acceptable excipients.

[0025] In certain embodiments, the Disclosure provides a method for modulating a cellular response to a TGFβ superfamily member, comprising the step of exposing the cell to a TβRII polypeptide or homodimer of the Disclosure.

[0026] In certain embodiments, the Disclosure provides a method for treating a disease or condition associated with a TGFβ superfamily member in a patient in need thereof, comprising the step of administering an effective amount of the TβRII polypeptide or homodimer of the Disclosure to the patient. In some embodiments, the TGFβ superfamily member is TGFβ1, TGFβ3, or GDF15.

[0027] In some embodiments, this disease or condition is cancer. In some embodiments, this cancer is selected from gastric cancer, intestinal cancer, skin cancer, breast cancer, melanoma, bone cancer, and thyroid cancer.

[0028] In some embodiments, this disease or condition is a fibrous or sclerosing disease or disorder. In some embodiments, this fibrous or sclerosing disease or disorder is selected from scleroderma, atherosclerosis, hepatic fibrosis, diffuse systemic sclerosis, glomerulonephritis, nerve scarring, cutaneous scarring, radiation-induced fibrosis, hepatic fibrosis, and myelofibrosis.

[0029] In some embodiments, this disease or condition is a heart disease.

[0030] In some embodiments, this disease or condition is selected from hereditary hemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, pre-eclampsia, atherosclerosis, restenosis, and hypertrophic cardiomyopathy / congestive heart failure.

[0031] In certain embodiments, the disclosure provides an antibody or an antigen-binding fragment thereof that binds to GDF15 and blocks the interaction between GDF15 and TβRII.

[0032] In certain embodiments, the Disclosure provides a GDF15 polypeptide or a fragment thereof that binds to TβRII, comprising the amino acid sequence of SEQ ID NO: 1, wherein the GDF15 polypeptide is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure with respect to protein impurities.

[0033] In certain embodiments, the Disclosure provides a GDF15 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof conjugated to the TβRII polypeptide of the Disclosure, wherein the GDF15 polypeptide is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure with respect to protein impurities.

[0034] In some embodiments, this GDF15 polypeptide is 10 -8 Equilibrium dissociation constants (K) less than or equal to M D ) binds to TβRII. In some embodiments, this GDF15 polypeptide is 10 -8 Equilibrium dissociation constants (K) less than or equal to M D ) binds to the TβRII polypeptide of this disclosure.

[0035] In some embodiments, this GDF15 polypeptide is produced by expression in CHO cells.

[0036] In certain embodiments, the Disclosure provides a method for concentrating or purifying GDF15, comprising the step of contacting a sample containing GDF15 with the TβRII polypeptide of the Disclosure. [Brief explanation of the drawing]

[0037] [Figure 1] Figure 1 shows the amino acid sequence of the native precursor of human GDF15 (NCBI reference sequence: NP_004855.2). The solid underline indicates mature GDF15 (residues 197-308) whose N-terminus was determined by sequencing. The dotted underline indicates the leader (residues 1-29).

[0038] [Figure 2] Figure 2 shows the nucleotide sequence encoding the native precursor of human GDF15. The solid underline indicates the sequence encoding mature GDF15 (nucleotides 589-924), and the dotted underline indicates the sequence encoding the leader (nucleotides 1-87). The silent mutation (G456A) used to disrupt the SfoI site in NM_004864.2 is double-underlined.

[0039] [Figure 3] Figure 3 shows the amino acid sequence (NP_035949.2) of the native precursor of mouse GDF15. The solid underline indicates the mature GDF15 (residues 192-303) whose N-terminus was determined by sequencing. The dotted underline indicates the leader (residues 1-30).

[0040] [Figure 4] Figure 4 shows the nucleotide sequence encoding the native precursor of mouse GDF15 (derived from NM_011819.2). The solid underline indicates the sequence encoding mature GDF15 (nucleotides 574-909), and the dotted underline indicates the sequence encoding the leader (nucleotides 1-90).

[0041] [Figure 5]Figure 5 shows the amino acid sequence (NP_003233.4) of the native precursor of the B (short) isoform of the human TGFβ receptor type II (hTβRII). The solid underline indicates the mature extracellular domain (ECD) (residues 23-159), and the double underline indicates the valine that is replaced in the A (long) isoform. The dotted underline indicates the leader (residues 1-22).

[0042] [Figure 6] Figure 6 shows the amino acid sequence (NP_001020018.1) of the native precursor of the A(long) isoform of human TβRII. The solid underline indicates the mature ECD (residues 23-184), and the double underline indicates the isoleucine substitution that resulted from splicing. The dotted underline indicates the leader (residues 1-22).

[0043] [Figure 7-1] Figure 7 shows the N-terminal alignment of hTβRII short truncations and their hTβRII long counterparts. The 25 amino acid insertions present in the hTβRII long truncations are underlined. Note that during the splicing process, the valine adjacent to the insertion site in the short isoform is replaced by isoleucine in the long isoform. Sequences enclosed in squares indicate the leader. [Figure 7-2] Figure 7 shows the N-terminal alignment of hTβRII short truncations and their hTβRII long counterparts. The 25 amino acid insertions present in the hTβRII long truncations are underlined. Note that during the splicing process, the valine adjacent to the insertion site in the short isoform is replaced by isoleucine in the long isoform. Sequences enclosed in squares indicate the leader. [Modes for carrying out the invention]

[0044] 1. Overview The proteins described herein are in human form unless otherwise specified. The NCBI references for these proteins are as follows: human TβRII isoform A (hTβRII 長 ), NP_001020018.1; Human TβRII isoform B (hTβRII 短 ), NP_003233.4; Human GDF15, NP_004855.2; Mouse GDF15, NP_035949.2. The sequences of native TβRII and GDF15 proteins from humans and mice are shown in Figures 1–6.

[0045] The TGFβ superfamily comprises various growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a wide variety of cell types in both vertebrates and nonvertebrates. Members of this superfamily play crucial roles during embryonic development in pattern formation and tissue specificity, and can influence various differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. Manipulating the activity of members of the TGFβ family can often induce significant physiological changes in organisms. For example, the Piedmontese and Belgian Blue cattle breeds possess loss-of-function mutations in the GDF8 (also known as myostatin) gene, which result in a significant increase in muscle mass. Grobet et al., Nat Genet. 1997, Vol. 17 (No. 1): pp. 71-74. Similarly, in humans, the inactive allele of GDF8 is associated with increased muscle mass and, according to reports, exceptional strength. Schuelke et al., N Engl J Med 2004, Vol. 350: pp. 2682-2688.

[0046] TGFβ signaling is mediated by heteromeric complexes of type I (e.g., TβRI) and type II (e.g., TβRII) serine / threonine kinase receptors, which phosphorylate and activate downstream SMAD proteins upon ligand stimulation (Massague, 2000, Nat. Rev. Mol. Cell Biol. Vol. 1: pp. 169-178). These type I and type II receptors are transmembrane proteins consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine / threonine specificity. The type I receptor is crucial for signaling; the type II receptor is required for ligand binding and for the expression of the type I receptor. The type I and type II receptors form a stable complex after ligand binding, resulting in phosphorylation of the type I receptor by the type II receptor. TGFβ has three mammalian isoforms, TGFβ1, TGFβ2, and TGFβ3, each with distinct functions in vivo. The binding of TGFβ to TβRII is a crucial step in initiating the activation of the TGFβ signaling pathway, leading to the phosphorylation of SMAD2 and the translocation of the activated SMAD2 / SMAD4 complex to the nucleus, thereby modulating gene expression.

[0047] Growth factor 15 (GDF15) is a member of the TGFβ family. Like other ligands in the TGFβ superfamily that contain a characteristic cysteine ​​knot motif, mature GDF15 is synthesized with a larger prodomain (Harrison et al., Growth Factors vol. 29: p. 174, 2011; Shi et al., Nature vol. 474: p. 343, 2011) that is removed by cleavage by a furin-like protease at the canonical RXXR site to produce the mature dimer GDF15. GDF15 has been described in the literature as macrophage inhibitory cytokine-1 (MIC-1), placental bone morphogenesis protein (PLAB), placental transforming growth factor beta (PTGFβ), prostate-derived factor (PDF), and nonsteroidal anti-inflammatory drug activator-1 (NAG-1), reflecting the different functions implied for this protein. GDF15 has been associated with several physiological and pathological conditions. For example, GDF15 is highly expressed in the placenta and is necessary for maintaining pregnancy. GDF15 concentrations are also significantly increased in the serum of patients with prostate, colorectal, or pancreatic cancer, as well as gliomas. It has not been biochemically demonstrated that GDF15 directly binds to or interacts with any receptor. This disclosure relates in part to the discovery that the TGFβ type II receptor TβRII binds to GDF15 with high affinity and is a functional receptor for GDF15. It is demonstrated herein that TβRII fusion polypeptides, and other polypeptides containing the ligand-binding moiety of TβRII, inhibit GDF15-inducible gene activation. Potent inhibition of GDF15 signaling provides evidence that TβRII is a functional type II receptor for GDF15 and opens new avenues for therapeutic interventions in this signaling pathway. Thus, in part, this disclosure identifies a physiologically high-affinity receptor for GDF15 polypeptides.

[0048] Surprisingly, the soluble TβRII polypeptide exhibits highly specific, high-affinity binding to GDF15, as is shown herein. TβRII is a known type II receptor for TGFβ, binding to TGFβ1 and TGFβ3 with high affinity. Human TβRII occurs naturally as at least two isoforms—A (long) and B (short)—produced by alternative splicing in the extracellular domain (ECD) (Figures 6 and 5, and SEQ ID NOs: 6 and 5). This long isoform has a 25-amino acid insertion, and the splicing process replaces the valine adjacent to the insertion site in the short isoform with isoleucine in the long isoform. The soluble receptor extradomain may function as a scavenger or ligand trap to inhibit ligand-receptor interactions. Ligand traps, such as soluble TβRII-Fc fusion proteins incorporating the native TβRII extracellular domain (external domain), function as general inhibitors against TβRII ligands, including TGFβ1, TGFβ3, and GDF15, based on the findings disclosed herein. In some therapeutic settings, this broader spectrum of ligand binding and signal inhibition may be advantageous, while in others, more selective molecules may be preferable. It is highly desirable for ligand traps, such as TβRII extracellular domain polypeptides, to exhibit a selective ligand binding profile. This disclosure relates to the surprising discovery that polypeptides containing truncated portions of the extracellular domain of TβRII and / or mutations within the extracellular domain have a differential inhibitory effect on cellular signaling mediated by GDF15, TGFβ1, or TGFβ3. In part, this disclosure provides ligand traps generated by a series of mutations and / or truncations in the extracellular domain of TβRII that exhibit a variable ligand binding profile distinct from that of the native TβRII extracellular domain. The variant TβRII polypeptides disclosed herein offer advantageous properties compared to native full-length extracellular domains and can be used to selectively inhibit pathways mediated by different TβRII ligands in vivo.

[0049] Accordingly, in certain embodiments, the present disclosure provides a TβRII polypeptide as an antagonist of GDF15, TGFβ1, or TGFβ3 for use in addressing various GDF15, TGFβ1, or TGFβ3-related disorders. While we do not wish to be bound to any particular mechanism of action, such polypeptides are expected to act by binding to GDF15, TGFβ1, or TGFβ3 and inhibiting the ability of these ligands to form a ternary signaling complex.

[0050] The terms used herein generally have their common meanings in the art within the context of the present invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide further guidance to practitioners in describing the compositions and methods of the present invention and how they are prepared and used. The scope or meaning of any use of a term is evident from the specific context in which it is used.

[0051] 2. TβRII polypeptide The naturally occurring TβRII protein is a transmembrane protein having an extracellular portion located outside the cell and an intracellular portion located inside the cell. Aspects of this disclosure encompass variant TβRII polypeptides containing mutations within the extracellular domain of TβRII and / or truncated portions of the extracellular domain. As described above, human TβRII occurs naturally as at least two isoforms—A (long) and B (short)—produced by alternative splicing in the extracellular domain (ECD) (Figures 6 and 5 and SEQ ID NOs: 6 and 5). SEQ ID NO: 7, corresponding to residues 23-159 of SEQ ID NO: 5, shows the native full-length extracellular domain of the short isoform of TβRII. SEQ ID NO: 13, corresponding to residues 23-184 of SEQ ID NO: 6, shows the native full-length extracellular domain of the long isoform of TβRII. Unless otherwise noted, the amino acid position numbering for variants based on the short and long isoforms of TβRII refers to the corresponding positions in the native precursors, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0052] In certain embodiments, the Disclosure provides variant TβRII polypeptides. The TβRII polypeptides of the Disclosure may bind to, but are not limited to, TGFβ15, TGFβ1, or TGFβ3, and inhibit their function. The TβRII polypeptides may include polypeptides having an amino acid sequence that is at least 80% identical to the truncated ECD domain of a naturally occurring TβRII polypeptide, whose C-terminus is located at any of amino acids 153-159 of SEQ ID NO: 5, and optionally, an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or containing such an amino acid sequence. TβRII polypeptides may include polypeptides consisting of or containing amino acid sequences that are at least 80% identical to the truncated ECD domain of a naturally occurring TβRII polypeptide, whose C-terminus is located at any of amino acids 178–184 of SEQ ID NO: 6, and optionally, amino acid sequences that are at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Optionally, TβRII polypeptides may not contain more than five consecutive amino acids, or more than 10, 20, 30, 40, 50, 52, 60, 70, 80, 90, 100, 150, or 200 consecutive amino acids, derived from the sequence of amino acids 160–567 of SEQ ID NO: 5 or the sequence of amino acids 185–592 of SEQ ID NO: 6. Unprocessed TβRII polypeptides may contain or exclude any signal sequence, as well as any N-terminal sequence relative to the signal sequence. As detailed herein, the N-terminus of a mature (processed) TβRII polypeptide may be located at either amino acids 23-35 of SEQ ID NO: 5 or amino acids 23-60 of SEQ ID NO: 6.Examples of mature TβRII polypeptides include, but are not limited to, amino acids 23-159 of SEQ ID NO: 5 (shown in SEQ ID NO: 7), amino acids 29-159 of SEQ ID NO: 5 (shown in SEQ ID NO: 9), amino acids 35-159 of SEQ ID NO: 5 (shown in SEQ ID NO: 10), amino acids 23-153 of SEQ ID NO: 5 (shown in SEQ ID NO: 11), amino acids 29-153 of SEQ ID NO: 5 (shown in SEQ ID NO: 48), amino acids 35-153 of SEQ ID NO: 5 (shown in SEQ ID NO: 47), amino acids 23-184 of SEQ ID NO: 6 (shown in SEQ ID NO: 13), amino acids 29-184 of SEQ ID NO: 6 (shown in SEQ ID NO: 15), amino acids 60-184 of SEQ ID NO: 6 (shown in SEQ ID NO: 10), amino acids 23-178 of SEQ ID NO: 6 (shown in SEQ ID NO: 16), amino acids 29-178 of SEQ ID NO: 6 (shown in SEQ ID NO: 49), and amino acids 60-178 of SEQ ID NO: 6 (shown in SEQ ID NO: 47). Similarly, TβRII polypeptides may include polypeptides encoded by nucleic acids that hybridize to their complements under stringent hybridization conditions (generally known in the art, such conditions may include, for example, hybridization overnight at 65°C in 50% v / v formamide, 5×SSC, 2% w / v blocking agent, 0.1% N-lauroyl sarcosine and 0.3% SDS, and washing in 5×SSC at approximately 65°C). It will be understood by those skilled in the art that the corresponding variants based on the long isoform of TβRII include the nucleotide sequence encoding the insertion, along with a conserved Val-Ile substitution at a position adjacent to the C-terminus for a 25-amino acid insertion.These TβRII polypeptides may include, as appropriate, both short and long isoforms, their variants (e.g., variants containing 2, 3, 4, 5, 10, 15, 20, 25, 30 or 35 or fewer amino acid substitutions in the sequences corresponding to amino acids 23-159 of SEQ ID NO: 5 or amino acids 23-184 of SEQ ID NO: 6), their fragments, and isolated extracellular components of TβRII polypeptides, including fusion proteins containing any of the above, but in each case, preferably, any of the above TβRII polypeptides retain substantial affinity for at least one of GDF15, TGFβ1, or TGFβ3. Generally, TβRII polypeptides are designed to be soluble in aqueous solutions at biologically appropriate temperatures, pH levels, and molar osmotic concentrations.

[0053] In some embodiments, the variant TβRII polypeptides of this disclosure include one or more mutations in the extracellular domain that confer an altered ligand-binding profile. The TβRII polypeptide may include one, two, five or more changes in its amino acid sequence compared to the corresponding portion of a naturally occurring TβRII polypeptide. In some embodiments, this mutation results in a substitution, insertion, or deletion at the position corresponding to position 70 of SEQ ID NO: 5. In some embodiments, this mutation results in a substitution, insertion, or deletion at the position corresponding to position 110 of SEQ ID NO: 5. Examples include, but are not limited to, N-to-D substitutions or D-to-K substitutions at the positions corresponding to positions 70 and 110, respectively, of SEQ ID NO: 5. Examples of such variant TβRII polypeptides include, but are not limited to, the sequences shown in SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 12, and SEQ ID NO: 17. The TβRII polypeptide may contain polypeptides or portions thereof encoded by nucleic acids that hybridize to their complements under stringent hybridization conditions, such as nucleotides 73-483 of SEQ ID NO: 26, nucleotides 73-465 of SEQ ID NO: 42, or their silent variants.

[0054] In some embodiments, the variant TβRII polypeptide of the present disclosure further includes a 36-amino acid insertion (SEQ ID NO: 18) between pairs of glutamic acid residues located near the C-terminus of human TβRII ECD (positions 151 and 152 of SEQ ID NO: 5, or positions 176 and 177 of SEQ ID NO: 6), as naturally occurring in human TβRII isoform C (Konrad et al., BMC Genomics 8: p. 318, 2007).

