Transforming growth factor beta (TGFβ) binding agents and uses thereof

Abstract

Tetravalent TGFβ receptor-extracellular domain-based traps with tailored isoform specificity profiles for neutralizing TGFβ ligands and methods for their use in treating diseases and conditions associated with TGFβ, particularly TGFβ1 and TGFβ3, are provided. In particular, TGFβ binding agents designed to tailor TGFβ isoform specificity to maximize therapeutic efficacy in specific disease indications while minimizing adverse effects are provided. The TGFβ binding agents comprise two polypeptides assembled via a multimerization domain, each polypeptide having two TGFβ II receptor (TGFβR) ligand-binding domains linked as a doublet, where a linker is selected to tailor isoform specificity.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 038,290, filed June 12, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to an electronically submitted sequence listing This application incorporates by reference the entirety of the computer readable form (CRF) of the Sequence Listing submitted herewith. The text file of the Sequence Listing submitted herewith, entitled "14247-632-228_SEQ_LISTING.txt," was created on June 7, 2021, and is 247,915 bytes in size.

[0003] The present disclosure relates to TGFβ binding agents comprising TGFβ receptor extracellular domain (TGFβR-ECD) derived fusion molecules and their use for binding to and neutralizing TGFβ ligands, particularly for the treatment of diseases or conditions associated with TGFβ. [Background technology]

[0004] Transforming growth factor β (TGFβ) is part of a superfamily of over 30 ligands that regulate several physiological processes, including cell proliferation, migration, and differentiation. Perturbations in their levels and / or signaling have significant pathological effects. TGFβ has been implicated in the pathogenesis of many human disorders (Non-Patent Document 1; Non-Patent Document 2). For example, TGFβ and activin ligands play a crucial pathogenic role in many diseases, including fibrosis and cancer. Examples of TGFβ-associated disorders include hematological malignancies, solid tumors, bone marrow failure states, and a wide variety of disorders characterized by uncontrolled fibrosis, such as pulmonary, hepatic, renal, and vascular fibrosis, pulmonary arterial hypertension, and systemic sclerosis (SSc; also known as scleroderma) (Non-Patent Document 3; Non-Patent Document 4).

[0005] Sustained activation of TGF-β signaling plays a central role in the development of fibrosis (Non-Patent Document 5). TGF-β canonical signaling stimulates the transition of fibroblasts into myofibroblasts (Non-Patent Document 6; Non-Patent Document 7), plays a central role in the production and deposition of collagen and other components of the extracellular matrix (ECM) (Non-Patent Document 8), and induces other mediators involved in fibrosis (Non-Patent Document 9). In patients with fibrosis, such as scleroderma and idiopathic pulmonary fibrosis (IPF), TGF-β increases collagen deposition in the skin and / or lung and stimulates fibroblast activation into myofibroblasts in the skin (Non-Patent Document 8; Non-Patent Document 10; Non-Patent Document 11). Furthermore, the non-canonical TGF-β pathway also contributes to the maintenance of the fibrotic phenotype (Non-Patent Document 12). Therefore, the TGF-β signaling pathway has emerged as the most obvious target for therapeutic intervention in fibrosis (Non-Patent Document 5; Non-Patent Document 13; Non-Patent Document 14).

[0006] TGF-β is also considered a key regulator of tumor progression and is overexpressed by most tumor types. It favors tumorigenesis, in part, by inducing epithelial-mesenchymal transition (EMT) in epithelial tumor cells, leading to aggressive metastasis. TGF-β also promotes tumorigenesis by acting as a potent suppressor of immune responses in the tumor microenvironment. Indeed, TGF-β is recognized as one of the most potent immunosuppressive factors present in the tumor microenvironment. TGF-β inhibits the differentiation, proliferation, and survival of many immune cell types, including dendritic cells, macrophages, NK cells, neutrophils, B cells, and T cells; it alters both innate and adaptive immunity. The importance of TGF-β in the tumor microenvironment is underscored by evidence showing that elevated levels of TGF-β ligands correlate with disease progression and recurrence, metastasis, and mortality in several tumor types, including melanoma, lung, pancreatic, colorectal, liver, and breast. Therefore, considerable effort has been devoted to devising antitumor therapies involving TGF-β inhibition. These approaches involve the use of polypeptide fusions based on the TGFβ receptor extracellular domain that bind to or "capture" the TGFβ ligand (see, e.g., U.S. Patent No. 5,623,299; ... and U

[0007] One approach to developing therapeutic agents that inhibit TGFβ function has been to use antibodies or soluble decoy receptors (also called receptor extracellular domain (ECD)-based ligand traps) to bind to and sequester the ligand, thereby blocking its access to its cell surface receptor. In general, receptor ECD-based traps are a class of therapeutic agents that can selectively sequester ligands and can be optimized using protein engineering techniques.

[0008] Previously, it has been shown that single-chain, bivalent TGFβ traps bearing two TGFβ receptor type II (TGFβRII) extracellular domains linked as a doublet can neutralize members of the TGFβ superfamily of ligands (Patent Documents 3 and 5). In such cases, bivalency was achieved by covalently linking two TGFβRII extracellular domains using intrinsically disordered regions (IDRs) adjacent to the structured ligand-binding domains of the TGFβRII extracellular domains. It has further been shown that potency increases when such bivalent doublets are linked side-by-side to a multimerization domain, such as an Fc moiety at the N- or C-terminus (Patent Documents 19 and 10).

[0009] To date, most therapeutic approaches to neutralizing TGFβ, particularly in cancer immunology, have focused on the TGFβ1 isoform. This is because TGFβ1 is the isoform predominantly expressed in the immune system (Non-Patent Document 15) and many types of human tumors (Non-Patent Document 16). While the intended target has usually been the TGFβ1 isoform, most therapeutic agents in development generally inhibit other TGFβ isoforms with varying potency. For example, fresolimumab is a monoclonal antibody that is a pan-inhibitor of all three TGFβ isoforms. While it neutralizes all isoforms, it inhibits the TGFβ1 isoform approximately 7-fold more potently than the TGFβ3 isoform and approximately 14-fold more potently than the TGFβ2 isoform (Non-Patent Document 17). This monoclonal antibody has been tested in clinical trials in cancer patients (Non-Patent Document 18; Non-Patent Document 19) and in patients with glomerulosclerosis (Non-Patent Document 20).

[0010] The TGFβ2 isoform has been implicated in cardiac homeostasis (Non-Patent Document 21; Non-Patent Document 22), control of tumor dormancy (Non-Patent Document 23), and positive regulation of hematopoiesis (Non-Patent Document 24), suggesting that this isoform should escape neutralization in order to play beneficial roles.

[0011] Therefore, it would be useful to provide a TGFβRII-ECD-based trap with tailored isoform specificity to maximize therapeutic efficacy in specific disease indications while minimizing adverse effects. In particular, it would be useful to provide a trap that neutralizes TGFβ3 with potency similar to that of TGFβ1. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 01 / 83525 Brochure [Patent Document 2] International Publication No. 2005 / 028517 Pamphlet [Patent Document 3] International Publication No. 2008 / 113185 Brochure [Patent Document 4] International Publication No. 2008 / 157367 Brochure [Patent Document 5] International Publication No. 2010 / 0031168 Pamphlet [Patent Document 6] International Publication No. 2010 / 099219 Brochure [Patent Document 7] International Publication No. 2012 / 071649 Brochure [Patent Document 8] International Publication No. 2012 / 142515 Brochure [Patent Document 9] International Publication No. 2013 / 000234 Brochure [Patent Document 10] International Publication No. 2018 / 158727 Brochure No. [Patent Document 11] U.S. Patent No. 5,693,607 [Patent Document 12] US Patent Application Publication No. 2005 / 0203022 [Patent Document 13] US Patent Application Publication No. 2007 / 0244042 [License 14] U.S. Patent No. 8318135 [License 15] U.S. Patent No. 8658135 [License 16] U.S. Patent No. 8815247 [License 17] U.S. Patent and Trademark Office Publication No. 2015 / 0225483 [License 18] U.S. Patent and Trademark Office Publication No. 2015 / 0056199 [License 19] International Publication No. 2017 / 037634 パンフレット [Non-licensed literature]

[0013] [Non-licensed Document 1] Akhurst, RJand Hata, A., 2012 [Non-licensed Document 2] Akhurst, RJ, 2017 [Non-licensed Document 3] Nanthakumar, DB et al., 2015 [Non-licensed Document 4] Meng, X.-M. et al., 2016 [Non-licensed Document 5] Varga, J. and Whitfield, ML, 2009 [Non-licensed Document 6] Desmouliere, A. et al., 1993 [Non-licensed Document 7] Midgley, AC et al., 2013 [Non-licensed Document 8] Prud'homme, GJ, 2007 [Non-licensed Document 9] Todd, NWet al., 2015 [Non-licensed Document 10] Lafyatis, R., 2014 [Non-licensed Document 11] Kissin, E.Y. et al., 2006 [Non-Patent Document 12] Leask, A., 2008 [Non-Patent Document 13] Hunzelmann, N. and Krieg, T., 2010 [Non-Patent Document 14] Varga, J. and Pasche, B., 2008 [Non-Patent Document 15] Li, M.O., et al., 2006 [Non-Patent Document 16] Martin, C.J. et al., 2020 [Non-Patent Document 17] Grutter, C. et al., 2008 [[ID=2,6]][Non-Patent Document 18] Morris, J.C. et al., 2014 [Non-Patent Document 19] Lacouture, M.E. and Morris, J.C., 2015 [Non-Patent Document 20] Vincenti, F. et al., 2017 [Non-Patent Document 21] Roberts, A.B. et al., 1992 [Non-Patent Document 22] Herbertz, S. et al., 2015 [Non-Patent Document 23] Bragado, P. et al., 2013 [Non-Patent Document 24] Langer, J.C. et al., 2004 [Summary of the Invention] [Means for Solving the Problems]

[0014] Provided herein are tetravalent TGFβ receptor-extracellular domain-based traps with tailored isoform specificity profiles for neutralizing TGFβ ligands, and methods for their use in treating TGFβ-related diseases and conditions. The tetravalent TGFβ binding agents provided herein comprise two polypeptides assembled via a multimerization domain, each polypeptide having two TGFβ II receptor (TGFβR) ligand-binding domains linked as a doublet. The TGFβ binding agents provided herein are designed to tailor TGFβ isoform specificity to maximize therapeutic efficacy in specific disease indications while minimizing adverse effects.

[0015] The present technology is based, at least in part, on the inventors' recognition that a TGFβ ligand trap with isoform specificity distinct from other known drugs under development may be advantageous for the treatment of certain TGFβ-related diseases and conditions. Recent reports have demonstrated the important role of the TGFβ3 isoform in certain TGFβ-related conditions, such as fibrosis. For example, a recent report identified TGFβ3 as an important therapeutic target in renal fibrosis by demonstrating that specific downregulation of TGFβ3 by miR-29 suppresses renal fibrosis (Wang, H. et al., 2019). The important role of TGFβ3 isoforms in immunity has also been suggested by recent reports of TGFβ3 production by immune cells (Komai, ID and Okamura, T., 2018). Regarding SSc, a genome-wide association study in African American patients identified TGFβ3 as a novel SSc susceptibility gene (Gourh, P. et al., 2017).

[0016] With regard to TGFβ2, the involvement of this isoform in cardiac homeostasis (Roberts, A.B. et al., 1992; Herbertz, S. et al., 2015), control of tumor dormancy (Bragado, P. et al., 2013), and positive regulation of hematopoiesis (Langer, J.C. et al., 2004) suggested that avoiding neutralization of this isoform would be desirable.

[0017] Taken together, these findings suggest that neutralizing TGFβ1 and TGFβ3 to a similar extent may be beneficial in treating certain disorders, particularly those involving TGFβ3. Achieving approximately equal inhibition of TGFβ1 and TGFβ3 may be useful in some cases to ensure that both TGFβ1 and TGFβ3 can be effectively neutralized, preventing compensatory mechanisms that may occur if one of these isoforms is preferentially neutralized and / or maximizing efficacy. It may also be desirable to similarly inhibit TGFβ1 and TGFβ3 without neutralizing TGFβ2 signaling (as it may be beneficial to avoid neutralizing this isoform).

[0018] In a broad aspect, provided herein are novel polypeptide constructs useful for inhibiting the effects of transforming growth factor β (TGFβ) isoforms. The polypeptides of the present disclosure comprise a TGFβ binding region and a multimerization domain, wherein the N-terminus of the multimerization domain is linked to the C-terminus of the TGFβ binding region. The TGFβ binding region comprises two TGFβ receptor ligand binding domains (TGFβR-LBDs) linked together by a first linker and by the multimerization domain via a second linker. In another broad aspect, provided are TGFβ binding agents comprising two such polypeptide chains assembled via the multimerization domains, thereby forming a tetravalent molecule with specific inhibitory specificity for TGFβ ligands (TGFβ1, TGFβ2, and TGFβ3).