[0055] This disclosure further demonstrates that the TβRII polypeptide can be modified to selectively antagonistize TβRII ligands. The data presented herein show that Fc fusion proteins, including shorter N-terminus and C-terminus truncated variants of the TβRII polypeptide, exhibit differential inhibitory effects on GDF15, TGFβ1, and TGFβ3-mediated cellular signaling. Specifically, N-terminus truncated variants, beginning at amino acid 29 or 35 of SEQ ID NO: 5 and possessing a 6-amino acid or 12-amino acid N-terminal truncation of the extracellular domain, respectively, were found to inhibit GDF15 most potently, TGFβ3 least strongly, and TGFβ1 to an intermediate degree compared to the full-length extracellular domain of the short isoform of TβRII. C-terminus truncated variants, ending at amino acid 153 of SEQ ID NO: 5 and possessing a 6-amino acid C-terminal truncation of the extracellular domain, have virtually no effect on ligand binding and can therefore be used interchangeably with the full-length version. A substitution of N to D at the position corresponding to position 70 of SEQ ID NO: 5 was found to potently inhibit TGFβ3, have an intermediate effect on GDF15, and have a negligible effect on TGFβ1. The N70 residue indicates a potential glycosylation site. Furthermore, an Fc fusion protein containing a substitution of D to K at the position corresponding to position 110 of SEQ ID NO: 5 was found to inhibit GDF15 most potently, TGFβ1 least strongly, and TGFβ3 to an intermediate degree compared to the full-length extracellular domain of the short isoform of TβRII. The region around position 110 has not been associated with selectivity for known TβRII ligands TGFβ1, TGFβ2, and TGFβ3. Therefore, unexpectedly, TβRII polypeptides containing mutations in the ECD, such as N70D and D110K (the residue numbering corresponds to the numbering in SEQ ID NO: 5), but not limited to these, as well as those beginning between amino acids 29 and 35 and ending between amino acids 153 and 159, are all expected to be active and exhibit broadly different inhibitory potentials against different ligands.Depending on the clinical or experimental setting, it may be desirable to use one of these truncated variant forms.

[0056] In certain embodiments, the TβRII polypeptide binds to GDF15, and this TβRII polypeptide shows no substantial binding to TGFβ1 or TGFβ3. In certain embodiments, the TβRII polypeptide binds to TGFβ1, and this TβRII polypeptide shows no substantial binding to GDF15 or TGFβ3. In certain embodiments, the TβRII polypeptide binds to TGFβ3, and this TβRII polypeptide shows no substantial binding to GDF15 or TGFβ1. Binding can be evaluated using purified proteins in solution or in a surface plasmon resonance system such as the Biacore® system.

[0057] In certain embodiments, the TβRII polypeptide inhibits GDF15 cell signaling, and this TβRII polypeptide has an intermediate or limited inhibitory effect on TGFβ1 or TGFβ3. In certain embodiments, the TβRII polypeptide inhibits TGFβ1 cell signaling, and this TβRII polypeptide has an intermediate or limited inhibitory effect on GDF15 or TGFβ3. In certain embodiments, the TβRII polypeptide inhibits TGFβ3 cell signaling, and this TβRII polypeptide has an intermediate or limited inhibitory effect on GDF15 or TGFβ1. The inhibitory effect on cell signaling can be assayed by methods known in the art.

[0058] In summary, the active moiety of the TβRII polypeptide may include variants of the amino acid sequences 23-153, 23-154, 23-155, 23-156, 23-157, or 23-158 of SEQ ID NO: 5, and any of the amino acids 24-35 of SEQ ID NO: 5. Similarly, the active moiety of the TβRII polypeptide may include variants of the amino acid sequences 23-178, 23-179, 23-180, 23-181, 23-182, or 23-183 of SEQ ID NO: 6, and any of the amino acids 24-60 of SEQ ID NO: 6. Exemplary TβRII polypeptides include amino acid sequences 29-159, 35-159, 23-153, 29-153, and 35-153 of SEQ ID NO: 5, or amino acid sequences 29-184, 60-184, 23-178, 29-178, and 60-178 of SEQ ID NO: 6. Variants within these ranges, in particular those having at least 80%, 85%, 90%, 95%, or 99% identity with the corresponding portion of SEQ ID NO: 5 or SEQ ID NO: 6, are also intended. TβRII polypeptides that do not contain sequences consisting of amino acids 160-567 of SEQ ID NO: 5 or amino acids 185-592 of SEQ ID NO: 6 may be selected.

[0059] As described above, this disclosure provides TβRII polypeptides that share a specified degree of sequence identity or similarity with naturally occurring TβRII polypeptides. To determine the percentage identity of two amino acid sequences, these sequences are aligned for the purpose of optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for the purpose of comparison). Then, amino acid residues at the corresponding amino acid positions are compared. If a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then these molecules are identical at that position (as used herein, amino acid "identity" is equivalent to amino acid "homology"). The percentage identity between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0060] The comparison of sequences between two sequences, as well as the determination of percent identity and similarity, can be achieved using mathematical algorithms (Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Develeux, J., M Stockton Press, New York, 1991).

[0061] In one embodiment, the percentage identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J Mol. Biol. (Vol. 48): pp. 444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com). In a specific embodiment, the following parameters are used in the GAP program: either a Blosum 62 matrix or a PAM250 matrix, as well as gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percentage identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J. et al., Nucleic Acids Res. Vol. 12 (No. 1): p. 387 (1984)) (available at http: / / www.gcg.com). Exemplary parameters include using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Unless otherwise noted, the percentage identity between two amino acid sequences is determined using the GAP program with the Blosum 62 matrix, 10 gap weights, and 3 length weights. If such an algorithm cannot calculate the desired percentage identity, an appropriate alternative method disclosed herein should be selected.

[0062] In another embodiment, the percentage identity between two amino acid sequences is determined using the PAM120 weight residue table, a 12-gap length penalty, and a 4-gap penalty, as incorporated in the ALIGN program (version 2.0) by E. Myers and W. It is determined using Miller's algorithm (CABIOS, Vol. 4: pp. 11-17 (1989)).

[0063] Another embodiment for determining the best overall alignment between two amino acid sequences can be determined using a FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., Vol. 6: pp. 237-245 (1990)). In sequence alignment, both the query sequence and the target sequence are amino acid sequences. The results of the comprehensive sequence alignment are expressed in units of percentage identity. In one embodiment, amino acid sequence identity is performed using a FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., Vol. 6: pp. 237-245 (1990)). In a specific embodiment, the parameters used to calculate the percentage identity and similarity of the amino acid alignment include matrix=PAM 150, k-tuple=2, mismatch penalty=1, joining penalty=20, randomization group length=0, cutoff score=1, gap penalty=5, and gap size penalty=0.05.

[0064] TβRII polypeptides may further contain one of various leader sequences at their N-terminus. Such sequences enable the peptide to be expressed in eukaryotic systems and targeted to secretory pathways. See, for example, Ernst et al., U.S. Patent No. 5,082,783 (1992). Alternatively, native TβRII signal sequences may be used to induce extrusion from cells. Possible leader sequences include native leaders, tissue plasminogen activator (TPA), and honeybee melittin (SEQ ID NOs. 22-24, respectively). Examples of TβRII-Fc fusion proteins incorporating TPA leader sequences include SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43. The processing of the signal peptide can vary depending on several variables, including the selected leader sequence, the cell type used, and the culture conditions. Therefore, the actual N-terminal start site for mature TβRII polypeptides can be shifted by 1, 2, 3, 4, or 5 amino acids in either the N-terminal or C-terminal direction. Examples of TβRII-Fc fusion proteins include SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62 shown herein, with the TβRII polypeptide portion underlined (see Examples). It will be understood by those skilled in the art that the corresponding variants based on the long isoform of TβRII include this insertion, along with a conservative Val-Ile substitution at an adjacent position at the C-terminus for the 25-amino acid insertion.

[0065] In certain embodiments, this disclosure intends to describe specific mutations of a TβRII polypeptide that alter the glycosylation of the polypeptide. Such mutations may be selected to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites. Asparagine-linked glycosylation recognition sites generally include a tripeptide sequence, asparagine-X-threonine (or asparagine-X-serine) (wherein "X" is any amino acid), that is specifically recognized by a suitable cellular glycosylation enzyme. This modification may also be made by adding or substituting one or more serine or threonine residues to the sequence of the wild-type TβRII polypeptide (for O-linked glycosylation sites). Substitutions or deletions of various amino acids at one or both of the first or third amino acid positions of the glycosylation recognition site (and / or amino acid deletions at the second position) result in deglycosylation in the modified tripeptide sequence. Another means of increasing the number of carbohydrate moieties on a TβRII polypeptide is by chemical or enzymatic coupling of glycosides to the TβRII polypeptide. Depending on the coupling method used, the sugar(s) may be coupled to (a) arginine and histidine; (b) a free carboxyl group; (c) a free sulfhydryl group such as that of cysteine; (d) a free hydroxyl group such as that of serine, threonine, or hydroxyproline; (e) an aromatic residue such as that of phenylalanine, tyrosine, or tryptophan; or (f) an amide group of glutamine. These methods are described in WO87 / 05330, published on 11 September 1987, incorporated herein by reference, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259–306. Removal of one or more carbohydrate moieties present on a TβRII polypeptide can be achieved chemically and / or enzymatically. Chemical deglycosylation may involve, for example, exposure of the TβRII polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound.This process results in the cleavage of almost all or all sugars, with the removal of the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while keeping the amino acid sequence intact. Chemical deglycosylation is further described by Hakimuddin et al. (1987) Arch. Biochem. Biophys. vol. 259: p. 52 and Edge et al. (1981) Anal. Biochem. vol. 118: p. 131. Enzymatic cleavage of the carbohydrate moiety on the TβRII polypeptide can be achieved using various endoglycosidases and exoglycosidases, as described by Thotakura et al. (1987) Meth. Enzymol. vol. 138: p. 350. Since mammalian, yeast, insect, and plant cells can all introduce different glycosylation patterns that can be influenced by the amino acid sequence of the peptide, the sequence of the TβRII polypeptide may be adjusted depending on the type of expression system used, as needed. Generally, TβRII polypeptides for human use are expressed in mammalian cell lines that provide adequate glycosylation, such as HEK293 or CHO cell lines, but other mammalian expression cell lines, yeast cell lines with engineered glycosylation enzymes, and insect cells are also expected to be equally useful.

[0066] This disclosure further intends to describe a set of combinatorial variants of the TβRII polypeptide, as well as methods for generating truncation variants; a pool of combinatorial variants is particularly useful for identifying functional variant sequences. The purpose of screening such combinatorial libraries may be, for example, to generate TβRII polypeptide variants that can act as either agonists or antagonists, or that possess all of the novel activities together. Various screening assays are provided below, and such assays may be used to evaluate variants. For example, TβRII polypeptide variants may be screened for their ability to bind to a TβRII ligand, their ability to prevent the binding of a TβRII ligand to a TβRII polypeptide, or their ability to interfere with signaling induced by a TβRII ligand. The activity of the TβRII polypeptide or its variants may also be tested in cell-based assays or in vivo assays, in particular any of the assays disclosed in the Examples.

[0067] Combinatorially induced variants may be generated that have selective or generally increased potential compared to TβRII polypeptides containing the extracellular domain of naturally occurring TβRII polypeptides. Similarly, mutagenesis can produce variants with dramatically different serum half-lives from the corresponding wild-type TβRII polypeptide. For example, the modified protein may be made more stable or less stable to proteolytic degradation or other processes that result in the disruption of the native TβRII polypeptide or its elimination or inactivation by other means. Such variants, and the genes encoding them, may be used to alter TβRII polypeptide levels by modulating the half-life of the TβRII polypeptide. For example, a shorter half-life may result in more transient biological effects and allow for tighter control of recombinant TβRII polypeptide levels in a patient. In Fc fusion proteins, mutations may be created in the linker (if present) and / or the Fc portion to alter the protein's half-life.

[0068] A combinatorial library can be produced by a degenerate library of genes encoding a library of polypeptides, each containing at least a portion of the potential TβRII polypeptide sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into a gene sequence so that the degenerate set of nucleotide sequences of the potential TβRII polypeptide can be expressed as individual polypeptides or as a larger set of fusion proteins (for example, for phage display).

[0069] Numerous methods exist for generating libraries of potential TβRII polypeptide variants from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in automated DNA synthesizers, and the synthetic genes can then be ligated into appropriate vectors for expression. The synthesis of degenerate oligonucleotides is well-known in this field (see, for example, Narang, SA (1983) Tetrahedron Vol. 39: p. 3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, AG Walton (ed.), Amsterdam: Elsevier pp. 273-289; Itakura et al., (1984) Annu. Rev. Biochem. Vol. 53: p. 323; Itakura et al., (1984) Science Vol. 198: p. 1056; Ike et al., (1983) Nucleic Acid Res. Vol. 11: p. 477). Such techniques have been used in the evolution of targeting in other proteins (see, for example, Scott et al., Science Vol. 249 (1990): pp. 386-390; Roberts et al., PNAS USA Vol. 89 (1992): pp. 2429-2433; Devlin et al., Science Vol. 249 (1990): pp. 404-406; Cwirla et al., PNAS USA Vol. 87 (1990): pp. 6378-6382; and U.S. Patents No. 5,223,409, 5,198,346 and 5,096,815).

[0070] Alternatively, other forms of mutagenesis may be used to generate combinatorial libraries. For example, TβRII polypeptide variants can be screened using methods such as alanine scanning mutagenesis (Ruf et al., (1994) Biochemistry 33: pp. 1565-1572; Wang et al., (1994) J. Biol. Chem. Vol. 269: pp. 3095-3099; Balint et al., (1993) Gene Vol. 137: pp. 109-118; Grodberg et al., (1993) Eur. J. Biochem. Vol. 218: pp. 597-601; Nagashima et al., (1993) J. Biol. Chem. Vol. 268: pp. 2888-2892; Lowman et al., (1991) Biochemistry Vol. 30: pp. 10832-10838; and Cunningham et al., (1989) Science Vol. 244: pp. 1081-1085; linker scanning mutagenesis (Gustin et al., (1993) Virology Vol. 193: pp. 653-660; Brown et al., (1992) Mol. Cell It can be generated and isolated from libraries by: Biol. Vol. 12: pp. 2644-2652; McKnight et al., (1982) Science Vol. 232: p. 316; by saturated mutagenesis (Meyers et al., (1986) Science Vol. 232: p. 613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol Vol. 1: pp. 11-19); or by random mutagenesis including chemical mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol Vol. 7: pp. 32-34). Linker scanning mutagenesis in particular in a combinatorial setting is an alternative method for identifying the truncated (bioactive) form of TβRII polypeptide.

[0071] A wide range of techniques are known in the art for screening gene products of combinatorial libraries constructed by point mutations and truncations, and, as such, for screening cDNA libraries for gene products of specific characteristics. Such techniques are generally adaptable to the rapid screening of gene libraries produced by combinatorial mutagenesis of TβRII polypeptides. The most widely used techniques for screening large gene libraries typically involve the steps of cloning the gene library into a replicable expression vector, transforming suitable cells with the resulting library in the vector, and expressing the combinatorial gene under conditions where the detection of desired activity facilitates the relatively easy isolation of the vector encoding the gene for which the product was detected. Preferred assays include TβRII ligand binding assays and ligand-mediated cell signaling assays.

[0072] In certain embodiments, the TβRII polypeptides of this disclosure may further include post-translational modifications in addition to any modifications naturally present in the TβRII polypeptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, pegylation (polyethylene glycol), and acylation. As a result, modified TβRII polypeptides may contain non-amino acid elements, such as polyethylene glycol, lipids, monosaccharides or polysaccharides, and phosphates. The effect of such non-amino acid elements on the functionality of the TβRII polypeptide can be tested as described herein for other TβRII polypeptide variants. If the TβRII polypeptide is produced in cells by cleaving the nascent form of the TβRII polypeptide, post-translational processing may also be important for the correct folding and / or function of the protein. Different cell types (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK-293) possess specific cellular machinery and characteristic mechanisms for such post-translational activity and can be selected to ensure precise modification and processing of TβRII polypeptides.

[0073] In certain embodiments, functional variants or modified forms of TβRII polypeptides include fusion proteins having at least a portion of a TβRII polypeptide and one or more fusion domains. Well-known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose-binding protein (MBP), or human serum albumin. Fusion domains may be selected to confer desired properties. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. For affinity purification, suitable matrices for affinity chromatography, such as glutathione, amylase, and nickel or cobalt conjugate resins, are used. Many of these matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpress® system (Qiagen), which are useful with (HIS6) fusion partners. As another example, the fusion domain may be selected to facilitate the detection of the TβRII polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and “epitope tags,” which are usually short peptide sequences against which specific antibodies are available. Well-known epitope tags against which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, these fusion domains have protease cleavage sites for factor Xa or thrombin, etc., which allow a suitable protease to partially digest the fusion protein, thereby releasing the recombinant protein therefrom. The released protein can then be isolated from the fusion domain by subsequent chromatographic separation. In a particular preferred embodiment, the TβRII polypeptide is fused with a domain ("stabilizer" domain) that stabilizes the TβRII polypeptide in vivo."Stabilization" means increasing the serum half-life, whether due to reduced disruption, reduced renal clearance, or other pharmacokinetic effects. Fusion with the Fc portion of immunoglobulins is known to confer desired pharmacokinetic properties to a wide range of proteins. Similarly, fusion with human serum albumin can confer desired properties. Other types of fusion domains that can be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains.

[0074] As a specific example, this disclosure provides a fusion protein comprising a variant of a TβRII polypeptide fused to one of three Fc domain sequences (e.g., SEQ ID NOs: 19, 20, and 21). Optionally, this Fc domain has one or more mutations at residues such as Asp-265, Lys-322, and Asn-434 (numbered according to the corresponding full-length IgG). In certain cases, the mutant Fc domain having one or more of these mutations (e.g., the Asp-265 mutation) reduced its ability to bind to the Fcγ receptor compared to the wild-type Fc domain. In other cases, the mutant Fc domain having one or more of these mutations (e.g., the Asn-434 mutation) increased its ability to bind to MHC class I-associated Fc receptors (FcRNs) compared to the wild-type Fc domain.

[0075] It is understood that the different elements of a fusion protein can be arranged in any manner that matches the desired functionality. For example, the TβRII polypeptide may be positioned at the C-terminus relative to the heterologous domain, or the heterologous domain may be positioned at the C-terminus relative to the TβRII polypeptide. The TβRII polypeptide domain and the heterologous domain do not need to be adjacent in the fusion protein, and further domains or amino acid sequences may be included at the C-terminus or N-terminus of either domain, or between the domains.

[0076] As used herein, the term “immunoglobulin Fc domain” or simply “Fc” is understood to mean the carboxyl-terminal portion of the constant region of an immunoglobulin chain, preferably the constant region of an immunoglobulin heavy chain, or a portion thereof. For example, an immunoglobulin Fc region may include 1) a CH1 domain, a CH2 domain, and a CH3 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment, this immunoglobulin Fc region includes at least the immunoglobulin hinge regions CH2 and CH3 domains, and preferably lacks a CH1 domain.

[0077] In one embodiment, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclass 1, 2, 3, or 4). Other classes of immunoglobulins, IgA (Igα), IgD (Igδ), IgE (Igε), and IgM (Igμ), may be used. The selection of an appropriate immunoglobulin heavy chain constant region is discussed in detail in U.S. Patents No. 5,541,087 and No. 5,726,044. The selection of a specific immunoglobulin heavy chain constant region sequence from a specific immunoglobulin class and subclass to achieve a particular result is considered to be within the realm of the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably includes at least a portion of the hinge domain and, preferably, at least a portion of the CH3 domain of Fc gamma or a homologous domain in any of IgA, IgD, IgE, or IgM.

[0078] Furthermore, it is intended that amino acid substitutions or deletions within the constant region of the immunoglobulin heavy chain may be useful in carrying out the methods and compositions disclosed herein. One example is the introduction of amino acid substitutions in the CH2 region above to create an Fc variant with reduced affinity for the Fc receptor (Cole et al. (1997) J.Immunol. 159:3613).

[0079] In certain embodiments, the disclosure makes available isolated and / or purified forms of TβRII polypeptides isolated from other proteins and / or other TβRII polypeptide species, or substantially free from other proteins and / or other TβRII polypeptide species in other ways (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%). TβRII polypeptides are generally produced by expression from recombinant nucleic acids.

[0080] In certain embodiments, the Disclosure relates to a nucleic acid encoding a soluble TβRII polypeptide, which includes the coding sequence for the extracellular portion of the TβRII protein. In further embodiments, the Disclosure also relates to a host cell containing such nucleic acid. This host cell may be any prokaryotic or eukaryotic cell. For example, the polypeptide of the Disclosure may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. Accordingly, some embodiments of the Disclosure further relate to methods for producing TβRII polypeptides.

[0081] 3. Nucleic acid encoding TβRII polypeptide In certain embodiments, this disclosure provides isolated and / or recombinant nucleic acids encoding any of the TβRII polypeptides, including the fragments, functional variants, and fusion proteins disclosed herein. Sequence IDs 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 encode variants of the TβRII extracellular domain fused to an IgG2 Fc domain or an IgG1 Fc domain with its N-terminus truncated. The nucleic acids in question may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for producing TβRII polypeptides or as direct therapeutic agents (e.g., in antisense, RNAi, or gene therapy approaches).

[0082] In certain embodiments, it is further understood that the target nucleic acid encoding the TβRII polypeptide includes nucleic acids that are variants of SEQ ID NOs. 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions, e.g., allelic variants.

[0083] In certain embodiments, the Disclosure provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44. Those skilled in the art will understand that nucleic acid sequences complementary to SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44, as well as variants of SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44, are also within the scope of the Disclosure. In further embodiments, the nucleic acid sequences of the Disclosure may be isolated, recombinant, and / or fused with heterologous nucleotide sequences, or may be in a DNA library.

[0084] In other embodiments, the nucleic acids of the present disclosure also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequences shown in SEQ ID NOs. 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44, the complementary sequences of SEQ ID NOs. 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44, or fragments thereof. As discussed above, those skilled in the art will readily understand that the appropriate stringency conditions for promoting DNA hybridization can vary. For example, hybridization can be carried out in 6.0 × sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing with 2.0 × SSC at 50°C. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 × SSC at 50°C to a high stringency of about 0.2 × SSC at 50°C. Furthermore, the temperature in the washing step can be increased from low stringency conditions at room temperature or about 22°C to high stringency conditions at about 65°C. Both temperature and salt can be varied, or temperature or salt concentration can be kept constant even if other variables change. In some embodiments, the disclosure provides nucleic acids that hybridize under low stringency conditions of washing 6×SSC at room temperature and then 2×SSC at room temperature.

[0085] Isolated nucleic acids that differ from those shown in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44 due to degeneracy in the genetic code are also within the scope of this disclosure. For example, some amino acids are specified by more than one triplet. Codons that specify the same amino acid or synonym (e.g., CAU and CAC are synonyms for histidine) may result in “silent” mutations that do not affect the amino acid sequence of a protein. However, DNA sequence polymorphisms that result in changes in the amino acid sequence of a protein of interest are expected to exist among mammalian cells. Those skilled in the art will understand that these variations in one or more nucleotides of a nucleic acid encoding a particular protein (up to about 3-5% of nucleotides) may exist among individuals of a given species due to natural allelic variations. Any such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.

[0086] It is understood by those skilled in the art that the corresponding variant based on the long isoform of TβRII contains a nucleotide sequence encoding the 25-amino acid insertion, along with a conserved Val-Ile substitution at a position adjacent to the C-terminus. It is also understood that the corresponding variant based on either the long (A) or short (B) isoform of TβRII contains a variant nucleotide sequence containing a 108-nucleotide insertion encoding a 36-amino acid insertion (SEQ ID NO: 18) at the same position as described for the naturally occurring TβRII isoform C (see illustration).

[0087] In certain embodiments, the recombinant nucleic acids of this disclosure may be operably ligated to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences are generally appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and appropriate regulatory sequences are known in the art for various host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, reader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated by this disclosure. These promoters may be either naturally occurring promoters or hybrid promoters combining elements of more than one promoter. The expression construct may reside in the cell on an episome, as in a plasmid, or the expression construct may be inserted into a chromosome. In a preferred embodiment, the expression vector includes a selectable marker gene that allows for the selection of transformed host cells. The selectable marker gene is known in the art and varies depending on the host cell used.

[0088] In certain embodiments disclosed herein, the nucleic acid of interest is provided in an expression vector comprising a nucleotide sequence encoding a TβRII polypeptide, operably ligated to at least one regulatory sequence. The regulatory sequence is art-recognized and selected to direct the expression of the TβRII polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression regulatory elements. Exemplary regulatory sequences are described in Goddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of the broad range of expression regulatory sequences that, when operably ligated to them, control the expression of a DNA sequence may be used in these vectors to express the DNA sequence encoding the TβRII polypeptide. Such useful expression regulatory sequences include, for example, the early and late promoters of SV40, the tet promoter, the earliest promoters of adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the regulatory regions of fd coat proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases, such as Pho5, the promoter of the yeast α-conjugation factor, the polyhedron promoter of baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It should be understood that the design of expression vectors may depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. Furthermore, the copy number of the vector, its ability to control its copy number, and the expression of any other proteins encoded by this vector, such as antibiotic markers, should also be considered.

[0089] Recombinant nucleic acids included in this disclosure may be produced by ligating a cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vehicles for the production of recombinant TβRII polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as E. coli.

[0090] Some mammalian expression vectors contain both prokaryotic sequences to promote vector reproduction in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. Examples of mammalian expression vectors suitable for eukaryotic cell transfection include pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors. Some of these vectors are modified with bacterial plasmid-derived sequences, such as pBR322, to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) may be used for transient protein expression in eukaryotic cells. Examples of other viral (including retrovirus) expression systems can be found below in the description of gene therapy delivery systems. Various methods used in plasmid preparation and host organism transformation are well known in the field. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombination procedures, see Molecular Cloning: A Laboratory Manual, 3rd edition, edited by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant polypeptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., β-gal-containing pBlueBac III).

[0091] In certain embodiments, vectors such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wisc.) are designed for the production of the target TβRII polypeptide in CHO cells. In preferred embodiments, the vectors are designed for the production of the target TβRII polypeptide in HEK-293 cells. As is obvious, these target gene constructs can be used to induce the expression of the target TβRII polypeptide in cells cultured in culture for the production of proteins, including fusion proteins or variant proteins, for purification.

[0092] This disclosure also relates to host cells transfected with a recombinant gene containing one or more coding sequences of the target TβRII polypeptide (e.g., SEQ ID NOs. 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44). This host cell may be any prokaryotic or eukaryotic cell. For example, the TβRII polypeptides disclosed herein may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0093] Therefore, the present disclosure further relates to a method for producing a target TβRII polypeptide. For example, a host cell transfected with an expression vector encoding a TβRII polypeptide can be cultured under appropriate conditions to produce the expression of the TβRII polypeptide. This TβRII polypeptide can be secreted and isolated from a mixture of cells and medium containing the TβRII polypeptide. Alternatively, this TβRII polypeptide can be retained in the cytoplasm or in the membrane fraction, the cells can be harvested, lysed, and the protein can be isolated. A cell culture comprises a host cell and a medium. Media suitable for cell culture are well known in the art. These target TβRII polypeptides can be isolated from cell culture media, host cells or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification using an antibody specific for a particular epitope of the TβRII polypeptide, and affinity purification using an agent that binds to a domain fused to the TβRII polypeptide (e.g., a protein A column can be used to purify a TβRII-Fc fusion). In a preferred embodiment, this TβRII polypeptide is a fusion protein comprising a domain that facilitates its purification. As an example, purification can be achieved by a series of column chromatography steps, including, for example, three or more of the following in any order: protein A chromatography, Q Sepharose chromatography, phenyl Sepharose chromatography, size exclusion chromatography and cation exchange chromatography. Purification can be completed using virus filtration and buffer exchange.

[0094] In another embodiment, a fusion gene encoding a purification leader sequence, such as a poly(His) / enterokinase cleavage site sequence, at the N-terminus of the desired portion of the recombinant TβRII polypeptide, is Ni 2+Affinity chromatography using a metal resin may enable the purification of the expressed fusion protein. This purified leader sequence can then be subsequently removed by treatment with enterokinase to provide a purified TβRII polypeptide (see, e.g., Hochuli et al., J. Chromatography 411, 1987: p. 177; and Janknecht et al., PNAS USA 88: p. 8972).

[0095] Techniques for constructing fusion genes are well known. Essentially, the joining of various DNA fragments encoding different polypeptide sequences is carried out according to conventional techniques using blunt or sticky ends for ligation, restriction enzyme digestion to provide suitable ends, packing of sticky ends as needed, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of the gene fragments may be carried out using anchor primers that create a complementary overhang between two consecutive gene fragments that can be subsequently annealed to generate a chimeric gene sequence (see, for example, *Current Protocols in Molecular Biology*, edited by Ausubel et al., John Wiley & Sons: 1992).

[0096] Examples of nucleic acid compounds that are antagonists of TβRII, TGFβ1, TGFβ3, and GDF15 include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. Nucleic acid compounds can be single-stranded or double-stranded. Double-stranded compounds may also include overhanging or non-complementary regions, where one or the other of these strands is single-stranded. Single-stranded compounds may include self-complementary regions, meaning that the compound forms a so-called "hairpin" or "stem-loop" structure having a double-helix region. Nucleic acid compounds may include nucleotide sequences complementary to a region of the full-length TβRII nucleic acid sequence or ligand nucleic acid sequence consisting of 1000 or fewer, 500 or fewer, 250 or fewer, 100 or fewer, or 50, 35, 30, 25, 22, 20, or 18 nucleotides or fewer. The complementary region is preferably at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, e.g., between 15 and 25 nucleotides. Complementary regions may be located within introns, coding sequences, or non-coding sequences of the target transcript, for example, within coding segments. Generally, nucleic acid compounds have a length of about 8 to about 500 nucleotides or base pair lengths, for example, about 14 to about 50 nucleotides. Nucleic acids can be DNA (especially for use as antisense), RNA, or RNA:DNA hybrids. Any single strand may contain a mixture of DNA and RNA, as well as modified forms that cannot be readily classified as either DNA or RNA. Similarly, double-stranded compounds can be DNA:DNA, DNA:RNA, or RNA:RNA, and any single strand may also contain a mixture of DNA and RNA, as well as modified forms that cannot be readily classified as either DNA or RNA. Nucleic acid compounds may contain any of a variety of modifications, including one or more modifications to the backbone (sugar-phosphate moieties in native nucleic acids, including nucleotide linkages) or the base moieties (purine or pyrimidine moieties in native nucleic acids).Antisense nucleic acid compounds preferably have a length of about 15 to about 30 nucleotides and often contain one or more modifications to improve characteristics such as stability in serum, in cells, or at the site where the compound is likely to be delivered, e.g., in the stomach in the case of orally delivered compounds, or in the lungs in the case of inhaled compounds. In the case of RNAi constructs, the strand complementary to the target transcript is generally RNA or a modified version thereof. The other strand may be RNA, DNA, or any other variation. The double-stranded portion of double-stranded or single-stranded "hairpin" RNAi constructs preferably has a length of 18 to 40 nucleotides and, optionally, about 21 to 23 nucleotides, insofar as it functions as a Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also include modified forms. When nucleic acid compounds are brought into contact with cells under physiological conditions and at concentrations in which nonsense or sense control has little or no effect, they may inhibit target expression by about 50%, 75%, 90%, or more. Preferred concentrations for testing the effects of nucleic acid compounds are 1, 5, and 10 micromoles. Nucleic acid compounds can also be tested for their effects on, for example, angiogenesis.

[0097] 4. Changes in Fc-fusion proteins This application further provides TβRII-Fc fusion proteins having an engineered or variant Fc region. Such antibody and Fc fusion proteins may be useful in modulating effector functions such as antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, these modifications may improve the stability of the antibody and Fc fusion proteins. Amino acid sequence variants of these antibody and Fc fusion proteins are prepared by introducing appropriate nucleotide changes into DNA or by peptide synthesis. Such variants include, for example, deletions from the amino acid sequences of the antibody and Fc fusion proteins disclosed herein, and / or insertions into such amino acid sequences, and / or substitutions of residues within such amino acid sequences. Any combination of deletions, insertions, and substitutions is created to arrive at a final construct, provided that the final construct has the desired characteristics. Amino acid changes may also alter the post-translational processes of the antibody and Fc fusion proteins, such as changing the number or location of glycosylation sites.

[0098] Antibodies and Fc fusion proteins with reduced effector function can be produced by introducing changes into the amino acid sequence that include, but are not limited to, Ala-Ala mutations described by Bluestone et al. (see WO94 / 28027 and WO98 / 47531; also see Xu et al. 2000 Cell Immunol vol. 200; pp. 16-26). Thus, in certain embodiments, antibodies and Fc fusion proteins of this disclosure having mutations including Ala-Ala mutations in the constant region can be used to reduce or disable effector function. According to these embodiments, the antibodies and Fc fusion proteins may include a mutation to alanine at position 234 or a mutation to alanine at position 235, or a combination thereof. In one embodiment, the antibody or Fc fusion protein comprises an IgG4 framework, where Ala-Ala mutation describes a mutation(s) from phenylalanine to alanine at position 234 and / or a mutation from leucine to alanine at position 235. In another embodiment, the antibody or Fc fusion protein comprises an IgG1 framework, where the Ala-Ala mutation describes a leucine-to-alanine mutation(s) at position 234 and / or at position 235. The antibody or Fc fusion protein may optionally or further harbor other mutations, including the point mutation K322A in the CH2 domain (Hezareh et al. 2001, J Virol. 75: pp. 12161-1218).

[0099] In certain embodiments, this antibody or Fc fusion protein may be modified to enhance or inhibit complement-dependent cell damage (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions into the Fc region (see, for example, U.S. Patent No. 6,194,551). Alternatively, or further, cysteine ​​residues may be introduced into the Fc region, thereby enabling interchain disulfide bond formation in this region. The homodimer antibodies thus produced may have improved or reduced internalization ability and / or increased or decreased complement-mediated cell death. See Caron et al., J. Exp Med. 176: pp. 1191-1195 (1992) and Shopes, BJ Immunol. 148: pp. 2918-2922 (1992), WO99 / 51642, Duncan and Winter Nature 322: pp. 738-740 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0100] 5. GDF15-TβRII signaling This disclosure relates in part to the discovery that the TGFβ type II receptor (TβRII) binds to GDF15 with high affinity. To date, direct binding or interaction of GDF15 with the receptor has not been biochemically demonstrated. Inappropriate or unsuitable ligand purification may be a potential reason for the inactivity of commercially available GDF15. Exemplary GDF15 polypeptides demonstrating TβRII binding activity, as well as methods for producing and purifying such polypeptides, are disclosed herein. The sequences of native precursor GDF15 proteins and nucleotides from human and mouse are shown in Figures 1–4. Mature human GDF15 occupies residues 197–308 of SEQ ID NO: 1. Similarly, mature mouse GDF15 occupies residues 192–303 of SEQ ID NO: 3. In certain embodiments, the Disclosure makes available isolated and / or purified forms of GDF15 polypeptides or fragments thereof that are isolated from other proteins and / or other GDF15 polypeptide species, or substantially free from other proteins and / or other GDF15 polypeptide species in other ways (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%). The GDF15 polypeptides of the Disclosure bind to TβRII with high affinity. Binding can be evaluated using the purified protein in solution or in a surface plasmon resonance system such as the Biacore® system. These GDF15 polypeptides bind to TβRII polypeptides with approximately 10% affinity. -6 , 10 -7 , 10 -8 , 10 -9 It has an affinity (dissociation constant) of M or less. Preferably, the GDF15 polypeptide of this disclosure is isolated and purified according to the method described herein. The GDF15 polypeptide is generally produced by expression from recombinant nucleic acids.

[0101] GDF15 polypeptides may include polypeptides consisting of, or containing, amino acid sequences or functional fragments thereof that are at least 80% identical to the GDF15 polypeptide of SEQ ID NO: 1 or SEQ ID NO: 3, and optionally, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. GDF15 polypeptides may include polypeptides consisting of, or containing, amino acid sequences or functional fragments thereof that are at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the GDF15 polypeptide containing residues 197-308 of SEQ ID NO: 1 or residues 192-303 of SEQ ID NO: 3, and optionally, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Unprocessed GDF15 polypeptides may either include or exclude any signal sequence, and any N-terminal sequence relative to the signal sequence. The GDF15 polypeptide may include variants of SEQ ID NO: 1 or SEQ ID NO: 3, or portions thereof, fragments thereof, corresponding to residues 197-308 of SEQ ID NO: 1 or residues 192-303 of SEQ ID NO: 3 (for example, variants containing 2, 3, 4, 5, 10, 15, 20, 25, 30 or 35 or more amino acid substitutions in the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, respectively), as well as fusion proteins containing any of the above, wherein preferably, any of the above GDF15 polypeptides has substantial affinity for the TβRII polypeptide.

[0102] In certain embodiments, the GDF15 polypeptides of this disclosure may further include post-translational modifications in addition to any modifications naturally present in the GDF15 polypeptide. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, pegylation (polyethylene glycol), and acylation. As a result, modified GDF15 polypeptides may contain non-amino acid elements, such as polyethylene glycol, lipids, monosaccharides or polysaccharides, and phosphates. The effect of such non-amino acid elements on the functionality of the GDF15 polypeptide can be tested as described herein for other GDF15 polypeptides. If the GDF15 polypeptide is produced in cells by cleaving a nascent form of the GDF15 polypeptide, post-translational processing may also be important for the correct folding and / or function of the protein. Different cell types (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK-293) possess specific cellular machinery and characteristic mechanisms for such post-translational activity and can be selected to ensure precise modification and processing of the GDF15 polypeptide.