[0019] Without wishing to be bound by theory, the present invention is based, at least in part, on the discovery that modifying one or more of the linkers in such TGFβ ligand traps (e.g., the linker linking two TGFβR-LBDs together and / or the linker linking the TGFβR-LBD to the multimerization domain) differentially affects the inhibitory potency of the binding agent against different TGFβ isoforms. In some cases, modifying one or both linkers can differentially affect the inhibitory potency of binding agents against different TGFβ isoforms without increasing undesired inhibition of TGFβ2 and without significantly decreasing overall potency (e.g., IC 50 remains in the low picomolar range), TGFβ3:TGFβ1 IC 50 It is shown herein that the ratio can be reduced or equalized (showing similar or equalized inhibitory potency against both isoforms).

[0020] The TGFβ binding agents provided herein generally comprise a first polypeptide and a second polypeptide linked together via a multimerization domain, each polypeptide comprising, from N-terminal to C-terminal: an N-terminal region; a first TGFβ receptor ligand-binding domain (TGFβR-LBD); a first linker; a second TGFβR-LBD; a second linker; and a multimerization domain. One embodiment of a TGFβ binding agent is shown generally in FIG. 1 (which illustrates an embodiment in which the TGFβ binding agent is a homodimer, i.e., the first and second polypeptides are the same). The first and second polypeptides can be linked to each other via their respective multimerization domains, for example, by a disulfide bond (cysteine ​​bridge), by coiled-coil interactions, etc.

[0021] The TGFβ binding agents of the present technology are TGFβ3:TGFβ1 IC 50The TGFβ binding agents provided herein are therefore characterized by their specificity profile for isoform inhibition: specifically, the relative inhibitory potency for TGFβ1 and TGFβ3 isoforms (referred to herein as TGFβ3:TGFβ1 IC 50 The ratio (expressed as a function of the agonist activity) is about 2.5:1 or less, and the activity of both the TGFβ3 and TGFβ1 isoforms is inhibited with much higher potency than that of the TGFβ2 isoform (e.g., in the picomolar range for TGFβ3 and TGFβ1, and in the nanomolar range for TGFβ2).

[0022] Furthermore, in certain embodiments, the polypeptides and TGFβ binding agents of the present technology may provide several advantages in addition to tailored isoform specificity. For example, and without limitation, the polypeptides and TGFβ binding agents may provide improved manufacturability, e.g., due to reduced glycosylation, increased homogeneity, ease of expression, etc. Thus, in certain embodiments, the polypeptides and TGFβ binding agents of the present technology provide one or more of the following advantages over previous TGFβ binding agents: improved therapeutic efficacy for a particular disease indication, e.g., a TGFβ3-mediated pathology; reduced glycosylation; increased homogeneity; improved manufacturability; and increased production.

[0023] In certain embodiments of the polypeptides and TGFβ binding agents of the present technology, the first and second linkers are designed to provide the desired relative isoform specificity of inhibition. For example, in certain embodiments, the length of the first and second linkers is such that the TGFβ3:TGFβ1 IC 50The ratio is selected to be about 2.5:1 or less, and both TGFβ3 and TGFβ1 isoform activity is inhibited with much higher potency than TGFβ2 isoform activity (e.g., in the picomolar range for TGFβ3 and TGFβ1, and in the nanomolar range for TGFβ2).

[0024] In certain embodiments of the polypeptides and TGFβ binding agents of the present technology, the first linker and the second linker are TGFβ3:TGFβ1 IC 50 In one embodiment, the ratio is selected to be about 2.5:1 or less. 50 In certain embodiments, the TGFβ3:TGFβ1 IC for the TGFβ binding agent is less than about 2.5:1, about 2.3:1 or less, about 2:1 or less, about 1.8:1 or less, about 1.5:1 or less, about 1.3:1 or less, about 1.1:1 or less, about 1:1 or less, about 0.8:1 or less, or about 0.5:1 or less. 50 In certain embodiments, the ratio of TGFβ3:TGFβ1 IC for the TGFβ binding agent is about 1:1 to about 2:1, or about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, or 1.9:1. 50 The ratio is about 1:1 to about 1.5:1, or about 1.4:1 to about 1.6:1, or about 1.4:1, 1.5:1, or 1.6:1. In certain such embodiments, the TGFβ binding agent inhibits both TGFβ1 and TGFβ3 isoform activity with at least 20-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold greater potency than TGFβ2 isoform activity.

[0025] In certain embodiments, the first linker is 33 amino acids or less in length. In one embodiment, the first linker is about 10 to 33 amino acids in length. In embodiments, the first linker can be about 15 to 33 amino acids in length, or about 18 to about 30 amino acids in length. In one embodiment, the first linker is 16, 18, 30, or 32 amino acids in length. In certain embodiments, the first linker is 16 amino acids in length. In other embodiments, the first linker is 18 amino acids in length. In other embodiments, the first linker is 30 amino acids in length. In other embodiments, the first linker is 32 amino acids in length.

[0026] In certain embodiments, the second linker is 10 or more amino acids in length. In one embodiment, the second linker is about 10 to about 35 amino acids in length. In embodiments, the second linker can be about 10 to about 34, or about 15 to about 34 amino acids in length. In one embodiment, the second linker is 16, 30, 32, or 34 amino acids in length. In certain embodiments, the second linker is 30 amino acids in length. In other embodiments, the second linker is 16 amino acids in length. In other embodiments, the second linker is 32 amino acids in length. In other embodiments, the second linker is 34 amino acids in length.

[0027] In one embodiment, the first linker is 18 amino acids and the second linker is 16 amino acids. In another embodiment, the first linker is 18 amino acids and the second linker is 30 amino acids. In another embodiment, the first linker is 18 amino acids and the second linker is 10 amino acids. In another embodiment, the first linker is 18 amino acids and the second linker is 32 amino acids. In another embodiment, the first linker is 18 amino acids and the second linker is 34 amino acids. In another embodiment, the first linker is 16 amino acids and the second linker is 18 amino acids. In another embodiment, the first linker is 16 amino acids and the second linker is 16 amino acids. In another embodiment, the first linker is 16 amino acids and the second linker is 30 amino acids. In another embodiment, the first linker is 16 amino acids and the second linker is 32 amino acids. In another embodiment, the first linker is 26 amino acids and the second linker is 26 amino acids. In another embodiment, the first linker is 32 amino acids and the second linker is 32 amino acids. In another embodiment, the first linker is 32 amino acids and the second linker is 34 amino acids. It should be understood that many other permutations are possible so long as the desired isoform specificity of inhibition is achieved.