[0103] In certain embodiments, the Disclosure includes nucleic acids encoding precursor and mature GDF15 polypeptides. In further embodiments, the Disclosure also relates to a host cell containing such nucleic acid. This host cell may be any prokaryotic or eukaryotic cell. For example, the polypeptides of the Disclosure may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. Accordingly, some embodiments of the Disclosure further relate to methods for producing GDF15 polypeptides.

[0104] In certain embodiments, the Disclosure provides isolated and / or recombinant nucleic acids encoding any of the GDF15 polypeptides, including the fragments, functional variants, and fusion proteins disclosed herein. The nucleic acids may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for producing GDF15 polypeptides, or as direct therapeutic agents (e.g., in antisense, RNAi, or gene therapy approaches).

[0105] In certain embodiments, it is further understood that the target nucleic acid encoding the GDF15 polypeptide includes a nucleic acid that is a variant of SEQ ID NO: 1 or SEQ ID NO: 3. The variant nucleotide sequence includes sequences that differ by one or more nucleotide substitutions, additions, or deletions, e.g., allelic variants.

[0106] In certain embodiments, the Disclosure provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3. Those skilled in the art will understand that nucleic acid sequences complementary to SEQ ID NO: 1 or SEQ ID NO: 3, and variants of SEQ ID NO: 1 or SEQ ID NO: 3, are also within the scope of the Disclosure. In further embodiments, the nucleic acid sequences of the Disclosure may be isolated, recombinant, and / or fused with heterologous nucleotide sequences, or may be in a DNA library.

[0107] In other embodiments, the nucleic acids of the present disclosure also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, the complementary sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or fragments thereof. As discussed above, those skilled in the art will readily understand that the appropriate stringency conditions for promoting DNA hybridization can be varied. For example, hybridization can be carried out in 6.0 × sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing with 2.0 × SSC at 50°C. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 × SSC at 50°C to a high stringency of about 0.2 × SSC at 50°C. Furthermore, the temperature in the washing step can be increased from low stringency conditions at room temperature and about 22°C to high stringency conditions at about 65°C. Both temperature and salt concentration may be varied, or temperature or salt concentration may be kept constant even if other variables change. In some embodiments, the disclosure provides nucleic acids that hybridize under low stringency washing conditions in 6×SSC at room temperature and subsequently in 2×SSC at room temperature.

[0108] Isolated nucleic acids that differ from those shown in SEQ ID NO: 1 or SEQ ID NO: 3 due to degeneracy in the genetic code are also within the scope of this disclosure. For example, some amino acids are specified by more than one triplet. Codons that specify the same amino acid or synonym (e.g., CAU and CAC are synonyms for histidine) may result in “silent” mutations that do not affect the amino acid sequence of a protein. However, DNA sequence polymorphisms that result in changes in the amino acid sequence of a protein in question are expected to exist among mammalian cells. Those skilled in the art will understand that these variations in one or more nucleotides of a nucleic acid encoding a particular protein (up to about 3–5% of nucleotides) may exist among individuals of a given species due to natural allele variations. Any such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.

[0109] In certain embodiments, the recombinant nucleic acids of this disclosure may be operably ligated to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences are generally appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and appropriate regulatory sequences are known in the art for various host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, reader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated by this disclosure. These promoters may be either naturally occurring promoters or hybrid promoters combining elements of more than one promoter. The expression construct may reside in the cell on an episome, as in a plasmid, or the expression construct may be inserted into a chromosome. In one preferred embodiment, the expression vector includes a selectable marker gene that allows for the selection of transformed host cells. The selectable marker gene is known in the art and varies depending on the host cell used.

[0110] In certain embodiments disclosed herein, the nucleic acid of interest is provided in an expression vector comprising a nucleotide sequence encoding the GDF15 polypeptide, operably ligated to at least one regulatory sequence. The regulatory sequence is art-recognized and selected to direct the expression of the GDF15 polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression regulatory elements. Exemplary regulatory sequences are described in Goddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of the broad range of expression regulatory sequences that control the expression of a DNA sequence when operably ligated thereto may be used in these vectors to express the DNA sequence encoding the GDF15 polypeptide. Such useful expression regulatory sequences include, for example, the early and late promoters of SV40, the tet promoter, the earliest promoters of adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the regulatory regions of fd coat proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases, such as Pho5, the promoter of the yeast α-conjugation factor, the polyhedron promoter of baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It should be understood that the design of expression vectors may depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. Furthermore, the copy number of the vector, its ability to control its copy number, and the expression of any other proteins encoded by this vector, such as antibiotic markers, should also be considered.

[0111] The recombinant nucleic acids included in this disclosure may be produced by ligating a cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression vehicles for the production of recombinant GDF15 polypeptide include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as E. coli.

[0112] Some mammalian expression vectors contain both prokaryotic sequences to promote vector reproduction in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. Examples of mammalian expression vectors suitable for eukaryotic cell transfection include pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors. Some of these vectors are modified with bacterial plasmid-derived sequences, such as pBR322, to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) may be used for transient protein expression in eukaryotic cells. Examples of other viral (including retrovirus) expression systems can be found below in the description of gene therapy delivery systems. Various methods used in plasmid preparation and host organism transformation are well known in the field. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombination procedures, see Molecular Cloning: A Laboratory Manual, 3rd edition, edited by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 2001). In some cases, it may be desirable to express recombinant polypeptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., β-gal-containing pBlueBac III).

[0113] In preferred embodiments, vectors such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), the pCI-neo vector (Promega, Madison, Wisc.), and the UCOE®-derived vector (Millipore) are designed for the production of the target TβRII polypeptide in CHO cells. As is evident, these target gene constructs may be used to induce the expression of the target GDF15 polypeptide in cells cultured in a culture medium to produce, for example, a fusion protein or variant protein for purification.

[0114] This disclosure also relates to host cells transfected with a recombinant gene containing one or more coding sequences (e.g., SEQ ID NO: 1 or SEQ ID NO: 3) of the GDF15 polypeptides in question. These host cells may be any prokaryotic or eukaryotic cells. For example, the GDF15 polypeptides disclosed herein may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art. In preferred embodiments, the GDF15 polypeptides disclosed herein are expressed in CHO cells.

[0115] Therefore, this disclosure further relates to a method for producing the target GDF15 polypeptide. For example, host cells transfected with an expression vector encoding the GDF15 polypeptide can be cultured under appropriate conditions to produce GDF15 polypeptide expression. This GDF15 polypeptide can be secreted and isolated from a mixture of cells and culture medium containing the GDF15 polypeptide. Alternatively, this TβRII polypeptide can be retained in the cytoplasm or in a membrane fraction, cells can be recovered, lysed, and the protein can be isolated. A cell culture comprises host cells and culture medium. Appropriate culture media for cell culture are well known in the art. These target GDF15 polypeptides can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification using antibodies specific to specific epitopes of the GDF15 polypeptide, and affinity purification using agents that bind to domains fused to the GDF15 polypeptide (for example, a Protein A column may be used to purify the GDF15-Fc fusion). In a preferred embodiment, the GDF15 polypeptide is a fusion protein containing a domain that facilitates its purification. As an example, purification can be achieved by a series of column chromatography steps, for example, including three or more of the following in any order: Protein A chromatography, Q Sepharose chromatography, Phenylen Sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be completed using viral filtration and buffer exchange.

[0116] In preferred embodiments, these target GDF15 polypeptides are purified from the culture medium using a series of cation exchange column chromatography steps. Examples of materials used for the cation exchange column may be resins having substituents such as carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Examples of materials used for cation exchange column chromatography include SP Sepharose® Fast Flow, Q Sepharose® Fast Flow, DEAE Sepharose® Fast Flow, Capto® S, Capto® DEAE (GE Healthcare), S HyperCel® (Pall), TOYOPEARL GigaCap S-650 (TOSOH), or weak cation exchangers such as carboxymethyl. SP Sepharose® Fast Flow and Q Sepharose® Fast Flow are preferred.

[0117] To initiate purification, the parameters of a conditioned medium derived from host cells stably expressing the GDF15 polypeptide, such as pH, ionic strength, and temperature, may be adjusted as needed. In some embodiments, the chromatography column is washed and equilibrated with one or more solutions before contact with the polypeptide-containing supernatant. Such solutions may include, for example, buffers (e.g., Tris, MES, HEPES, histidine, phosphate, or sodium acetate, e.g., between 1–500 mM, between 25–100 mM, between 15–30 mM, or 20 mM) and / or salts (e.g., NaCl, NaPO4, sodium acetate, or CaCl2, e.g., between 0–2 M, between 1–2 M, or between 500 mM and 1 M). The pH of the equilibration solution is generally in the range of 3.5–10 (e.g., between pH 3.5–6, between 4.0–5.5, between 4.5–4.8, or 4.7). After contacting the column with a polypeptide-containing fluid, the bound column can be washed. The washing solution may contain a buffer (e.g., Tris, MES, HEPES, histidine, phosphate, or sodium acetate, e.g., between 1 and 500 mM, between 25 and 100 mM, between 15 and 30 mM, or between 20 mM) and / or a salt (e.g., NaCl, NaPO4, sodium acetate, or CaCl2, e.g., between 0 and 2 M, between 1 and 2 M, between 100 mM and 1 M, or between 100 mM and 500 mM) and / or an additive (e.g., guanidine, urea, sucrose, arginine, or an arginine derivative) and / or a solvent (e.g., ethanol, acetonitrile, or polyethylene glycol). The washing solution generally has a pH between 3.5 and 10 (e.g., between 4.5 and 8.0). Polypeptides can be eluted from a column using stepwise or gradient changes in pH, salt type, salt concentration, solvent type, solvent concentration, displacer type, displacer concentration, or a combination thereof. Generally, to elute polypeptides from a column, the medium is brought into contact with an elution buffer.In some embodiments, the elution buffer comprises a buffer (e.g., HEPES or Tris, e.g., 10–100 mM, 25–75 mM, or 50 mM) and / or a salt (e.g., NaCl or CaCl2, e.g., 0–2 M, e.g., 10–100 mM). In some embodiments, the elution buffer may comprise glycine, acetic acid, or citric acid (e.g., 20–250 mM, or 150 mM). The elution buffer may also comprise acetic acid (e.g., 20 mM to about 50 mM), additives (e.g., guanidine, urea, or sucrose, e.g., 1–10 M, 2–8 M, or 6 M) and / or a solvent (e.g., ethanol, acetonitrile, polyethylene glycol, e.g., 1–10% solvent, e.g., 5% solvent). The pH of the elution buffer may be in the range of about 5.0 to about 10.0. In some embodiments, the pH may be changed (e.g., gradually) to produce gradient elution. In some embodiments, the pH of the elution buffer is approximately 8.0. In some embodiments, a series of column chromatography steps are performed.

[0118] The data presented herein demonstrate that TβRII polypeptides act as antagonists of GDF15 signaling. While soluble TβRII polypeptides, and in particular TβRII-Fc, are preferred antagonists, other types of GDF15 antagonists are predicted to be useful, including anti-GDF15 antibodies, anti-TβRII antibodies, antisense antibodies, RNAi or ribozyme nucleic acids that inhibit the production of GDF15 or TβRII, and other inhibitors of GDF15 or TβRII, especially those that disrupt the GDF15-TβRII binding.

[0119] Antibodies that are specifically reactive to the GDF15 polypeptide and bind to it in a way that competes with its binding to the TβRII polypeptide (competitive binding), or that otherwise inhibit GDF15-mediated signaling, can be used as antagonists of GDF15 polypeptide activity. Similarly, antibodies that are specifically reactive to the TβRII polypeptide and disrupt GDF15 binding can be used as antagonists.

[0120] Antiprotein / antipeptide antiserum or monoclonal antibodies can be prepared by standard protocols using immunogens derived from GDF15 polypeptide or TβRII polypeptide (see, for example, Antibodies: A Laboratory Manual edited by Harlow and Lane (Cold Spring Harbor Press: 1988)). Mammals, such as mice, hamsters, or rabbits, can be immunized with immunogenic forms of GDF15 polypeptide, antigenic fragments capable of eliciting an antibody response, or fusion proteins. Techniques for conferring immunogenicity to proteins or peptides include conjugation to a carrier or other techniques well known in the art. The immunogenic portion of GDF15 or TβRII polypeptide may be administered in the presence of an adjuvant. The progress of immunization can be monitored by detecting antibody titers in plasma or serum. Standard ELISA or other immunoassays may be used with the immunogen as an antigen to assess antibody levels.

[0121] After immunization of animals with antigenic preparations of the GDF15 polypeptide, antiserum can be obtained, and if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be recovered from the immunized animals and fused with immortal cells such as myeloma cells by standard somatic cell fusion procedures to obtain hybridoma cells. Such techniques are well known in the field and include, for example, hybridoma techniques (originally developed by Kohler and Milstein, (1975) Nature, vol. 256: pp. 495-497), human B-cell hybridoma techniques (Kozbar et al., (1983) Immunology Today, vol. 4: p. 72), and EBV-hybridoma techniques for producing human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be immunochemically screened for the production of antibodies and monoclonal antibodies specifically reactive with GDF15 polypeptide, isolated from cultures containing such hybridoma cells.

[0122] As used herein, the term “antibody” is intended to include fragments of its own that are also specifically reactive with the target polypeptide. Antibodies may be fragmented using conventional techniques, and these fragments may be screened for utility in the same manner as described above for the whole antibody. For example, an F(ab)2 fragment may be produced by treating an antibody with pepsin. The resulting F(ab)2 fragment may be processed to reduce disulfide crosslinks in order to produce a Fab fragment. Antibodies of the present invention are further intended to include bispecific, single-chain, chimeric, humanized, and fully human molecules having affinity for the TβRII or GDF15 polypeptide conferred by at least one CDR region of the antibody. Antibodies may further include labels that can bind to and be detected (for example, the labels may be radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors).

[0123] In certain embodiments, this antibody is a recombinant antibody, and this term is used to refer to CDR-transplanted antibodies or chimeric antibodies, human or other antibodies assembled from library-selected antibody domains, single-chain antibodies and single-domain antibodies (e.g., human V H Protein or camelid (camelid) V HH The present invention encompasses any antibody, including proteins, that is partially generated by molecular biology techniques. In certain embodiments, the antibody of the present invention is a monoclonal antibody, and in certain embodiments, the present invention creates a usable method for generating novel antibodies. For example, a method for generating a monoclonal antibody that specifically binds to a GDF15 polypeptide or a TβRII polypeptide may include the steps of: administering to a mouse an amount of an immunogenic composition containing an antigen polypeptide effective to stimulate a detectable immune response; obtaining antibody-producing cells (e.g., spleen-derived cells) from the mouse; fusing these antibody-producing cells with myeloma cells to obtain an antibody-producing hybridoma; and testing the antibody-producing hybridoma to identify a hybridoma that produces a monoclonal antibody that specifically binds to this antigen. Once obtained, the hybridoma can be propagated in a cell culture, optionally under culture conditions in which the hybridoma-derived cells produce a monoclonal antibody that specifically binds to this antigen. This monoclonal antibody can be purified from the cell culture.

[0124] The adjective "specifically reactive with" when used in reference to antibodies is intended to mean, as is commonly understood in the field, that the antibody is sufficiently selective between the target antigen (e.g., GDF15 polypeptide) and other non-target antigens, and that the antibody is useful, at a minimum, for detecting the presence of the target antigen in a particular type of biological sample. In certain applications of antibodies, such as therapeutic use, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally tend to be more efficient at distinguishing between desired antigens and cross-reactive polypeptides (compared to polyclonal antibodies). One feature that influences the specificity of antibody-antigen interactions is the antibody's affinity for the antigen. While the desired specificity can be achieved within a range of different affinities, generally preferred antibodies have an affinity of approximately 10. -6 , 10 -7 , 10 -8 , 10 -9 Or it has an affinity (dissociation constant) of less than that. Given the high affinity between GDF15 and TβRII, neutralizing anti-GDF15 or anti-TβRII antibodies are generally 10 -9 It is predicted to have a dissociation constant of or less than that.

[0125] Furthermore, the techniques used to screen antibodies to identify a desired antibody can influence the characteristics of the acquired antibody. For example, if the antibody is used to bind to an antigen in solution, it may be desirable to test its solution binding. Various different techniques are available to test antibody-antigen interactions, particularly to identify a desirable antibody. Such techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore® binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., paramagnetic bead systems from IGEN International, Inc., Gaithersburg, Maryland), Western blotting, immunoprecipitation assays, and immunohistochemistry.

[0126] Examples of nucleic acid compounds that are GDF15 or TβRII antagonists include antisense nucleic acids, RNAi constructs, and catalytic nucleic acid constructs. Nucleic acid compounds can be single-stranded or double-stranded. Double-stranded compounds may also include overhangs or non-complementary regions, where one or the other of these strands is single-stranded. Single-stranded compounds may include self-complementary regions, meaning that the compound forms a so-called "hairpin" or "stem-loop" structure having a double-helix region. Nucleic acid compounds may include nucleotide sequences complementary to regions of the full-length GDF15 nucleic acid sequence or TβRII nucleic acid sequence consisting of 1000 or fewer, 500 or fewer, 250 or fewer, 100 or fewer, or 50, 35, 30, 25, 22, 20 or 18 nucleotides or fewer. The complementary region is preferably at least 8 nucleotides, and optionally at least 10 or at least 15 nucleotides, e.g., between 15 and 25 nucleotides. Complementary regions may be located within introns, coding sequences, or non-coding sequences of the target transcript, for example, within coding segments. Generally, nucleic acid compounds have a length of about 8 to about 500 nucleotides or base pair lengths, for example, about 14 to about 50 nucleotides. Nucleic acids can be DNA (especially for use as antisense), RNA, or RNA:DNA hybrids. Any single strand may contain a mixture of DNA and RNA, as well as modified forms that cannot be readily classified as either DNA or RNA. Similarly, double-stranded compounds can be DNA:DNA, DNA:RNA, or RNA:RNA, and any single strand may also contain a mixture of DNA and RNA, as well as modified forms that cannot be readily classified as either DNA or RNA. Nucleic acid compounds may contain any of a variety of modifications, including one or more modifications to the backbone (sugar-phosphate moieties in native nucleic acids, including nucleotide linkages) or the base moieties (purine or pyrimidine moieties in native nucleic acids).Antisense nucleic acid compounds preferably have a length of about 15 to about 30 nucleotides and often contain one or more modifications to improve characteristics such as stability in serum, in cells, or at the site where the compound is likely to be delivered, e.g., in the stomach in the case of orally delivered compounds, or in the lungs in the case of inhaled compounds. In the case of RNAi constructs, the strand complementary to the target transcript is generally RNA or a modified version thereof. The other strand may be RNA, DNA, or any other variation. The double-stranded portion of double-stranded or single-stranded "hairpin" RNAi constructs preferably has a length of 18 to 40 nucleotides and, optionally, about 21 to 23 nucleotides, insofar as it functions as a Dicer substrate. Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also include modified forms. When nucleic acid compounds are brought into contact with cells under physiological conditions and at concentrations in which nonsense or sense control has little or no effect, they may inhibit target expression by about 50%, 75%, 90%, or more. The preferred concentrations for testing the effects of nucleic acid compounds are 1, 5, and 10 micromoles.

[0127] 6. Screening assay In certain embodiments, the present invention relates to the use of TβRII polypeptides (e.g., soluble TβRII polypeptides) and GDF15 polypeptides for identifying compounds (pharmaceuticals) that are agonists or antagonists of the GDF15-TβRII signaling pathway. Compounds identified by this screening may be tested to evaluate their ability to modulate GDF15 signaling activity in vitro. Optionally, these compounds may be further tested in animal models to evaluate their ability to modulate tissue growth in vivo.

[0128] Numerous approaches exist for screening therapeutic agents for modulating tissue growth by targeting GDF15 and TβRII polypeptides. In certain embodiments, high-throughput screening of compounds may be performed to identify agents that disrupt GDF15 or TβRII-mediated cellular signaling. In certain embodiments, this assay is performed to screen and identify compounds that specifically inhibit or reduce the binding of TβRII polypeptides to GDF15. Alternatively, this assay may be used to identify compounds that enhance the binding of TβRII polypeptides to GDF15. In further embodiments, these compounds may be identified by their ability to interact with GDF15 or TβRII polypeptides.

[0129] Various assay formats are sufficient in light of this disclosure; nevertheless, assay formats not expressly described herein will be understood by those skilled in the art. As described herein, the test compounds (pharmaceuticals) of the present invention can be created by any combinatorial chemical method. Alternatively, these target compounds may be synthesized in vivo or in These may be naturally occurring biomolecules synthesized in vitro. The compounds (agents) tested for their ability to act as modulators of tissue growth may be produced by bacteria, yeast, plants, or other organisms (e.g., natural products), chemically produced (e.g., small molecules including peptide mimes), or recombinantly produced. The test compounds intended by this invention include non-peptidyl organic molecules, peptides, polypeptides, peptide mimes, sugars, hormones, and nucleic acid molecules. In specific embodiments, the test agent is a small organic molecule having a molecular weight of less than about 2,000 daltons.

[0130] The test compounds of the present invention may be provided as single, individual entities or in a higher-complexity library prepared by combinatorial chemistry or the like. These libraries may include, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers, and other classes of organic compounds. Presentation of the test compounds to the test system may be either as isolated forms of the compounds or as mixtures of the compounds, particularly in the initial screening step. Optionally, these compounds may be optionally derivatized with other compounds and may have derivatizing groups that facilitate the isolation of the compounds. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidines, magnetic beads, glutathione S-transferase (GST), photoactivatable crosslinkers, or any combination thereof.

[0131] In many drug screening programs testing libraries of compounds and natural extracts, high-throughput assays are desirable to maximize the number of compounds investigated over a given period. Assays performed in cell-free systems, such as those that can be induced using purified or semi-purified proteins, are often preferred as “primary” screenings in that they can be produced to allow for rapid development and relatively easy detection of changes in molecular targets mediated by the test compound. Furthermore, the effects of cytotoxicity or bioavailability of the test compound can generally be ignored in in vitro systems; instead, this assay focuses primarily on the drug’s effects on molecular targets, which may manifest as changes in the binding affinity between TβRII polypeptide and GDF15.

[0132] For illustrative purposes only, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified TβRII polypeptide, which is typically capable of binding to GDF15. A composition containing a TβRII ligand is then added to the mixture of the compound and the TβRII polypeptide. Detection and quantification of the TβRII / GDF15 complex provides a means for determining the potency of the compound in inhibiting (or enhancing) complex formation between the TβRII polypeptide and GDF15. The potency of this compound can be evaluated by generating a dose-response curve from data obtained using various concentrations of the test compound. Furthermore, a control assay may also be performed to provide a baseline for comparison. For example, in a control assay, isolated and purified GDF15 is added to a composition containing the TβRII polypeptide, and the formation of the TβRII / GDF15 complex is quantified in the absence of the test compound. Generally, it is understood that the order in which the reactants may be mixed may vary, and they may be mixed simultaneously. Furthermore, instead of purified proteins, cell extracts and lysates may be used to provide suitable cell-free assay systems.

[0133] The complex formation between TβRII polypeptide and GDF15 can be detected by various techniques. For example, the modulation of complex formation can be detected by immunoassay or chromatographic detection, for example, by detectably labeled proteins, for example, radiolabeled proteins (e.g., 32 P, 35 S, 14 C or 3 Quantification can be performed using fluorescently labeled (e.g., FITC) or enzymatically labeled TβRII polypeptide or GDF15.

[0134] In certain embodiments, the present invention intends to utilize fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays to directly or indirectly measure the degree of interaction between TβRII polypeptides and their binding proteins. Furthermore, other forms of detection, such as those based on optical waveguides (PCT Publication WO96 / 26432 and U.S. Patent No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the present invention.

[0135] Furthermore, the present invention intends to use an interaction trap assay, also known as a "two-hybrid assay," to identify agents that disrupt or enhance the interaction between TβRII polypeptides and their binding proteins. See, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72: pp. 223-232; Madura et al. (1993) J Biol Chem 268: pp. 12046-12054; Bartel et al. (1993) Biotechniques 14: pp. 920-924; and Iwabuchi et al. (1993) Oncogene 8: pp. 1693-1696). In specific embodiments, the present invention intends to use an inverse two-hybrid system to identify compounds (e.g., small molecules or peptides) that dissociate the interaction between TβRII polypeptides and their binding proteins. See, for example, Vidal and Legrain, Nucleic Acids Res Vol. 27: pp. 919-929 (1999); Vidal and Legrain, Trends Biotechnol Vol. 17: pp. 374-381 (1999); and U.S. Patents No. 5,525,490, 5,955,280, and 5,965,368.

[0136] In certain embodiments, these target compounds are identified by their ability to interact with the TβRII or GDF15 polypeptides of the present invention. The interaction between the compound and the TβRII or GDF15 polypeptide may be covalent or non-covalent. For example, such interactions can be identified at the protein level using in vitro biochemical methods including photocrosslinking, radioactively labeled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology, Vol. 46: p. 1). In specific cases, these compounds may be screened in mechanism-based assays, such as assays for detecting compounds that bind to GDF15 or TβRII polypeptides. This may include solid-phase or liquid-phase binding events. Alternatively, the gene encoding the GDF15 or TβRII polypeptide may be transfected into cells with a reporter system (e.g., β-galactosidase, luciferase, or green fluorescent protein), preferably screened against a library by high-throughput screening, or screened using individual members of the library. Other mechanism-based binding assays, such as those detecting changes in free energy, may be used. Binding assays can be performed using targets immobilized on wells, beads, or tips, or captured by immobilized antibodies, or separated by capillary electrophoresis. Binding compounds can typically be detected using colorimetric, fluorescence, or surface plasmon resonance.

[0137] In certain embodiments, the present invention provides methods and agents for modulating (stimulating or inhibiting) GDF15-mediated cellular signaling. Therefore, any identified compounds can be tested in vitro or in vivo in cells or tissues to confirm their ability to modulate GDF15 signaling. Various methods known in the art can be utilized for this purpose.

[0138] 7. Exemplary therapeutic use As used herein, a therapeutic agent that “prevents” a disorder or condition means a compound that, in a statistical sample, reduces the incidence of the disorder or condition in a treated sample compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample. The term “to treat” as used herein includes remission or elimination of a condition once it has been established. In either case, prevention or treatment may be identified as the intended result of the administration of a diagnostic and therapeutic agent provided by a physician.

[0139] This disclosure provides methods for treating or preventing diseases or conditions associated with TGFβ superfamily members by targeting an effective amount of a TβRII polypeptide, collectively referred to herein as “therapeutic agents,” including the aforementioned TβRII-Fc fusion protein or nucleic acid antagonist (e.g., antisense or siRNA). In some embodiments, the disease or condition is associated with dysregulated GDF15, TGFβ1, or TGFβ3 signaling. Methods and compositions for treating specific cardiovascular or vascular disorders are also provided. Furthermore, this disclosure provides methods and compositions for treating or preventing cancer. Furthermore, this disclosure provides methods and compositions for treating or preventing fibrous disorders and conditions.

[0140] In particular, the polypeptide therapeutic agents of this disclosure are useful for treating or preventing chronic vascular or cardiovascular diseases. Exemplary disorders of this type include, but are not limited to, cardiac disorders (including cardiomyopathy, myocardial infarction, angina pectoris, and valvular heart disease); renal disorders (including chronic glomerulitis, diabetic nephritis, and lupus-associated nephritis); disorders associated with atherosclerosis or other types of arteriosclerosis (including stroke, cerebral hemorrhage, subarachnoid hemorrhage, angina pectoris, and renal arteriosclerosis); thrombotic disorders (including cerebral thrombosis and thrombotic bowel necrosis); complications of diabetes (including diabetes-associated retinal diseases, cataracts, diabetes-associated kidney diseases, diabetes-associated neuropathology, diabetes-associated gangrene, and diabetes-associated chronic infections); vascular inflammatory disorders (systemic lupus erythematosus, rheumatoid arthritis, arteriovenous arthritis, large cell arteritis, Kawasaki disease, Takayasu arteritis, Churg-Strauss syndrome, and Henoch-Schönlein purpura); diabetic vascular disorders; and cardiac disorders, such as congenital heart disease, cardiomyopathy (e.g., dilated, hypertrophic, and restrictive cardiomyopathy), and congestive heart failure. Exemplary disorders include, but are not limited to, hereditary hemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, tortuosomatic artery syndrome, pre-eclampsia, and restenosis.

[0141] TβRII polypeptides may be administered to a subject alone or in combination with one or more agents or therapeutic modalities useful for treating TGFβ-related cardiovascular disorders and / or conditions. In certain embodiments, the second agent or therapeutic modality is selected from one or more of the following: angioplasty, beta-blockers, antihypertensives, cardiotonic agents, antithrombotic agents, vasodilators, hormone antagonists, endothelin antagonists, calcium channel blockers, phosphodiesterase inhibitors, angiotensin type II antagonists, and / or cytokine blockers / inhibitors.

[0142] In particular, the polypeptide therapeutics of this disclosure are useful for treating or preventing cancer (tumors). The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. Examples of cancer or neoplasm disorders include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, stomach cancer, intestinal cancer, skin cancer, bone cancer, gastric cancer, melanoma, and various types of head and neck cancers, including head and neck squamous cell carcinoma. Other examples of neoplasms and related conditions include esophageal cancer, theca cell tumor, masculinizing tumor, endometrial hyperplasia, endometriosis, fibrosarcoma, choriocarcinoma, nasopharyngeal cancer, laryngeal cancer, hepatoblastoma, Kaposi's sarcoma, skin cancer, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, glioblastoma, Schwann cell tumor, oligodendroglioma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract cancer, Wilms' tumor, renal cell carcinoma, prostate cancer, phakomatoses and associated abnormal angiogenesis, and Meggs syndrome. Cancers particularly suitable for treatment with the therapeutic agents described herein may be characterized by one or more of the following: the cancer has a detectable elevated TβRII level, increased GDF15, TGFβ1, or TGFβ3 expression level or biological activity in the tumor or serum, and is metastatic, at risk of becoming metastatic, or any combination thereof.

[0143] In certain embodiments of such methods, one or more polypeptide therapeutic agents may be administered together (simultaneously) or at different time points (sequentially). Furthermore, polypeptide therapeutic agents may be administered together with other types of compounds to treat cancer or inhibit angiogenesis.

[0144] In certain embodiments, the methods covered by this disclosure may be used alone. Alternatively, these methods may be used in combination with other conventional anti-cancer treatment approaches directed towards the treatment or prevention of proliferative disorders (e.g., tumors). For example, such methods may be used in prophylactic cancer prevention, prevention of cancer recurrence and metastasis after surgery, and as adjuvants to other conventional cancer treatments. This disclosure recognizes that the effectiveness of conventional cancer treatments (e.g., chemotherapy, radiotherapy, phototherapy, immunotherapy, and surgery) may be enhanced through the use of the polypeptide therapeutic agents covered by this disclosure.

[0145] A variety of conventional compounds have been shown to possess antineoplastic or anticancer activity. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant cells in leukemia or bone marrow malignancies. While chemotherapy is effective in treating various types of malignancies, many antineoplastic compounds induce undesirable side effects. When two or more different treatments are combined, these treatments can act synergistically, allowing for a reduction in the dosage of each treatment, thereby reducing the adverse side effects caused by each compound at higher dosages. In other cases, treatment-resistant malignancies may respond to combination therapy of two or more different treatments.

[0146] When the therapeutic agents disclosed herein are administered in combination with or sequentially with another conventional antineoplastic agent, such therapeutic agents may enhance the therapeutic effect of the antineoplastic agent or overcome cellular resistance to such antineoplastic agent. This allows for a reduction in the dosage of the antineoplastic agent, thereby reducing undesirable side effects or restoring the effectiveness of the antineoplastic agent in resistant cells.

[0147] According to this disclosure, the polypeptide therapeutic agents described herein may be used in combination with other compositions and procedures for the treatment of diseases. For example, a tumor may be conventionally treated with surgery, radiotherapy or chemotherapy in combination with the TβRII polypeptide, and the TβRII polypeptide may then be subsequently administered to the patient to prolong the dormancy of micrometastases and stabilize any residual primary tumor.

[0148] In certain aspects of the present invention, other therapeutic agents useful for combination tumor treatment with TβRII polypeptide include other cancer treatments: for example, surgery, cytotoxic agents, radiological procedures involving irradiation or administration of radioactive materials, chemotherapeutic agents, antihormone agents, growth inhibitors, antineoplastic compositions, and treatment with anticancer agents listed herein and known in the art, or combinations thereof.

[0149] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. This term is not limited to radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 This includes radioactive isotopes of Lu, chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and their fragments, e.g., nucleases, antibiotics, and toxins, e.g., small molecule toxins, or their fragments and / or variants, enzymatically active toxins of bacterial, fungal, plant or animal origin, as well as various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Tumorkillers cause the destruction of tumor cells.

[0150] "Chemotherapy agents" are chemical compounds that are useful in treating cancer. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (specifically, bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-rapacon; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogues), topotecan (HYCAMTIN®), CPT-11 (irinoteca Including CAMPTOSAR®, acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (including its adzelesin, karzelesin, and biceresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including its synthetic analogs, KW-2189 and CB1-TM1); erytherovin; pankratis Tatin; sarcodicin; sponge statins; nitrogen mustards, e.g., chlorambucil, chlornafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine hydrochloride, melphalan, nobenbitin, fenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimnustine;Antibiotics, such as engine antibiotics (e.g., caritimycin, in particular caritimycin gamma and caritimycin omegar (see, e.g., Agnew, Chem Intl. Ed. Engl., Vol. 33: pp. 183-186 (1994)); dinemycin including dinemycin A; esperamicin; and neocardinostatin chromophores and related pigment proteins engine antibiotic chromophores), aclasinomycin, actinomycin, ausuramycin, azaserin, bleomycin, kakutinomycin, carabicin, carminomycin, cartinophylline, chromomycinis, dactinomycin Syn, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, purpuramycin - Romycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6 - Azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren;Edatraxate; Demecoltin; Diadicone; Eflornithine; Erliptinium acetate; Epotilon; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamine; Mytansinoids, e.g., Mytansine and Ansamitosine; Mitoguazone; Mitoxantrone; Mopidammol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK (Registered Trademark) Polysaccharide Complex (JHS Natural) Products, Eugene, OR); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2”-Trichlorotriethylamine; Trichothecin (especially T-2 toxin, Beraclin A, Loridine A and Angidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Paclitaxel Cremophor-free Albumin-Modified Nanoparticle Formulations (American Pharmaceuticals) Partners, Schaumberg, Illinois) and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate;This includes the topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine (XELODA®); any pharmaceutically acceptable salt, acid, or derivative of any of the above; and combinations of two or more of the above, e.g., the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, abbreviated as CHOP; and the treatment regimen using oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin, abbreviated as FOLFOX.

[0151] Antihormone agents, which act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth, and which are often in the form of systemic or systemic treatments, are also included in this definition. These can be hormones themselves. Examples include, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifen, droloxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, and LYl 17018, anti-estrogen agents and selective estrogen receptor modulators (SERMs), including onapristone and FARESTON® toremifene; anti-progesterone agents; estrogen receptor downmodulators (ERDs); agents that function to suppress or stop the ovaries, e.g., luteinizing hormone-releasing hormone (LHRH) agonists, e.g., LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens, e.g., flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland, e.g., 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestan, fadrozol, RIVIS This includes OR® borozol, FEMARA® letrozole, and ARIMIDEX® anastrozole, among others.Furthermore, such definitions of chemotherapeutic agents include bisphosphonates, e.g., clodronate (e.g., BONEFOS® or OSTAC®), DIDROC AL® etidronate, NE-58095, ZOMET A® zoledronic acid / zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tildronate, or ACTONEL® risedronate; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, in particular those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, e.g., PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines This includes, for example, THERATOPE® vaccine, and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib nitosylate (also known as GW572016, an ErbB-2 and EGFR bityrosine kinase small molecule inhibitor); and any pharmaceutically acceptable salts, acids, or derivatives of the above.

[0152] As used herein, "growth inhibitor" refers to a compound or composition that inhibits cell growth either in vitro or in vivo. Therefore, a growth inhibitor may be a drug that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitors include drugs that block cell cycle progression (at locations other than the S phase), such as those that induce G1 arrest and M phase arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. G1-stopping agents, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also extend to S phase arrest. Further information can be found in Chapter 1 of *The Molecular Basis of Cancer*, edited by Mendelsohn and Israel, in the section titled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), particularly on page 13. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting microtubule assembly from tubulin dimers and preventing depolymerization, thereby inhibiting mitosis in cells.