[0028] In certain embodiments, one or more of the first linker and the second linker comprise or consist of an IDR linker, IDR linker mutant, hybrid linker, hybrid linker mutant, truncated linker, truncated linker mutant, or extended linker disclosed herein. For example, one or more of the first linker and the second linker can independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 8-26, or a sequence 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 thereto. In certain embodiments, the first linker comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 16, 21, 22, 23, and 26, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In one embodiment, the second linker comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 4, 9, 11, 15, 17, 18, 19, 20, 22, 23, 24, 25, and 26, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0029] In an exemplary embodiment, the first linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, and / or the second linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11. In another exemplary embodiment, the first linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, and / or the second linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9. It should be understood that other embodiments using combinations of linkers provided herein are encompassed, so long as the desired isoform specificity of inhibition is achieved.

[0030] In certain embodiments of the polypeptides and TGFβ binding agents of the present technology, the N-terminal region comprises or consists of an IDR linker, IDR linker mutant, hybrid linker, hybrid linker mutant, truncated linker, truncated linker mutant, or extended linker. For example, the N-terminal region can comprise or consist of the amino acid sequence set forth in SEQ ID NO: 3, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0031] In certain embodiments of the polypeptides and TGFβ-binding agents of the present technology, the first TGFβR-LBD and / or second TGFβR-LBD comprise or consist of the amino acid sequence set forth in SEQ ID NO:2, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the first TGFβR-LBD and second TGFβR-LBD are the same or substantially the same. In other embodiments, the first TGFβR-LBD and second TGFβR-LBD may have different amino acid sequences.

[0032] In certain embodiments of the polypeptides and TGFβ binding agents of the present technology, the multimerization domain allows for dimerization of two polypeptides according to the present disclosure in a non-covalent manner, such as by coiled-coil interactions.

[0033] In other embodiments, the multimerization domain allows for the dimerization of two polypeptides according to the present disclosure in a covalent manner, such as by disulfide bridging.

[0034] In one embodiment, the multimerization domain comprises one or more constant regions of an antibody, e.g., the second constant domain of an antibody heavy chain (C H 2) and / or a third constant domain (C H3), or the Fc region of an antibody heavy chain. The antibody may be an IgG antibody, such as, for example and without limitation, an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a human antibody, e.g., the multimerization domain comprises the constant region of a human IgG1, IgG2, IgG3, or IgG4 heavy chain. In certain embodiments, the multimerization domain has 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% sequence identity with the human IgG1, IgG2, IgG3, or IgG4 constant region. In certain embodiments, the multimerization domain comprises or consists of the Fc region of a human IgG1 antibody. In other embodiments, the multimerization domain comprises or consists of the Fc region of a human IgG4 antibody.

[0035] In some embodiments, the multimerization domain contains one or more cysteine ​​residues for crosslinking a first polypeptide construct with a second polypeptide construct. For example, the multimerization domain may contain at least two cysteine ​​residues for forming disulfide bridges between two polypeptide constructs, thereby forming a dimer.

[0036] In certain embodiments, the multimerization domain is engineered to reduce aggregation or modulate the dimeric or multimeric stability of the polypeptide construct. For example, the Fc region may contain one or more amino acid substitutions relative to a native Fc sequence that reduce aggregation and / or increase the stability of the TGFβ binding agent. In certain embodiments, the multimerization domain is selected to provide one or more effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement activation (complement-dependent cytotoxicity or CDC), opsonization, etc.

[0037] In certain embodiments, the multimerization domain comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 49-80, or a sequence 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 thereto. In certain embodiments, the multimerization domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49, or a sequence 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 thereto. In other embodiments, the multimerization domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, or a sequence 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 thereto.

[0038] In certain embodiments of the polypeptides and TGFβ-binding agents of the present technology, the TGFβ-binding region (including the N-terminal domain, two LBDs, and two linkers) comprises or consists of the sequence set forth in any one of SEQ ID NOS: 27-48, or a sequence 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 thereto. In specific embodiments, the TGFβ-binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27. In other embodiments, the TGFβ-binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29. In other embodiments, the TGFβ-binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32. In other embodiments, the TGFβ-binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41.

[0039] In some alternative embodiments, the TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO:40.

[0040] In certain embodiments of the polypeptides and TGFβ-binding agents of the present technology, the polypeptide construct comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 81-103 and 105, or a sequence 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 thereto. In certain embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 81. In other embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 84. In other embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87. In other embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96.

[0041] In some other embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95, or a sequence 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 thereto. In some other embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95.

[0042] In one embodiment, the polypeptide construct provided herein is a polypeptide construct comprising, from the N-terminus to the C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) an Fc region of human IgG1.

[0043] In certain embodiments of the present technology, the TGFβ binding agent is a heterodimer, i.e., the first and second polypeptides are different. In such embodiments, the first and second polypeptides may differ in one or more regions or domains, for example, by the sequence of the first linker, second linker, LBD, multimerization domain, and combinations thereof. Thus, each of the following may independently be the same or different in the two polypeptides: the N-terminal region; the first linker; the second linker; the first LBD; the second LBD; and the multimerization domain. Many combinations are possible, as long as the desired isoform specificity of inhibition is provided.

[0044] In some embodiments of the TGFβ binding agent, the first polypeptide construct and the second polypeptide construct comprise or consist of the sequence set forth in SEQ ID NO: 95, or a sequence 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 thereto. In some embodiments of the TGFβ binding agent, the first polypeptide construct and the second polypeptide construct comprise or consist of the sequence set forth in SEQ ID NO: 95.

[0045] In some embodiments of the TGFβ binding agent, the different TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95, or a 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 thereto.

[0046] In certain embodiments of the TGFβ binding agent, the TGFβ binding agent is: A first polypeptide construct comprising, from the N-terminus to the C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) a first Fc region of human IgG1, and A second polypeptide construct comprising, from the N-terminus to the C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) a second Fc region of human IgG1. Includes; Here, the first polypeptide construct and the second polypeptide construct are linked together via a first and a second Fc region of human IgG1.

[0047] In certain embodiments, the inhibitory potency of a TGFβ binding agent against both TGFβ1 isoform activity and TGFβ3 isoform activity is higher than against TGFβ2 isoform activity; where the relative inhibitory potency of a TGFβ binding agent against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3 vs TGFβ1) is 50 The ratio is about 2.5:1 or less.

[0048] In certain embodiments, the TGFβ binding agents provided herein are homodimers of the polypeptide constructs provided herein.

[0049] In an alternative embodiment, the TGFβ binding agent is a homodimer, ie, the first and second polypeptides are the same or substantially the same.

[0050] In certain embodiments, the polypeptide or TGFβ binding agent may be conjugated to a targeting agent, a therapeutic moiety, a detectable moiety, and / or a diagnostic moiety.