[0153] In other embodiments, TβRII polypeptides may be useful in the treatment or prevention of fibrosis. As used herein, the term “fibrosis” refers to the abnormal formation or development of excessive fibrous connective tissue by cells in an organ or tissue. Processes associated with fibrosis may occur as part of normal tissue formation or repair, but dysregulation of these processes can result in altered cellular composition and excessive connective tissue deposition that progressively impairs the function of the tissue or organ. The formation of fibrous tissue may arise from repair or reactive processes. Fibrous disorders or conditions include, but are not limited to, vascular diseases, e.g., heart disease, cerebral disease and peripheral vascular disease, as well as fibroproliferative disorders associated with tissue and organ systems, including the heart, skin, kidneys, peritoneum, intestines and liver (disclosed, e.g., in Wynn, 2004, Nat Rev 4: pp. 583-594, incorporated herein by reference). Illustrative disorders that can be treated include, but are not limited to, nephropathy associated with injury / fibrosis, such as chronic nephropathy associated with diabetes (e.g., diabetic nephropathy), lupus, scleroderma, glomerulonephritis, focal segmental glomerulosclerosis and IgA nephropathy, renal fibrosis; intestinal fibrosis, such as scleroderma and radiation-induced intestinal fibrosis; hepatic fibrosis, such as cirrhosis, alcohol-induced hepatic fibrosis, biliary injury, primary biliary cirrhosis, infection or virus-induced hepatic fibrosis, congenital hepatic fibrosis and autoimmune hepatitis; and other fibrotic conditions, such as cystic fibrosis, endocardial fibrosis, mediastinal fibrosis, sarcoidosis, scleroderma, spinal cord injury / fibrosis, myelofibrosis, vascular restenosis, atherosclerosis, injection fibrosis (which can occur as a complication of intramuscular injections, particularly in children), endocardial fibrosis, retroperitoneal fibrosis and nephrogenic systemic fibrosis.

[0154] As used herein, the terms “fibrous disorder,” “fibrous condition,” and “fibrous disease” are interchangeable to refer to a disorder, condition, or disease characterized by fibrosis. Examples of fibrotic disorders include, but are not limited to, sclerotic disorders (e.g., scleroderma, atherosclerosis, diffuse systemic sclerosis), vascular fibrosis, pancreatic fibrosis, hepatic fibrosis (e.g., cirrhosis), renal fibrosis, musculoskeletal fibrosis, cardiac fibrosis (e.g., endocardial fibrosis, idiopathic cardiomyopathy), cutaneous fibrosis (e.g., scleroderma, post-traumatic, surgical skin scars, keloids and cutaneous keloid formation), ocular fibrosis (e.g., glaucoma, ocular sclerosis, conjunctival and corneal scars, and pterygium), myelofibrosis, progressive systemic sclerosis (PSS), chronic graft-versus-host disease, Peyronie's disease, post-cystoscopy urethral stricture, idiopathic and pharmacologically induced retroperitoneal fibrosis, mediastinal fibrosis, proliferative fibrosis, neoplastic fibrosis, Dupuytren's disease, strictures, nerve scars, skin scars and radiation-induced fibrosis.

[0155] As used herein, inhibition of the fibrous response of cells includes, but is not limited to, inhibition of the fibrous response of one or more cells in the liver (or liver tissue); one or more cells in the kidney (or kidney tissue); one or more cells in muscle tissue; one or more cells in the heart (or heart tissue); one or more cells in the pancreas; one or more cells in the skin; one or more cells in the bone, one or more cells in the vascular structure, one or more stem cells, or one or more cells in the eye.

[0156] The present invention intends to utilize TβRII polypeptides in combination with one or more other therapeutic modalities. Thus, in addition to the use of TβRII polypeptides, one or more "standard" treatments for treating fibrotic disorders may also be administered to the subject. For example, these TβRII polypeptides may be administered in combination with (i.e., together with) cytotoxic agents, immunosuppressants, radiotoxic agents, and / or therapeutic antibodies. Specific co-therapeutic agents intended by the present invention include, but are not limited to, steroids (e.g., corticosteroids, e.g., prednisone), immunosuppressants and / or anti-inflammatory agents (e.g., gamma-interferon, cyclophosphamide, azathioprine, methotrexate, penicillamine, cyclosporine, colchicine, antithymocyte globulin, mycophenolate mofetil, and hydroxychloroquine), cytotoxic drugs, calcium channel blockers (e.g., nifedipine), angiotensin-converting enzyme inhibitor (ACE) inhibitors, para-aminobenzoic acid (PABA), dimethyl sulfoxide, transforming growth factor beta (TGFβ) inhibitors, interleukin-5 (IL-5) inhibitors, and general caspase inhibitors.

[0157] Further antifibrotic agents that may be used in combination with TβRII polypeptides include, but are not limited to, lectins (for example, as described in U.S. Patent No. 7,026,283, the full text of which is incorporated herein by reference) and antifibrotic agents described by Wynn et al. (as described in the full text of which is incorporated herein by reference, J Clin Invest, Vol. 117, pp. 524-529, 2007).For example, further antifibrotic agents and treatments include various anti-inflammatory / immunosuppressive / cytotoxic drugs (including colchicine, azathioprine, cyclophosphamide, prednisone, thalidomide, pentoxifylline, and theophylline), TGFβ signaling modifiers (relaxin, SMAD7, HGF, and BMP7, as well as TGFβI, TβRI, TβRII, EGR-I, and CTGF inhibitors), cytokines, and cytokine receptor antagonists (IL-1β, IL-5, IL-6, IL-13, I). Inhibitors for L-21, IL-4R, IL-13Rα1, GM-CSF, TNF-α, oncostatin M, WISP-I, and PDGF), cytokines and chemokines (IFN-γ, IFN-α / β, IL-12, IL-10, HGF, CXCL10, and CXCL11), chemokine antagonists (inhibitors for CXCL1, CXCL2, CXCL12, CCL2, CCL3, CCL6, CCL17, and CCL18), chemokine receptor antagonists (CCR2, CCR3, CCL2, CCL3, CCL2, CCL2, CCL3, CCL2, CCL17, and CCL18), Inhibitors of CR5, CCR7, CXCR2, and CXCR4; TLR antagonists (inhibitors of TLR3, TLR4, and TLR9); angiogenesis antagonists (VEGF-specific antibodies and adenosine deaminase replacement therapy); antihypertensive drugs (beta-blockers and inhibitors of ANG11, ACE, and aldosterone); vasoactive substances (ET-1 receptor antagonists and bosentan); inhibitors of enzymes that synthesize and process collagen (prolylhydroxyl This includes, but is not limited to, sylase inhibitors, B cell antagonists (rituximab), integrin / adhesion molecule antagonists (molecules that block α1β1 and αvβ6 integrins, as well as integrin-related kinase inhibitors, and antibodies specific to ICAM-I and VCAM-I), pro-apoptotic drugs that target myofibroblasts, MMP inhibitors (inhibitors of MMP2, MMP9, and MMP12), and TIMP inhibitors (antibodies specific to TIMP-1).

[0158] TβRII polypeptides and co-therapeutic agents or co-therapeutics may be administered in the same formulation or separately. In the case of separate administrations, the TβRII polypeptide may be administered before, after, or concurrently with the co-therapeutic agent or co-therapeutics. One agent may precede or follow the administration of the other agent by an interval ranging from several minutes to several weeks. In embodiments in which two or more different types of therapeutic agents are applied separately to the target, it is generally ensured that a significant period does not expire between each delivery so that these different types of agents can still exert a favorably combined effect on the target tissue or cells.

[0159] 8. Pharmaceutical Compositions The therapeutic agents described herein (e.g., TβRII polypeptides) can be formulated into pharmaceutical compositions. Pharmaceutical compositions for use according to this disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Such formulations are generally substantially pyrogenically free, in accordance with most regulatory requirements.

[0160] In certain embodiments, the therapeutic methods of the present disclosure include the step of administering the composition systemically or topically as an implant or device. When administered, the therapeutic compositions for use in the present disclosure are in a physiologically acceptable form that does not contain pyrogens. Other therapeutically useful agents, other than TβRII signaling antagonists which may also be optionally included in the composition as described above, may be administered simultaneously with or sequentially with the compound of interest (e.g., TβRII polypeptide) in the methods disclosed herein.

[0161] Typically, the protein therapeutics disclosed herein are administered parenterally, particularly intravenously or subcutaneously. Suitable pharmaceutical compositions for parenteral administration may include one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, in combination with antioxidants, buffers, bacteriostatic agents, solutes or suspending agents or thickeners to make them isotonic with the blood of the recipient to whom the formulation is intended, and one or more TβRII polypeptides. Examples of suitable aqueous and nonaqueous carriers that may be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, e.g., olive oil, and injectable organic esters, e.g., ethyl oleate. Adequate fluidity may be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0162] These compositions and formulations may be presented in a pack or dispenser device that may contain, if desired, one or more unit dosage forms containing the active ingredient. The pack may include, for example, metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0163] Furthermore, this composition may be encapsulated or injected in a form for delivery to a target tissue site. In certain embodiments, the composition of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., TβRII polypeptides) to a target tissue site and providing structure to developing tissue, and which is optimally absorbed throughout the body. For example, this matrix may provide a slow release of TβRII polypeptides. Such a matrix may be formed from materials currently used for other implanted medical applications.

[0164] The selection of matrix materials is based on biocompatibility, biodegradability, mechanical properties, surface appearance, and interfacial properties. The specific application of the composition in question dictates the appropriate formulation. Potential matrices for a composition may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, and polyacid anhydride. Other potential materials, such as bone or skin collagen, are biodegradable and biologically well-defined. Further matrices consist of pure proteins or extracellular matrix components. Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminate, or other ceramics. The matrix may consist of any combination of the above types of materials, e.g., polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be modified in composition, e.g., calcium-aluminate-phosphate, and processed to alter pore size, particle size, particle shape, and biodegradability.

[0165] In certain embodiments, the methods of the present invention may be administered orally, for example, in the form of capsules, sachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a lozenge (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc. The drug may also be administered as a bolus, lick or paste.

[0166] In solid dosage forms for oral administration (capsules, tablets, pills, sugar-coated tablets, powders, granules, etc.), one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, e.g., sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) hydrating agents, e.g., glycerol (4) Disintegrants, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) Dissolution retarders, e.g., paraffin; (6) Absorption accelerators, e.g., quaternary ammonium compounds; (7) Wetting agents, e.g., cetyl alcohol and glycerol monostearate; (8) Absorbents, e.g., kaolin and bentonite clay; (9) Lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) Colorants. In the case of capsules, tablets and pills, these pharmaceutical compositions may also contain buffers. Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycol and the like.

[0167] Liquid formulations for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, these liquid formulations may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as mixtures thereof. In addition to inert diluents, these oral compositions may also contain adjuvants, such as humectants, emulsifiers, and suspending agents, sweeteners, flavorings, colorants, fragrances, and preservatives.

[0168] The suspension may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, crystalline cellulose, aluminum methhydroxyl, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0169] The compositions of the present invention may also contain adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, extended absorption of injectable pharmaceutical forms can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin.

[0170] It is understood that the drug regimen will be determined by the attending physician, taking into account various factors that modify the action of the compound of the present invention (e.g., TβRII polypeptide). These factors include, but are not limited to, the patient's age, sex and diet, disease severity, timing of administration, and other clinical factors. Optionally, the dosage may vary depending on the type of matrix used in the reconstitution and the type of compound in the composition. The addition of other known growth factors to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of bone growth and / or repair, e.g., by X-ray (including DEXA), histomorphometric determination, and tetracycline labeling.

[0171] In certain embodiments, the present invention also provides gene therapy for the in vivo production of TβRII polypeptides. Such therapy achieves its therapeutic effect by introducing the TβRII polynucleotide sequence into cells or tissues having the disorders listed above. Delivery of the TβRII polynucleotide sequence can be achieved using recombinant expression vectors such as chimeric viruses or colloidal dispersion systems. The use of targeted liposomes is preferred for the therapeutic delivery of the TβRII polynucleotide sequence.

[0172] Various viral vectors that may be used in gene therapy as taught herein include RNA viruses such as adenoviruses, herpesviruses, vaccinia, or, preferably, retroviruses. Preferably, the retroviral vector is a derivative of a mouse or bird retrovirus. Examples of retroviral vectors into which a single foreign gene may be inserted include, but are not limited to, Moloney's mouse leukemia virus (MoMuLV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), and Rous sarcoma virus (RSV). Several further retroviral vectors may incorporate multiple genes. All of these vectors may be transfected with or incorporate genes for selectable markers so that transduced cells can be identified and produced. Retroviral vectors may be made target-specific, for example, by conjugating sugars, glycolipids, or proteins. Preferred targeting is achieved by using antibodies. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or bound to the viral envelope to enable targeted delivery of retroviral vectors containing TβRII polynucleotides. In preferred embodiments, the vector is targeted to bone or cartilage.

[0173] Alternatively, tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol, and env by conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the target gene. The resulting cells release the retroviral vector into the culture medium.

[0174] Another targeted delivery system for TβRII polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include polymer complexes, nanoencapsulants, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of the present invention is liposomes. Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated within aqueous interiors and delivered to cells in a biologically active form (see, e.g., Fraley et al., Trends Biochem. Sci., Vol. 6: p. 77, 1981). Methods for efficient gene transfer using liposome vehicles are known in the art (see, e.g., Mannino et al., Biotechniques, Vol. 6: p. 682, 1988). The composition of liposomes is typically a combination of phospholipids, usually combined with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0175] Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Liposome targeting is also possible, for example, based on organ specificity, cell specificity, and organelle specificity, and is well known in the art.

[0176] This disclosure provides formulations that may vary to include acids and bases for adjusting pH, as well as buffers for maintaining pH within a narrow range. [Examples]

[0177] Example Although the present invention has been described in general terms up to this point, it will be more readily understood by referring to the following examples, which are included merely to illustrate certain embodiments of the present invention and are not intended to limit the invention.

[0178] (Example 1) Production of bioactive GDF15 It has not been biochemically demonstrated that GDF15 (also known as macrophage inhibitory cytokine-1) directly binds to or interacts with any receptor. The applicants initially attempted to identify native receptors with high affinity binding to GDF15 using commercially available human GDF15 (R&D Systems) produced in mammalian CHO cells, but were unsuccessful. Like other ligands in the TGFβ superfamily containing a characteristic cysteine ​​knot motif, mature GDF15 is synthesized with a larger prodomain (Harrison et al., Growth Factors vol. 29: p. 174, 2011; Shi et al., Nature vol. 474: p. 343, 2011) that is removed by cleavage by a furin-like protease at the canonical RXXR site to generate mature dimer GDF15. Since inappropriate or unsuitable ligand purification could be a potential reason for the inactivity of commercially available GDF15, the applicants tested different purification procedures for GDF15.

[0179] Stable expression of GDF15 in CHO cells For further research, the applicants used CHO cells to express human GDF15 (hGDF15) and mouse GDF15 (mGDF15). The amino acid sequence of the native precursor of hGDF15 is shown in Figure 1, and the corresponding nucleotide sequence (with a silent single nucleotide substitution compared to the native sequence) is shown in Figure 2. The native amino acid and nucleotide sequences of the mGDF15 precursor are shown in Figures 3 and 4, respectively. For expression in CHO cells, UCOE®-based constructs encoding human or mouse GDF15 precursors were stably transfected into CHO-PACE cell lines. Clones were selected at 10 nM, 20 nM, and 50 nM methotrexate levels, and any clones that formed colonies (one or two per methotrexate concentration) were then pooled. Gene amplification was not performed because it is difficult to amplify the UCOE® pool while maintaining expression stability. Instead of dilution cloning, we identified high-expression pools and used them to generate hGDF15 and mGDF15.

[0180] Purification of human GDF15 To initiate purification, a conditioned medium derived from CHO cells stably expressing hGDF15 was adjusted to pH 4.7 with acetic acid. After incubation of the medium at ambient temperature for 10 minutes, the precipitate was removed by centrifugation. The supernatant was filtered through a 0.8 μm disposable filter. An SP Sepharose® Fast Flow column (GE Healthcare) was equilibrated with buffer A (20 mM sodium acetate, pH 4.7) and buffer B (20 mM sodium acetate, 1 M NaCl, pH 4.7). Loading was performed at 100 cm / hour. The column was washed with 20% B (200 mM NaCl) until no more protein was eluted from the column, and then washed back with 0% B to remove any residual salts. The proteins were eluted from the column with 50 mM Tris, 6 M urea, pH 8.0 until no more protein was eluted (Tris + urea pool), and then eluted again with 50 mM Tris, 6 M urea, 1 M NaCl, pH 8.0 (Tris + urea + salt pool). Each pool was dialyzed overnight at 4°C in 50 mM 4-morpholine ethanesulfonic acid (MES, pH 6.5).

[0181] GDF15 found in the Tris+urea+salt pool was degraded based on Western blot analysis, and the pool was discarded. The Tris+urea pool was loaded onto a Q Sepharose® Fast Flow column (GE Healthcare) pre-equilibrated with buffer A (50 mM MES, pH 6.5) and buffer B (50 mM MES, 1 M NaCl, pH 6.5). The flow-through was collected, and the column was washed with 10% B (100 mM NaCl), followed by washing over 5 column volumes at 120 cm / hour with a 10–50% B gradient (100–500 mM NaCl). After evaluation of the flow-through and wash fractions by Western blot, the protein was found primarily in the flow-through. This flow-through was injected onto a reverse-phase preparative C4 column (Vydac) mounted on an HPLC using buffer A (water / 0.1% TFA) and buffer B (acetonitrile / 0.1% TFA). A 25-40% B gradient over 1 hour at 4.5 mL / min yielded the best resolution. The collected fractions were processed on an SDS-PAGE gel (Sypro). They were evaluated by Ruby and Western blotting and selected for concentration in a centrifugal evaporator.

[0182] Purification of mouse GDF15 The pH of the acclimatization medium was adjusted to pH 4.7 with acetic acid. After incubation of the medium at ambient temperature for 10 minutes, the precipitate was removed by centrifugation. The supernatant was filtered through a 0.8 μm disposable filter. An SP Sepharose® Fast Flow column (GE Healthcare) was equilibrated with buffer A (20 mM sodium acetate, pH 4.7) and buffer B (20 mM sodium acetate, 1 M NaCl, pH 4.7). Loading was performed at 100–150 cm / hour, and the column was washed with buffer A until no more protein was eluted from the column. Washing was performed with 60% B (600 mM NaCl) over 3–4 column volumes, followed by elution with 100% B (1 M NaCl) over 3–4 column volumes. Elution was continued with 50 mM Tris, 6 M urea, pH 8.0 to remove any proteins still bound to the resin.

[0183] The unreduced samples from the SP column fraction were analyzed by Western blotting. Most of the protein was found in the Tris elution fraction, but since previous experiments have shown that the mGDF15 found in these fractions is essentially inactive, it was not used for further purification. Instead, purification was continued using the protein found in 100% B elution (salt-elution pool). This pool was injected onto a reverse-phase preparative C4 column (Vydac) attached to an HPLC. Buffer A was water / 0.1% TFA, and buffer B was acetonitrile / 0.1% TFA. The protein was eluted at 4.5 mL / min over 1 hour with a 25-40% B gradient. After evaluation of the reverse-phase column fraction by SDS-PAGE gel (Sypro Ruby) and Western blotting, the fraction containing pure mGDF15 was pooled and concentrated in a centrifugal evaporator.