[0051] In another broad aspect, nucleic acids encoding the polypeptides and TGFβ-binding agents of the present technology are provided. Also provided are vectors and plasmids containing such nucleic acids and / or for expressing the polypeptides and TGFβ-binding agents. For example, in one embodiment, nucleic acids having the sequence set forth in any one of SEQ ID NOS: 106-109, as well as vectors and plasmids containing these nucleic acids, are provided. In another embodiment, nucleic acids having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NOS: 106-109, or capable of hybridizing thereto under high stringency conditions, are provided. Cells expressing the polypeptides and TGFβ-binding agents of the present technology are also provided.

[0052] In a further aspect, methods are provided for producing the polypeptides and TGFβ-binding agents of the present technology, comprising expressing one or more polypeptides provided herein in a cell, followed by isolation and / or purification thereof. In certain embodiments, the polypeptide constructs and TGFβ-binding agents are expressed in a form that is secretable by the cell, e.g., using a signal peptide at the N-terminus, allowing recovery of the polypeptide or TGFβ-binding agent from the culture medium.

[0053] In another broad aspect, there is provided a pharmaceutical composition comprising a polypeptide construct or TGFβ binding agent according to the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is formulated for administration by injection or infusion, e.g., for intravenous, subcutaneous, intraperitoneal, or intramuscular administration. In some embodiments, the pharmaceutical composition is provided in unit dosage form.

[0054] In yet another broad aspect, there is provided a method of preventing or treating a TGFβ-associated disease or condition, comprising administering to a subject a therapeutically effective amount of a polypeptide, TGFβ-binding agent, or pharmaceutical composition of the present technology, such that the TGFβ-associated disease or condition is prevented or treated.

[0055] Examples of TGFβ-associated diseases or conditions that can be prevented or treated in accordance with the present disclosure include, but are not limited to, fibrosis (e.g., fibrosis, fibrotic scarring, fibroproliferative disorders); cancer (e.g., malignant tumors, solid tumors, metastases); and bone marrow failure (e.g., Shwachman-Bodian-Diamond syndrome, Fanconi anemia). In certain embodiments, a polypeptide or TGFβ-binding agent described herein is used to treat or prevent fibrosis, including, but not limited to, fibrosis and fibrotic scarring of tissues and / or organs, such as pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), renal fibrosis, liver fibrosis (e.g., cirrhosis), systemic sclerosis, scleroderma, skin fibrosis, cardiac fibrosis, myelofibrosis, etc.

[0056] In certain embodiments, there is provided a method of preventing or treating a disease or condition mediated by TGFβ1 and / or TGFβ3, comprising administering to a subject a therapeutically effective amount of a polypeptide, TGFβ-binding agent, or pharmaceutical composition of the present technology, such that the disease or condition mediated by TGFβ1 and / or TGFβ3 is treated. In one embodiment, there is provided a method of preventing or treating a disease or condition mediated by TGFβ3 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide, TGFβ-binding agent, or pharmaceutical composition of the present technology, such that the disease or condition mediated by TGFβ3 is prevented or treated.

[0057] In certain embodiments, there is provided a method of preventing or treating fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide, TGFβ binding agent, or pharmaceutical composition of the present technology, such that fibrosis is prevented or treated.

[0058] In a further broad aspect, kits and packages are provided for treating a TGFβ-related disease or condition in a subject in need thereof, comprising a polypeptide, TGFβ-binding agent, or pharmaceutical composition according to the present disclosure; optionally, one or more additional ingredients, such as an acid, base, buffer, inorganic salt, solvent, antioxidant, preservative, or metal chelator, and / or tools for its administration, such as a syringe, needle, etc. Instructions for administration or use may also be included.

[0059] In yet another aspect, there is provided a method of producing a polypeptide construct or TGFβ binding agent provided herein, the method comprising culturing a host cell provided herein under conditions suitable for protein expression; and harvesting the polypeptide construct or TGFβ binding agent.

[0060] In yet another aspect, provided herein is a polypeptide construct or a TGFβ binding agent produced by the manufacturing methods provided herein.

[0061] Further scope, applicability, and advantages of the present disclosure will become apparent from the non-limiting detailed description set forth hereinafter. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the present disclosure, is given by way of example only, with reference to the accompanying drawings, in which:

[0062] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0063] For a better understanding of the technology, and to show more clearly how it may be put into practice, reference is now made to the accompanying drawings, which illustrate, by way of example, aspects and features of non-limiting embodiments of the technology. [Brief explanation of the drawings]

[0064] [Figure 1]

[0023] Figure 1 shows a schematic structure of the domain organization of a tetravalent TGFβ binding agent according to certain embodiments. The embodiment shown herein is a homodimer of a first polypeptide (left) and a second polypeptide (right) linked by a disulfide bridge in the multimerization domain (shown as two lines). The ligand binding domain (LBD) is shown as a circle, the multimerization domain is shown as an oval, and the N-terminal region and linker are shown as a rectangle. In embodiments where the binder is a heterodimer, the first and second polypeptides differ in one or more regions or moieties (not shown).

[0065] [Figure 2] Figure 2A shows an overlay of the monomeric structures of TGFβ1 (blue) and TGFβ3 (green).

[0066] Figure 2B shows an overlay of the TGFβ1 dimer (blue) and the TGFβ3 dimer (green). Corresponding monomers in the region are superimposed to show the differences in the dimer angles of the monomers.

[0067] Figure 2C shows a representative model of T22d35-Fc-IgG1-v1(CC) (SEQ ID NO: 6) bound to a TGFβ ligand, showing the second ligand binding domain, second linker, and multimerization domain (Fc) region.

[0068] [Figure 3] Figure 3A shows polyacrylamide gel electrophoresis analysis of representative TGFβ binders under non-reducing conditions. After expression and purification, 2 μg of each protein was loaded onto a gel as indicated: Ctl: control; p61: protein 61; p96: protein 96; p101: protein 101; p107: protein 107; p112: protein 112.

[0069] Figure 3B shows polyacrylamide gel electrophoresis analysis under reducing conditions of representative TGFβ binders. After expression and purification, 2 μg of each protein was loaded onto a gel as indicated: Ctl: control; p61: protein 61; p96: protein 96; p101: protein 101; p107: protein 107; p112: protein 112.

[0070] [Figure 4] Figure 4A shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ1 for proteins 61, 96, 101, 107, and 112, as well as controls, as indicated. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error of the mean (SEM).

[0071] Figure 4B shows representative results in an A549 / IL-11 cell-based assay for the inhibition of TGFβ3 for proteins 61, 96, 101, 107, and 112, as well as controls, as indicated. The table shows the calculated IC values ​​calculated in Graphpad Prism. 50 Values ​​are listed and error bars indicate standard error of the mean (SEM).