[0184] The identities of hGDF15 and mGDF15 were confirmed by N-terminal sequencing. Purified GDF15 of both types stimulated SMAD2 / 3 phosphorylation in two different cell lines, thereby providing confirmation of ligand activity.

[0185] (Example 2) Identification of TGFβ superfamily receptors with high affinity binding to GDF15 After obtaining the active GDF15 protein, receptor-Fc fusion proteins, including those of the TGFβ superfamily receptors, were screened for binding to human or mouse GDF15, which was generated and purified as described in Example 1. These fusion proteins incorporated the IgG1 Fc domain and were either purchased from R&D Systems or generated in-house. Of the five type II receptors (TGFβ receptor type II, activin receptor type IIA, activin receptor type IIB, BMP receptor type II, and MIS receptor type II), only TGFβ receptor type II (TβRII) showed detectable binding to GDF15, as determined by surface plasmon resonance using the captured receptor-Fc fusion protein (k a = 2.92 × 10 5 M -1 s -1 ;k d =0.001s -1 ). hGDF15 has an equilibrium dissociation constant of 9.56 nM (K D At 37°C, it bound to the captured hTβRII-Fc. None of the seven type I receptors (ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, and ALK7) showed detectable binding to GDF15 (mGDF15 at 20 nM or 200 nM).

[0186] Human TβRII naturally occurs as at least two isoforms—A (long) and B (short)—generated by alternative splicing in the extracellular domain (ECD) (Figures 5 and 6). The hTβRII-hG1Fc fusion protein (R&D Systems) used in the above screening is wild-type TβRII 短 It incorporates isoforms. In follow-up analysis, wild-type TβRII 長 The affinity of mGDF15 binding to fusion proteins that incorporate isoforms (R&D Systems) is determined by surface plasmon resonance, specifically TβRII.短 It was found to be very similar to fusion proteins that incorporate isoforms (at 37°C K D (These were 2.7 nM and 4.8 nM, respectively). Since a general equivalence of these short and long isoforms with respect to GDF15 binding was observed, the applicants then proposed hTβRII fused with the human IgG2 Fc domain at its C-terminus via a minimal linker. 短 A receptor-Fc fusion protein consisting of the wild-type ECD (SEQ ID NO: 7) was generated. Unless otherwise noted, amino acid position numbering for variants based on the short and long isoforms of TβRII refers to the corresponding positions in the native precursor SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0187] Considering the high affinity binding of TβRII to GDF15, the inventors tested whether TβRII could be used as an inhibitor of GDF15. Fusion protein hTβRII 短 When (23~159)-hG2Fc was tested in A549 cells transfected with a reporter gene containing the CAGA-12 promoter construct, it was found to induce hGDF15-induced gene activation in these cells, resulting in an IC50 of 0.15~0.5 nM. 50 It was found that it inhibits hTβRII. 短Potent inhibition of GDF15 signaling by ECD provides further evidence that TβRII is a high-affinity receptor for GDF15. Although GDF15 did not show detectable binding to ALK5 under cell-free conditions, repression of endogenous ALK5 mRNA by siRNA methodology significantly reduced mGDF15-mediated signaling in A549 cells (human lung epithelial cell line) compared to control treatment. In contrast, repression of other type I receptors (ALK2, ALK3, ALK4, and ALK7) by siRNA methodology did not alter GDF15-mediated signaling in A549 cells. These results indicate that the GDF15 ternary signaling complex includes ALK5 (TGFβ receptor type I) as its type I receptor, and therefore provide supporting evidence for TβRII as a functional type II receptor for GDF15.

[0188] (Example 3) Receptor fusion protein variant generation TβRII ECD variant Since TβRII also binds to TGFβ1 and TGFβ3 with high affinity, native TβRII-Fc fusion proteins influence the signaling of these ligands as well as GDF15. In some therapeutic settings, this broader spectral ligand binding may be advantageous, while in others, a more selective molecule may be preferable. Therefore, the applicants searched for polypeptides with enhanced or reduced selectivity for GDF15 by generating fusion proteins containing variants of human TβRII ECD. Wild-type hTβRII is shown below. 短 The (23-159) sequence (sequence number 7) served as the basis for the following five receptor ECD variants (sequence numbers 8-12): wild-type hTβRII 短 (23-159) were fused to the Fc portion of IgG2 to generate a novel base Fc fusion construct. See Sequence IDs 50, 51, and 52 below. [ka] (1) The following hTβRII 短 (23~159 / D110K) Amino acid sequence (SEQ ID NO: 8). Substituted residues in the sequence are underlined. [ka] (2) The following hTβRII with the N-terminus truncated 短 (29-159) Amino acid sequence (SEQ ID NO: 9). [ka] (3) hTβRII with the N-terminus truncated as shown below 短 (35-159) Amino acid sequence (SEQ ID NO: 10). [ka] (4) hTβRII with the C-terminus truncated as shown below 短 (23-153) Amino acid sequence (SEQ ID NO: 11). [ka] (5) hTβRII with the C-terminus truncated as shown below 短 (23~153 / N70D) Amino acid sequence (SEQ ID NO: 12). Substituted residues in the sequence are underlined. [ka]

[0189] The applicants identified the following wild-type hTβRII 長 Five corresponding variants (sequence numbers 14-17) based on the (23-184) sequence (sequence number 13) are also assumed, with 25 amino acid insertions underlined in the sequence. Note that splicing will result in a conservative amino acid substitution (Val→Ile) at a position adjacent to the C-terminus for this insertion. Several hTβRII 短 Variants and their hTβRII 長 Figure 7 shows the relationships in the arrangement between corresponding objects. [ka] (1) The following hTβRII 長 (23~184 / D135K) Amino acid sequence (SEQ ID NO: 14). Substituted residues in the sequence are double-underlined. [ka] (2) The following hTβRII with the N-terminus truncated 長 (29-184) Amino acid sequence (SEQ ID NO: 15). [ka] [ka] (3) hTβRII with the N-terminus truncated as shown below 長 (60-184) Amino acid sequence (same as SEQ ID NO: 10). [ka] (4) hTβRII with the C-terminus truncated as shown below 長 (23-178) Amino acid sequence (SEQ ID NO: 16). [ka] (5) hTβRII with the C-terminus truncated as shown below 長 (23~178 / N95D) Amino acid sequence (SEQ ID NO: 17), substituted residues in the sequence are double-underlined. [ka]

[0190] Further TβRII ECD variants include: (A) The following hTβRII with the N-terminus and C-terminus truncated. 短 (35~153) or hTβRII 長 (60-178) Amino acid sequence (SEQ ID NO: 47). [ka] (B) hTβRII with the N-terminus and C-terminus truncated as shown below 短 (29-153) Amino acid sequence (SEQ ID NO: 48). [ka] [ka] (C) hTβRII with the N-terminus and C-terminus truncated as shown below 長 (29-178) Amino acid sequence (SEQ ID NO: 49). [ka]

[0191] Any of the above variants (SEQ ID NOs: 8-12, 14-17, and 47-49) may incorporate a 36-amino acid insertion (SEQ ID NO: 18) between pairs of glutamic acid residues located near the C-terminus of hTβRII ECD (positions 151 and 152 of SEQ ID NO: 5, or positions 176 and 177 of SEQ ID NO: 6), similar to the naturally occurring hTβRII isoform C (Konrad et al., BMC Genomics 8: p. 318, 2007). [ka]

[0192] As an example, a pair of glutamic acid residues adjacent to an optional insertion site is hTβRII 短 The variants (29-159) (sequence number 9) are shown below (underlined). [ka]

[0193] Fc domain variant The five hTβRIIs mentioned above 短Each variant was fused (via a minimal linker) at the C-terminus of a human IgG2 Fc domain having the following amino acid sequence (SEQ ID NO: 19), generating an hTβRII-hFc fusion protein: [ka]

[0194] The applicants have developed full-length human IgG1 Fc(hG1Fc) (SEQ ID NO: 20, hereafter) and human IgG1 Fc(hG1Fc) with the N-terminus truncated. 短 We envision an hTβRII-hFc fusion protein containing an alternative Fc domain, including (SEQ ID NO: 21, hereafter). Optionally, a polypeptide unrelated to the Fc domain can be conjugated in its place. [ka]

[0195] Leader array variant The following three leader sequences were considered: [ka]

[0196] Expression of hTβRII-hFc fusion protein The selected hTβRII-hFc protein variants incorporate the TPA reader and have the unprocessed amino acid sequences shown in SEQ ID NOs. 25, 29, 33, 37, and 41 (see Example 5). The corresponding nucleotide sequences for these variants are SEQ ID NOs. 26, 30, 34, 38, and 42. The selected hTβRII-hFc variants, each possessing a G2Fc domain (SEQ ID NO: 19), were expressed in HEK-293 cells and purified from conditioned medium by filtration and protein A chromatography. The purity of the samples for the reporter gene assay was assessed by SDS-PAGE and Western blotting.

[0197] The applicants envision additional hTβRII-hFc protein variants having the unprocessed amino acid sequences shown in SEQ ID NOs: 27, 31, 35, 39 and 43, as well as the corresponding nucleotide sequences shown in SEQ ID NOs: 28, 32, 36, 40 and 44.

[0198] Wild-type short construct hTβRII 短 (23-159)-hG2Fc amino acid sequence (SEQ ID NO: 50) is shown below.

Chemical Structure

[0199] This protein was expressed from a construct (SEQ ID NO: 52) containing a TPA leader sequence as shown below. The dotted underline indicates the leader and the solid underline indicates the linker.

Chemical Structure

[0200] The nucleic acid sequence encoding SEQ ID NO: 52 is shown below:

Chemical Structure

Chemical Structure

[0202] On the first day of the assay, A549 cells (ATCC®: CCL-185®) were divided into 6.5 × 10⁶ wells. 4 The cells were distributed into 48-well plates. On day 2, a solution containing 10 μg pGL3 (CAGA)12, 100 ng pRLCMV, 30 μl X-tremeGENE 9 (Roche Applied Science), and 970 μl OptiMEM (Invitrogen) was pre-incubated for 30 minutes and then added to Eagle's Minimal Essential Medium (EMEM, ATCC®) supplemented with 0.1% BSA. This was then applied to the plated cells (500 μl / well) over an overnight incubation at room temperature. On day 3, the medium was removed and the cells were incubated overnight at 37°C with a mixture of ligands and inhibitors prepared as described below.

[0203] Serial dilutions of the test material were performed in 200 μl volumes of assay buffer (EMEM + 0.1% BSA) in a 48-well plate. Equivolutes of assay buffer containing the test ligand were added to obtain a final ligand concentration equal to the predetermined EC50. Human GDF15 and mouse GDF15 were generated in-house (see above), while human TGFβ1, human TGFβ2, and human TGFβ3 were obtained from PeproTech. The test solution was incubated at 37°C for 30 minutes, and then 250 μl of the mixture was added to all wells. The test material at each concentration was determined in double dilutions. After overnight incubation with the test solution, the cells were rinsed with phosphate-buffered saline, and then lysed in passive lysis buffer (Promega). The cells were dissolved in E1941 and stored overnight at -70°C. On the final day (day 4), the plates were gently warmed to room temperature with shaking. The cell lysates were transferred in double rows to a chemiluminescent plate (96 wells), and the normalized luciferase activity was determined by luminometer analysis using reagents from the Dual-Luciferase Reporter Assay system (Promega E1980).

[0204] This assay was used to screen receptor fusion protein variants for potential inhibitory effects of TβRII ligands on cellular signaling. (Wild-type TβRII) 短 -Fc and TβRII 長 Consistent with previous reports on -Fc (del Re et al., J Biol Chem vol. 279: p. 22765, 2004), none of the variants tested, even at high concentrations, were able to inhibit TGFβ2. However, the hTβRII-hFc variant unexpectedly showed differential inhibition of cellular signaling mediated by GDF15, TGFβ1, and TGFβ3. Wild-type TβRII 短 Compared to (23~159)-G2Fc, TβRII 短The (23~159 / D110K)-G2Fc variant showed potent inhibition of GDF15 but loss of inhibition of TGFβ1 and significantly reduced inhibition of TGFβ3 (approximately 50-fold) (see table below). Position 110 is located within the "hook" region of TβRII (Radaev et al., J Biol Chem 285: p. 14806, 2010), but it has not been suggested that it confers selectivity among the recognized TβRII ligands TGFβ1, TGFβ2, and TGFβ3. Therefore, this variant exhibits a differential ligand inhibition profile, with GDF15 being most potently inhibited, TGFβ1 being the weakest inhibited, and TGFβ3 being inhibited to an intermediate degree. [Table 1]

[0205] In the second experiment, the potential of a variant with a truncated N-terminus TβRII ECD was compared to that of a full-length wild-type TβRII ECD. As shown in the table below, TβRII 短 (29~159)-G2Fc and TβRII 短 (35~159)-G2Fc is TβRII 短 Compared to (23~159)-G2Fc (wild-type), the variants showed a significantly reduced ability to inhibit TGFβ3, but no reduction (N'Δ6) or only a slight reduction (N'Δ12) in their ability to inhibit GDF15. The effect of N-terminal truncation on TGFβ1 inhibition was intermediate in magnitude compared to the wild-type. Therefore, these two variants exhibit a differential ligand inhibition profile in which GDF15 is most strongly inhibited, TGFβ3 is most weakly inhibited, and TGFβ1 is inhibited to an intermediate degree. [Table 2]

[0206] In the third experiment, the inventors determined the effect on the potential of the N70D substitution in the C-terminally truncated TβRII ECD. This asparagine residue represents a potential glycosylation site. As shown in the following table, TβRII 短 (23~153 / N70D)-G2Fc showed a greatly diminished ability to inhibit TGFβ1 and a virtually undiminished ability to inhibit TGFβ3 compared to TβRII 短 (23~153)-G2Fc. The effect of the N70D substitution on the inhibition of GDF15, compared to both TβRII 短 (23~153)-G2Fc and the wild type, was intermediate in magnitude. Thus, the C-terminally truncated variant with the N70D substitution shows a differential ligand inhibition profile where TGFβ3 is most potently inhibited, TGFβ1 is least inhibited, and GDF15 is inhibited to an intermediate extent.

Table 3

[0207] Taken together, these results demonstrate that the applicants have generated truncations and mutations of TβRII ECD that exhibit greatly different ligand binding profiles. In particular, this demonstration reveals that properly expressed and purified GDF15 can interact directly with TβRII and can be differentially inhibited by fusion proteins containing variants of TβRII ECD. The activity profiles of these variants can be summarized in the following table.

Table 4

[0208] The inventors predict that the TβRII 短 ECD counterparts of these TβRII 長 ECD variants will exhibit similar ligand selectivity. Furthermore, the C’Δ6-truncated ECDs (e.g., TβRII 短 and TβRII 長The sequences of SEQ ID NO: 11 and 16 for the isoform introduce mutations and N-terminal truncations of TβRII 短 or TβRII 長 can be used as the nucleotide sequences for it.

[0209] (Example 5) Exemplary hTβRII-hFc nucleic acids and proteins This example summarizes nucleic acid constructs that can be used to express the TβRII construct in HEK-293 or CHO cells according to the methods provided herein to provide proteins isolated from cell cultures. In each case, the mature protein isolated from the cell culture has the leader sequence (dotted underline in each of the following sequences) removed.

[0210] Item 1 shows the amino acid sequence of hTβRII 短 (23-159 / D110K)-hG2Fc (SEQ ID NO: 25). The double underline indicates the D110K substitution. The dotted underline indicates the leader, and the solid underline indicates the linker. [Chemical formula] [Chemical formula]

[0211] Item 2 shows the nucleotide sequence encoding hTβRII 短 (23-159 / D110K)-hG2Fc (SEQ ID NO: 26). The double underline indicates the D110K substitution. The dotted underline indicates the leader, and the solid underline indicates the linker. [Chemical formula]

[0212] Item 3 is hTβRII 短 (23-159 / D110K)-hG1Fc 短shows the amino acid sequence (SEQ ID NO: 27). The double underline indicates the D110K substitution. The dotted underline indicates the leader, and the solid underline indicates the linker. [Chemical formula]

[0213] Item 4 shows the nucleotide sequence (SEQ ID NO: 28) encoding hTβRII 短 (23~159 / D110K)-hG1Fc 短 The double underline indicates the D110K substitution. The dotted underline indicates the leader, and the solid underline indicates the linker. [Chemical formula] [Chemical formula]

[0214] Item 5 shows the amino acid sequence (SEQ ID NO: 29) of hTβRII 短 (29~159)-hG2Fc. The dotted underline indicates the leader, and the solid underline indicates the linker. [Chemical formula]

[0215] Item 6 shows the nucleotide sequence (SEQ ID NO: 30) encoding hTβRII 短 (29~159)-hG2Fc. The dotted underline indicates the leader, and the solid underline indicates the linker. [Chemical formula] [Chemical formula]

[0216] Item 7 shows hTβRII 短 (29~159)-hG1Fc 短The amino acid sequence (SEQ ID NO: 31) is shown. Dotted underlines indicate leaders, and solid underlines indicate linkers. [ka]

[0217] Item 8 is hTβRII 短 (29~159)-hG1Fc 短 The nucleotide sequence encoding (SEQ ID NO: 32) is shown. The dotted underline indicates the leader, and the solid underline indicates the linker. [ka] [ka]

[0218] Item 9 is hTβRII 短 The amino acid sequence of (35~159)-hG2Fc (SEQ ID NO: 33) is shown. Dotted lines indicate leaders, and solid lines indicate linkers. [ka]

[0219] Item 10 is hTβRII 短 The nucleotide sequence encoding (35~159)-hG2Fc (SEQ ID NO: 34) is shown. The dotted underline indicates the leader, and the solid underline indicates the linker. [ka] [ka]

[0220] Item 11 is hTβRII 短 (35~159)-hG1Fc 短 The amino acid sequence (SEQ ID NO: 35) is shown. Dotted underlines indicate leaders, and solid underlines indicate linkers. [ka]

[0221] Item 12 is hTβRII 短 (35~159)-hG1Fc 短 The nucleotide sequence encoding (SEQ ID NO: 36) is shown. The dotted underline indicates the leader, and the solid underline indicates the linker. [ka] [ka]

[0222] Item 13 is hTβRII 短 The amino acid sequence of (23~153)-hG2Fc (SEQ ID NO: 37) is shown. Dotted lines indicate leaders, and solid lines indicate linkers. [ka]

[0223] Item 14 is hTβRII 短 The nucleotide sequence encoding (23~153)-hG2Fc (SEQ ID NO: 38) is shown. The dotted underline indicates the leader, and the solid underline indicates the linker. [ka] [ka]