[0072] [Figure 5] Figure 5A shows polyacrylamide gel electrophoresis analysis under non-reducing conditions of the following representative TGFβ binders: p112, p111, p108, p105, p104, p101, p99, and p71. Error bars indicate standard error of the mean (SEM).

[0073] Figure 5B shows polyacrylamide gel electrophoresis analysis under reducing conditions of the following representative TGFβ binders: p112, p111, p108, p105, p104, p101, p99, and p71. Error bars indicate standard error of the mean (SEM).

[0074] [Figure 6]Figure 6A shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ1 for proteins 113, 115, and 116, as well as controls, as indicated. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error of the mean (SEM).

[0075] Figure 6B shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ3 for proteins 113, 115, and 116, and controls, as indicated. The table shows the calculated IC values ​​calculated in Graphpad Prism. 50 Values ​​are listed and error bars indicate standard error of the mean (SEM).

[0076] [Figure 7] Figure 7A shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ1 for proteins 101, 129, and 130, as well as controls, as indicated. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error of the mean (SEM).

[0077] Figure 7B shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ3 for proteins 101, 129, and 130, and controls, as indicated. The table shows the calculated IC values ​​calculated in Graphpad Prism. 50 Values ​​are listed and error bars indicate standard error of the mean (SEM).

[0078] [Figure 8] Figure 8A shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ1 for proteins 101, 131, 132, and 133, as well as controls, as indicated. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error of the mean (SEM).

[0079] Figure 8B shows representative results in an A549 / IL-11 cell-based assay for the inhibition of TGFβ3 for proteins 101, 131, 132, and 133, as well as controls, as indicated. The table shows the calculated IC values ​​calculated in Graphpad Prism. 50 Values ​​are listed and error bars indicate standard error of the mean (SEM).

[0080] [Figure 9] Figure 9A shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ1 for proteins 96, 134, and 135, as well as controls, as indicated. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error of the mean (SEM).

[0081] Figure 9B shows representative results in an A549 / IL-11 cell-based assay for the inhibition of TGFβ3 for proteins 96, 134, and 135, as well as controls, as indicated. The table shows the calculated IC values ​​calculated in Graphpad Prism. 50 Values ​​are listed and error bars indicate standard error of the mean (SEM).

[0082] [Figure 10] Figure 10 shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ1 for proteins 101 and 128, and controls, as indicated. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error of the mean (SEM).

[0083] Figure 10B shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ3 for proteins 101 and 128, and controls, as indicated. The table shows the calculated IC calculated in Graphpad Prism. 50 Values ​​are listed and error bars indicate standard error of the mean (SEM).

[0084] [Figure 11] Figure 11 shows representative results in an A549 / IL-11 cell-based assay for inhibition of TGFβ2 for proteins 61, 96, and 101, as well as controls, as indicated. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error of the mean (SEM). DETAILED DESCRIPTION OF THE INVENTION

[0085] The technology of the present invention is described in more detail below. This description is not intended to be a detailed listing of all the various ways in which the technology may be implemented or all the functions that may be added to the technology of the present invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be omitted from that embodiment. Furthermore, many modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in view of this disclosure, and these modifications and additions do not depart from the technology of the present invention. Therefore, the following description is intended to illustrate certain embodiments of the technology, and is not intended to exhaustively describe all permutations, combinations, and variations thereof.

[0086] definition In order to provide a clear and consistent understanding of the terms used herein, several definitions are provided below. Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0087] The use of the terms "a," "an," and "the," when used in conjunction with the term "comprising" in the claims and / or this specification, may mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more." Similarly, the term "another" may mean at least a second or more. These terms should be construed to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0088] As used herein, the terms "comprising" (and any form of "comprising," such as "comprise" and "comprises"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "include" and "includes"), or "containing" (and any form of "containing," such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. The term "consisting of" is to be construed as closed-ended.

[0089] The term "about" is used to indicate that a value or amount refers to the actual given value and also to approximations of such a given value that can be reasonably estimated by one of ordinary skill in the art (including equivalents and approximations of such a given value depending on experimental and / or measurement conditions). For example, the term "about" with respect to a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[0090] The term "and / or" as used herein should be construed as a specific disclosure of each of the specified features or components, with or without other features or components. For example, "A and / or B" should be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually described herein. Unless otherwise stated or clear from the context, as used herein, the term "or" is understood to be inclusive and encompasses both "or" and "and." For example, an embodiment of "a composition comprising A or B" would typically refer to an aspect using a composition containing both A and B. However, "or" should be construed as excluding stated aspects that cannot be consistently combined (e.g., a composition pH of 9-10 or 7-8).

[0091] It should be understood herein that terms such as "1 to 20" include any individual value within and including 1 and 20. Thus, the term "1 to 20" should be understood herein to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20. Terms such as "1 to 20" also include individual subranges within and including 1 to 20. Thus, the term "1 to 20" also includes subranges such as "1 to 9," "2 to 9," "3 to 5," "5 to 9," "5 to 20," and "8 to 20." The same applies to similar expressions such as, but not limited to, "1 to 19," "1 to 18," "1 to 10," "1 to 9," and "5 to 15."

[0092] It should be understood herein that terms such as "about 15 to about 35" are within and include 15 and 35. Thus, terms such as "about 15 to about 35" include any number between and including 15 and 35, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and / or 35. Terms such as "about 15 to about 35" also include any individual subranges within and including 15 to 35, such as "about 16 to about 34," "about 16 to about 24," "about 24 to about 34," etc. The term "about" with respect to the number of amino acids means that the specified number of amino acids is specifically included, allowing for a + / - 2 degree variation in the number of amino acid residues. Thus, terms such as "about 15 to about 35" also include "13 to 37," "13 to 35," "17 to 37," "17 to 35," etc. The same applies to similar expressions such as, but not limited to, "about 16 to about 34," "about 16 to about 24," and "about 24 to about 34."

[0093] It should be understood herein that terms such as "at least 80% identical" include any individual value between and including 80% and 100%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. The term "at least 80% identical" also includes any individual subrange between and including 80% and 100%, e.g., "85% to 99%," "97% to 100%," "90% to 100%," etc. The same applies to similar expressions, such as, but not limited to, "at least 70% identical," "at least 90% identical," etc.

[0094] As used herein, the term "inhibition potency" refers to the effectiveness of a substance in inhibiting a particular biological or biochemical function, such as, but not limited to, the binding between a protein receptor and its ligand, or the activation of a cellular receptor by its ligand. In certain embodiments, the potency of inhibition is determined by measuring the IC50 of the inhibitor for a particular ligand or substrate. In that case, the relative inhibitory potency for various inhibitors and / or ligands can be expressed as the IC 50 For example, a relative inhibitory potency of 3:1 can be assessed by comparing IC 50 This means that the ratio of the values ​​is 3: 1. The terms "inhibition potency", "inhibitory potency", "inhibitory efficacy" and "neutralizing efficacy" are used interchangeably herein.