[0224] Item 15 is hTβRII 短 (23~153)-hG1Fc 短 The amino acid sequence (SEQ ID NO: 39) is shown. Dotted underlines indicate leaders, and solid underlines indicate linkers. [ka]

[0225] Item 16 is hTβRII 短 (23~153)-hG1Fc 短 The nucleotide sequence encoding (SEQ ID NO: 40) is shown. The dotted underline indicates the leader, and the solid underline indicates the linker. [ka] [ka]

[0226] Item 17 is hTβRII 短 The amino acid sequence of (23~153 / N70D)-hG2Fc (SEQ ID NO: 41) is shown. Double underlines indicate N70D substitutions. Dotted underlines indicate leaders, and solid underlines indicate linkers. [ka]

[0227] Item 18 is hTβRII 短 The nucleotide sequence encoding (23~153 / N70D)-hG2Fc (SEQ ID NO: 42) is shown. Double underlines indicate N70D substitutions. Dotted underlines indicate leaders, and solid underlines indicate linkers. [ka] [ka]

[0228] Item 19 is hTβRII 短 (23~153 / N70D)-hG1Fc 短 The amino acid sequence (SEQ ID NO: 43) is shown. Double underlines indicate N70D substitutions. Dotted underlines indicate leaders, and solid underlines indicate linkers. [ka]

[0229] Item 20 is hTβRII短 (23~153 / N70D)-hG1Fc 短 The nucleotide sequence encoding (SEQ ID NO: 44) is shown. A double underline indicates an N70D substitution. A dotted underline indicates a leader, and a solid underline indicates a linker. [ka] [ka]

[0230] Item 21 is hTβRII 短 The mature amino acid sequence of (23~159 / D110K)-hG2Fc (i.e., without a leader sequence) (SEQ ID NO: 53) is shown. Double underlines indicate D110K substitutions. Single underlines indicate linkers. [ka]

[0231] Item 22 is hTβRII 短 (23~159 / D110K)-hG1Fc 短 The mature amino acid sequence (i.e., without a leader sequence) (SEQ ID NO: 54) is shown. Double underlines indicate D110K substitutions. Single underlines indicate linkers. [ka] [ka]

[0232] Item 23 is hTβRII 短 The mature amino acid sequence of (29~159)-hG2Fc (i.e., without a leader sequence) (SEQ ID NO: 55) is shown. The single underline indicates a linker. [ka]

[0233] Item 24 is hTβRII 短(29~159)-hG1Fc 短 The mature amino acid sequence (i.e., without the leader sequence) (SEQ ID NO: 56) is shown. The single underline indicates a linker. [ka]

[0234] Item 25 is hTβRII 短 The mature amino acid sequence of (35~159)-hG2Fc (i.e., without a leader sequence) (SEQ ID NO: 57) is shown. The single underline indicates a linker. [ka] [ka]

[0235] Item 26 is hTβRII 短 (35~159)-hG1Fc 短 The mature amino acid sequence (i.e., without a leader sequence) (SEQ ID NO: 58) is shown. A single underline indicates a linker. [ka]

[0236] Item 27 is hTβRII 短 The mature amino acid sequence of (23~153)-hG2Fc (i.e., without a leader sequence) (SEQ ID NO: 59) is shown. The single underline indicates a linker. [ka]

[0237] Item 28 is hTβRII 短 (23~153)-hG1Fc 短 The mature amino acid sequence (i.e., without the leader sequence) (SEQ ID NO: 60) is shown. The single underline indicates a linker. [ka] [ka]

[0238] Item 29 is hTβRII 短 The mature amino acid sequence of (23~153 / N70D)-hG2Fc (i.e., without a leader sequence) (SEQ ID NO: 61) is shown. Double underlines indicate N70D substitutions. Single underlines indicate linkers. [ka]

[0239] Item 30 is hTβRII 短 (23~153 / N70D)-hG1Fc 短 The mature amino acid sequence (i.e., without a leader sequence) (SEQ ID NO: 62) is shown. Double underlines indicate N70D substitutions. Single underlines indicate linkers. [ka]

[0240] Built-in by reference All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference.

[0241] While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not limiting. Many variations will become apparent to those skilled in the art through a review of this specification and the following claims. The full scope of the invention should be determined by referring to the claims together with the full scope of their equivalents and to the specification together with such variations. For example, the present invention provides the following items: (Item 1) A TβRII fusion polypeptide comprising a first amino acid sequence derived from the extracellular domain of TβRII and a heterologous amino acid sequence, wherein the first amino acid sequence is a) A sequence that begins at any position between 23 and 35 of sequence number 5 and ends at any position between 153 and 159 of sequence number 5, or b) A sequence that begins at position 23 to 60 of sequence number 6 and ends at position 178 to 184 of sequence number 6. A TβRII fusion polypeptide having at least 80% identical amino acid sequence to [the given molecule]. (Item 2) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 23 of SEQ ID NO: 5 and ends at position 159 of SEQ ID NO: 5. (Item 3) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 29 of SEQ ID NO: 5 and ends at position 159 of SEQ ID NO: 5. (Item 4) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 35 of SEQ ID NO: 5 and ends at position 159 of SEQ ID NO: 5. (Item 5) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 23 of SEQ ID NO: 5 and ends at position 153 of SEQ ID NO: 5. (Item 6) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 29 of SEQ ID NO: 5 and ends at position 153 of SEQ ID NO: 5. (Item 7) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 35 of SEQ ID NO: 5 and ends at position 153 of SEQ ID NO: 5. (Item 8) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 23 of SEQ ID NO: 6 and ends at position 184 of SEQ ID NO: 6. (Item 9) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 29 of SEQ ID NO: 6 and ends at position 184 of SEQ ID NO: 6. (Item 10) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 23 of SEQ ID NO: 6 and ends at position 178 of SEQ ID NO: 6. (Item 11) The TβRII fusion polypeptide described in item 1, wherein the first amino acid sequence consists of a sequence that begins at position 29 of SEQ ID NO: 6 and ends at position 178 of SEQ ID NO: 6. (Item 12) The TβRII fusion polypeptide according to any one of items 1 to 11, wherein the first amino acid sequence comprises a sequence having D at the position corresponding to position 36 of SEQ ID NO: 47 and / or K at the position corresponding to position 76 of SEQ ID NO: 47. (Item 13) A TβRII fusion polypeptide comprising a first amino acid sequence or its active fragment that is at least 80% identical to the sequence of SEQ ID NO: 7 or SEQ ID NO: 13, and a second heterologous portion, wherein the first amino acid sequence has D at the position corresponding to position 36 of SEQ ID NO: 47 and / or K at the position corresponding to position 76 of SEQ ID NO: 47. (Item 14) The TβRII fusion polypeptide described in item 13, wherein the first amino acid sequence includes an N-terminal truncation of 1 to 12 amino acids corresponding to amino acids 1 to 12 of SEQ ID NO: 7 or 1 to 37 amino acids corresponding to amino acids 1 to 37 of SEQ ID NO: 13. (Item 15) The TβRII fusion polypeptide described in item 13 or 14, wherein the first amino acid sequence includes a six-amino acid N-terminal truncation corresponding to amino acids 1-6 of SEQ ID NO: 7 or SEQ ID NO: 13. (Item 16) The TβRII fusion polypeptide described in item 13 or 14, wherein the first amino acid sequence comprises an N-terminal truncation of 12 amino acids corresponding to amino acids 1-12 of SEQ ID NO: 7 or 37 amino acids corresponding to amino acids 1-37 of SEQ ID NO: 13. (Item 17) The TβRII fusion polypeptide according to any one of items 13 to 16, wherein the first amino acid sequence includes a C-terminal truncation of 1 to 6 amino acids corresponding to amino acids 137 to 132 of SEQ ID NO: 7 or amino acids 162 to 157 of SEQ ID NO: 13. (Item 18) A TβRII fusion polypeptide according to any one of items 13 to 17, wherein the first amino acid sequence includes a six-amino acid C-terminal truncation corresponding to amino acids 132-137 of SEQ ID NO: 7 or amino acids 157-162 of SEQ ID NO: 13. (Item 19) The TβRII fusion polypeptide described in any one of items 1 to 18, wherein the first amino acid sequence includes an insertion corresponding to SEQ ID NO: 18 between the residues corresponding to positions 117 and 118 of SEQ ID NO: 47. (Item 20) The TβRII fusion polypeptide according to any one of items 1 to 19, wherein the heterogeneous portion comprises one or more polypeptide portions that enhance one or more of the following: in vivo stability, in vivo half-life, uptake / administration, tissue localization or distribution, protein complex formation, and / or purification. (Item 21) The TβRII fusion polypeptide according to any one of items 1 to 20, wherein the heterogeneous portion comprises an immunoglobulin Fc domain and a polypeptide portion selected from serum albumin. (Item 22) The TβRII fusion polypeptide according to item 21, wherein the immunoglobulin Fc domain is linked to the TβRII polypeptide by a linker. (Item 23) The TβRII fusion polypeptide according to any one of items 1 to 22, wherein the polypeptide comprises one or more modified amino acid residues selected from glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid portion, and amino acids conjugated to an organic derivatizer. (Item 24) The TβRII fusion polypeptide described in item 23, wherein the polypeptide is glycosylated. (Item 25) A TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. (Item 26) A TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. (Item 27) A TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. (Item 28) A TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. (Item 29) A TβRII fusion polypeptide comprising a first amino acid sequence consisting of a portion of the extracellular domain of TβRII containing an amino acid sequence selected from SEQ ID NOs. 7-17 and 47-49, and a second heterologous portion. (Item 30) A polypeptide containing an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. (Item 31) A polypeptide containing an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. (Item 32) A polypeptide containing an amino acid sequence that is at least 98% identical to an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. (Item 33) A polypeptide containing an amino acid sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. (Item 34) A polypeptide containing an amino acid sequence selected from SEQ ID NOs. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. (Item 35) A TβRII polypeptide comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to a complement of a nucleotide sequence selected from SEQ ID NOs. 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44. (Item 36) 1 x 10 -8 Equilibrium dissociation constant (K) less than M D A polypeptide according to any one of items 1 to 35, which binds to human GDF15. (Item 37) A polypeptide according to any one of items 1 to 36, having a glycosylation pattern characteristic of the expression of the polypeptide in CHO cells. (Item 38) A homodimer containing two polypeptides described in any of items 1 through 37. (Item 39) An isolated polynucleotide containing the coding sequence of a polypeptide described in any one of items 1 through 37. (Item 40) Recombinant polynucleotides comprising a promoter sequence operably ligated to the polynucleotide described in item 39. (Item 41) Cells transformed with isolated polynucleotides as described in item 35 or recombinant polynucleotides as described in item 40. (Item 42) A mammalian cell, as described in item 41. (Item 43) Cells as described in item 42, which are CHO cells or human cells. (Item 44) A pharmaceutical preparation comprising a polypeptide as described in any of items 1 to 37 or a homodimer as described in item 38 and a pharmaceutically acceptable excipient. (Item 45) A method for modulating a cellular response to a TGFβ superfamily member, comprising the step of exposing the cells to a polypeptide described in any one of items 1 to 37 or a homodimer described in item 38. (Item 46) A method for treating a disease or condition associated with a TGFβ superfamily member in a patient in need thereof, comprising the step of administering to the patient an effective amount of a polypeptide described in any of items 1 to 37 or a homodimer described in item 38. (Item 47) The method according to item 46, wherein the TGFβ superfamily member is TGFβ1, TGFβ3, or GDF15. (Item 48) The method described in item 46 or 47, wherein the disease or condition is cancer. (Item 49) The method according to item 48, wherein the cancer is selected from gastric cancer, intestinal cancer, skin cancer, breast cancer, melanoma, bone cancer, and thyroid cancer. (Item 50) The method according to item 46 or 47, wherein the disease or condition is a fibrous or sclerotic disease or disorder. (Item 51) The method according to item 50, wherein the fibrous or sclerotic disease or disorder is selected from scleroderma, atherosclerosis, hepatic fibrosis, diffuse systemic sclerosis, glomerulonephritis, nerve scarring, skin scarring, radiation-induced fibrosis, hepatic fibrosis, and myelofibrosis. (Item 52) The method described in item 46 or 47, wherein the disease or condition is a heart disease. (Item 53) The method according to item 46 or 47, wherein the disease or condition is selected from hereditary hemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, tortuosomatic artery syndrome, pre-eclampsia, atherosclerosis, restenosis, and hypertrophic cardiomyopathy / congestive heart failure. (Item 54) An antibody or its antigen-binding fragment that binds to GDF15 and blocks the interaction between GDF15 and TβRII. (Item 55) A GDF15 polypeptide containing the amino acid sequence of SEQ ID NO: 1, or a fragment thereof bound to TβRII, which is at least 95% pure with respect to protein impurities. (Item 56) A GDF15 polypeptide containing the amino acid sequence of SEQ ID NO: 1, or a fragment thereof conjugated to a polypeptide described in any one of items 1 to 37, which is at least 95% pure with respect to protein impurities. (Item 57) 10 -8 Equilibrium dissociation constants (K) less than or equal to M D The GDF15 polypeptide described in item 55, which binds to TβRII. (Item 58) 10 -8 Equilibrium dissociation constants (K) less than or equal to M D ) a GDF15 polypeptide as described in item 56, which binds to a polypeptide as described in any one of items 1 to 37. (Item 59) A GDF15 polypeptide, as described in any of items 55 to 58, produced by expression in CHO cells. (Item 60) A method for concentrating or purifying GDF15, comprising the step of contacting a sample containing GDF15 with a polypeptide described in any of items 1 to 37. (Item 61) A polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 50. (Item 62) A polypeptide as described in item 61, comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 50. (Item 63) A polypeptide as described in item 61, comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 50. (Item 64) A polypeptide as described in item 61, containing an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 50. (Item 65) A polypeptide described in item 61, comprising an amino acid sequence identical to the amino acid sequence of sequence number 50. (Item 66) A nucleic acid encoding a polypeptide as described in any of items 61 to 65. (Item 67) A nucleic acid containing the nucleic acid sequence of sequence number 51. (Item 68) A polypeptide produced by expressing the nucleic acid described in item 67 in mammalian cells. (Item 69) The polypeptide described in item 68, wherein the mammalian cells are Chinese hamster ovary cells (CHO cells).

Claims

1. a) The first portion derived from the extracellular domain of the human TβRII A isoform, consisting of the amino acid sequence of SEQ ID NO: 13; b) A heterogeneous portion which is an immunoglobulin Fc domain, comprising an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 19, 20, and 21; c) A linker containing 3 to 50 amino acids connecting the first portion to the dissimilar portion A TβRII fusion polypeptide comprising, The aforementioned different portion is the C-terminus of the first portion, Here, the fusion polypeptide does not contain amino acids corresponding to amino acids 1-22 and 185-592 of SEQ ID NO: 6, and in homodimer form, binds to transforming growth factor β1 and transforming growth factor β3, and is a TβRII fusion polypeptide.

2. The TβRII fusion polypeptide according to claim 1, wherein the heterogeneous portion comprises one of the amino acid sequences of SEQ ID NOs: 19, 20, and 21.

3. The TβRII fusion polypeptide according to claim 1 or 2, wherein the heterogeneous portion consists of one amino acid sequence from sequence numbers 19, 20, and 21.

4. The TβRII fusion polypeptide according to claim 2, wherein the linker comprises 3 to 10 amino acids.

5. The TβRII fusion polypeptide according to any one of claims 1 to 4, wherein the polypeptide comprises one or more modified amino acid residues selected from glycosylated amino acids, PEG-modified amino acids, farnesyl-modified amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid portion, and amino acids conjugated to an organic derivatizer.

6. The TβRII fusion polypeptide according to claim 5, wherein the polypeptide is glycosylated.

7. The TβRII fusion polypeptide according to any one of claims 1, 4 to 6, wherein the heterogeneous portion includes an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO:

21.

8. The TβRII fusion polypeptide according to claim 7, wherein the heterogeneous portion includes the amino acid sequence of SEQ ID NO:

21.

9. In the homodimer form, the TβRII fusion polypeptide according to any one of claims 1 to 8 binds to transforming growth factor β1, transforming growth factor β3, and GDF15.

10. The polypeptide according to any one of claims 1 to 9, having a glycosylation pattern characteristic of the expression of the polypeptide in CHO cells.

11. A homodimer comprising two polypeptides according to any one of claims 1 to 10.

12. An isolated polynucleotide comprising the coding sequence of a polypeptide according to any one of claims 1 to 10.

13. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide according to claim 12.

14. Cells transformed with the isolated polynucleotide described in claim 12 or the recombinant polynucleotide described in claim 13.

15. The cell according to claim 14, which is a mammalian cell.

16. The cell according to claim 15, which is a CHO cell or a human cell.

17. A pharmaceutical preparation comprising a polypeptide according to any one of claims 1 to 10 or a homodimer according to claim 11 and a pharmaceutically acceptable excipient.

18. A composition for use in modulating a cellular response to a TGFβ superfamily member in a patient requiring such use, comprising a polypeptide according to any one of claims 1 to 10 or a homodimer according to claim 11.

19. A composition for use in treating a disease or condition associated with a TGFβ superfamily member in a patient who requires it, comprising an effective amount of the polypeptide according to any one of claims 1 to 10 or the homodimer according to claim 11.

20. The composition according to claim 19, wherein the TGFβ superfamily member is TGFβ1, TGFβ3, or GDF15.

21. The composition according to claim 19 or 20, wherein the disease or condition is cancer.

22. The composition according to claim 21, wherein the cancer is selected from gastric cancer, intestinal cancer, skin cancer, breast cancer, melanoma, bone cancer, and thyroid cancer.

23. The composition according to claim 19 or 20, wherein the disease or condition is a fibrous or sclerotic disease or disorder.

24. The composition according to claim 23, wherein the fibrous or sclerotic disease or disorder is selected from scleroderma, atherosclerosis, hepatic fibrosis, diffuse systemic sclerosis, glomerulonephritis, nerve scarring, skin scarring, radiation-induced fibrosis, hepatic fibrosis, and myelofibrosis.

25. The composition according to claim 19 or 20, wherein the disease or condition is a heart disease.

26. The composition according to claim 19 or 20, wherein the disease or condition is selected from hereditary hemorrhagic telangiectasia (HHT), Marfan syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, tortuosomatic syndrome, pre-eclampsia, atherosclerosis, restenosis, and hypertrophic cardiomyopathy / congestive heart failure.

27. The composition according to claim 19, wherein the disease or condition is a fibrous disorder.

28. The composition according to any one of claims 19 to 23 or 27, wherein the disease or condition affects lung epithelial cells.

29. The composition according to any one of claims 19 to 23 or 27, wherein the disease or condition affects lung cells.

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