[0095] As used herein, "IC 50 The term "half maximal inhibitory concentration" refers to the concentration of a substance required for 50% inhibition in vitro. It is a measure of the potency or effectiveness of a substance in inhibiting a specific biological or biochemical function. IC 50 Values ​​are typically expressed as molar concentrations. IC of inhibitor 50 can be determined by constructing a dose-response curve and examining the effect of various concentrations of the inhibitor on a particular biological or biochemical function of interest.

[0096] As used herein, the term "avidity" refers to the overall strength of the binding interaction between a protein receptor and its ligand. Avidity generally refers to the cumulative strength of multiple individual non-covalent interactions, for example, between a protein receptor and its ligand, and differs from "affinity," which describes the strength of a single binding interaction. It should be understood that avidity is rarely a simple sum of its component affinities, as many factors (such as local concentration or proximity, multimerization, 3D structure or conformation) can affect a biomolecular interaction.

[0097] As used herein, the term "functionally equivalent" refers to a variant sequence that has the same or substantially the same biological activity or function as the original sequence from which it is derived, e.g., has no significant changes in physiological, chemical, physicochemical, or functional properties compared to the original sequence. The term "substantially identical" refers to a sequence that is functionally equivalent to and has a high degree of sequence identity with the original or reference sequence. Generally, a substantially identical sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the original or reference sequence and has the same function. In some cases, when referring to nucleic acid sequences, a substantially identical sequence hybridizes to the original sequence under high stringency conditions, e.g., salt and temperature conditions substantially equivalent to 0.5×SSC to about 5×SSC and 65°C for both hybridization and washing. Generally, variant sequences that are substantially identical or functionally equivalent to the sequences provided in accordance with the present disclosure are intended to be encompassed.

[0098] As used herein, the term "multimerization domain" refers to an amino acid sequence that allows a polypeptide chain to assemble into a multimer. The term "multimer" refers to a molecule made up of multiple monomers. The term "multimer" includes, but is not limited to, dimers, trimers, tetramers, pentamers, hexamers, octamers, decamers, etc.

[0099] The term "dimeric" refers to the presence of two polypeptides described herein in a TGFβ binding agent. "Homodimeric" means that the two polypeptides have the same sequence, while "heterodimeric" means that the two polypeptides have different sequences.

[0100] The term "doublet" refers to the presence of two copies of the TGFβR ligand binding domain (LBD) linked together side-by-side in a polypeptide.

[0101] The term "tetravalent" refers to the presence of four copies of the TGFβR ligand binding domain (LBD) in the TGFβ binding agent.

[0102] Polypeptides and TGFβ Binding Agents Provided herein are novel polypeptide constructs comprising a TGFβ binding region and a multimerization domain, as well as TGFβ binding agents comprising two such polypeptide constructs assembled via the multimerization domain. The TGFβ binding region comprises two TGFβRII-LBDs linked side-by-side by a first linker, which is linked to the multimerization domain by a second linker. The polypeptide constructs and TGFβ binding agents of the present disclosure are optimized by improving their binding to TGFβ. Specifically, the linker is designed to bind to TGFβ2 without increasing undesired inhibition and without significantly reducing overall potency (e.g., IC 50 remains in the picomolar range), TGFβ3:TGFβ1 IC 50 The ratio is chosen to optimize TGFβ isoform specificity so that it is about 2.5:1 or less (showing similar inhibitory potency against both isoforms).

[0103] In exemplary embodiments, the polypeptide constructs and TGFβ-binding agents of the present disclosure comprise two polypeptide chains linked via an antibody Fc region, or via a constant CH2 domain, a constant CH3 domain, and / or a combination of CH2 and CH3. The antibody constant region can be derived from or substantially identical to a human IgG1, IgG2, IgG3, or IgG4 antibody. Linkage of both polypeptide chains typically occurs during protein expression and secretion, e.g., in mammalian cells. In certain exemplary embodiments, the TGFβ-binding agent can comprise a homodimer, i.e., a dimer of a polypeptide construct having a sequence set forth in any one of SEQ ID NOS: 81-103 and 105, or a sequence 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 thereto. In other embodiments, the TGFβ binding agent comprises a heterodimer, i.e., a dimer of two different polypeptide constructs, at least one of which has a sequence set forth in any one of SEQ ID NOs: 81-103 and 105, or a sequence 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 thereto.

[0104] Generally, the polypeptide constructs and TGFβ binding agents are organized such that the multimerization domain is linked to the TGFβ binding region from its N-terminus to its C-terminus, such that for each polypeptide, the orientation of the construct is, from N-terminus to C-terminus, a single chain of (N-terminal region)-(first TGFβR-LBD)-(first linker)-(second TGFβR-LBD)-(second linker)-multimerization domain.

[0105] In exemplary embodiments, the multimerization domain allows for the assembly of two or more polypeptide chains in a covalent manner, for example, by disulfide bonds between cysteine ​​residues. Alternatively, the multimerization domain may allow the polypeptide chains to assemble in a non-covalent manner, for example, and without limitation, by a coiled-coil structure (De Crescenzo, G. et al., 2004).

[0106] In one embodiment, the multimerization domain is a dimerization domain, i.e., it allows the assembly of two polypeptide chains to form a dimer. According to the present disclosure, such a dimer generally comprises two polypeptides, each comprising two TGFβR-LBDs linked together and linked to a dimerization domain described herein, thereby forming a tetravalent TGFβ binding agent. Homodimers and heterodimers of the polypeptide constructs provided herein are encompassed.

[0107] In certain embodiments, the multimerization or dimerization domain of the polypeptide comprises the constant region of an immunoglobulin heavy chain, e.g., including the CH2 and / or CH3 domains. The Fc portion of an immunoglobulin is typically used. However, coiled-coil structures have also been found to be suitable for dimerization. Exemplary embodiments of Fc portions include, for example and without limitation, those that have lost the ability to interact with specific Fc receptors. In further embodiments, the multimerization domain may comprise an IgG-like dimerization domain, e.g., an IgG1, IgG2, IgG3, or IgG4 dimerization domain. In certain embodiments, the multimerization domain may provide one or more effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement activation (complement-dependent cytotoxicity, CDC), or opsonization.

[0108] In certain embodiments, the multimerization or dimerization domain comprises a CH2, a CH3, or a CH2 and a CH3 from an antibody heavy chain of human origin. For example, but not by way of limitation, the antibody heavy chain may be selected from the group consisting of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the constant domain in the construct is CH2 itself, or CH3 itself, or CH2-CH3. The antibody heavy chain components typically provide disulfide bridges between the same or different single-chain polypeptide constructs. In one embodiment, the multimerization domain provides at least one disulfide bond between the single-chain polypeptide constructs. In another embodiment, the multimerization domain provides at least two disulfide bonds between the single-chain polypeptide constructs. In some cases, the antibody heavy chain also provides for Protein A-based isolation of dimeric polypeptides, for example, after production in a host cell.

[0109] Thus, in certain embodiments, the multimerization or dimerization domain is an antibody constant domain that provides a bridge between two polypeptide constructs of the present invention. This is achieved, for example, when the expressed polypeptide constructs are secreted from their expression host. Thus, production of a single-chain polypeptide can provide a dimeric construct in which two polypeptide chains are crosslinked via disulfide bridges involving one or more cysteine ​​residues in each of the antibody constant domains present in each of the polypeptides. In certain embodiments, the multimerization domain (e.g., constant region) has no specific activity other than serving as a structure through which multimers (e.g., dimers) can be formed. Such minimal constant regions can also be modified to provide some benefit by incorporating a corresponding hinge region and, optionally, altering the cysteine ​​residue composition. For example, some or all of the cysteine ​​residues involved in bridging two Fc fragments or naturally used to bridge between the heavy and light chains of a complete antibody can be substituted or deleted. One advantage of minimizing the number of cysteine ​​residues is that it reduces the tendency for disulfide bond scrambling, which promotes aggregation. While noting that the stability of an Fc dimer may depend on the number of intermolecular disulfide bridges, it should be noted that not all of the native inter-hinge disulfide bonds need to be formed for Fc dimerization to occur.

[0110] As used herein, the terms "antibody" and "immunoglobulin (Ig)" are used interchangeably to refer to proteins constructed from paired heavy and light polypeptide chains. The respective structures of antibodies and domains are well established and known to those skilled in the art and will only be briefly summarized herein. When an antibody is properly folded, each chain folds into several distinct globular domains connected by a more linear polypeptide sequence. In particular, Ig light chains fold into a variable (VL) and a constant (CL) domain, while heavy chains fold into a variable (VH) and three constant (CH1, CH2, CH3) domains. Upon pairing, the interaction of the heavy and light chain variable domains (VH and VL) and the first constant domain (CL and CH1) results in the formation of Fab (Fragment Antigen Binding) containing the binding region (Fv); the interaction of two heavy chains results in the pairing of the CH2 and CH3 domains, resulting in the formation of Fc (Fragment Crystallizable). The properties described herein for the CH2 and CH3 domains also apply to the Fc.

[0111] In certain embodiments and aspects of the present disclosure, the multimerization or dimerization domain may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with an IgG1, IgG2, IgG3, or IgG4 constant region or CH2 and / or CH3 domain. The IgG1, IgG2, IgG3, or IgG4 may be of human origin. In certain embodiments, the TGFβ-binding agents described herein include those having an IgG1 dimerization domain. In some other embodiments, the TGFβ-binding agents described herein include those having an IgG4 dimerization domain.

[0112] The multimerization or dimerization domain can be engineered to reduce aggregation or modulate the stability of a TGFβ binding agent formed by assembly of two or more polypeptides disclosed herein. Thus, for example, Fc portions with mutations in the hinge region are encompassed by the present disclosure. Exemplary embodiments of Fc variants and modified hinge regions are provided, for example, in patent applications published under the numbers WO 2018 / 158727 and WO 2017 / 037634. ​​When referring to the hinge portion of a multimerization or dimerization domain, it should be understood that the hinge is considered to be part of the multimerization domain and not part of the second linker.

[0113] In exemplary embodiments, the multimerization or dimerization domain has a sequence set forth in SEQ ID NOs: 49-80, or a functionally equivalent variant thereof, or a sequence at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% identical thereto. In certain embodiments, the multimerization domain may comprise SEQ ID NO: 49 or a sequence at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% identical thereto. In another embodiment, the multimerization domain may comprise SEQ ID NO: 50 or a sequence at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% identical thereto.

[0114] According to the present disclosure, the first multimerization or dimerization domain and the second multimerization or dimerization domain of a TGFβ binding agent can have the same or substantially the same amino acid sequence in certain embodiments, or in certain embodiments, the multimerization or dimerization domains can be different so long as multimerization is not adversely affected.

[0115] It should be understood that the multimerization domain is not intended to be particularly limiting: any amino acid sequence that allows for the association of polypeptide chains to form a tetravalent TGFβ binding agent according to the present disclosure can be used, so long as the desired function and isoform specificity are maintained.

[0116] According to the present disclosure, the linker in the polypeptide constructs and TGFβ binding agents can comprise or consist of an IDR linker, an IDR linker variant, a hybrid linker, a hybrid linker variant, a truncated linker, a truncated linker variant, or an extended linker, as disclosed herein.

[0117] The human TGFβRII extracellular domain (TGFβRII-ECD; SEQ ID NO: 1) comprises a 102 amino acid structured ligand-binding domain (SEQ ID NO: 2; also referred to herein as "TGFβR-LBD") flanked by two intrinsically disordered regions: a 24 amino acid region at the N-terminus (SEQ ID NO: 3) and a 10 amino acid region at the C-terminus (SEQ ID NO: 4).

[0118] As used herein, the term "intrinsically disordered region (IDR) linker" refers to a linker comprising or consisting of at least a portion of one or both of the intrinsically disordered regions (IDRs) flanking the structured ligand-binding domain of the TGFβRII extracellular domain. An IDR linker generally has substantial sequence identity with the sequence of at least one of the intrinsically disordered regions of the TGFβRII extracellular domain, and it may have substantial sequence identity with both the N- and C-terminal IDRs of the TGFβRII extracellular domain or portion thereof. It should be understood that an IDR linker may comprise the entire IDR of a TGFβR, only a portion thereof, or multiple portions linked together.

[0119] In certain embodiments, the IDR linker comprises or consists of portions of one or both of the IDRs (SEQ ID NOS: 3 and 4) of human TGFβRII-ECD (SEQ ID NO: 1). In embodiments in which portions of each of the two IDRs of TGFβR are included, the portions may be linked together directly or via an intervening linker sequence. The IDR portions may comprise the entire IDR sequence or variants thereof (e.g., substitutions, truncations).

[0120] In one embodiment, the IDR linker comprises or consists of portions of each IDR directly linked together. In some embodiments, the C-terminal IDR or portion thereof is directly linked to the N-terminus of the N-terminal IDR or portion thereof. Non-limiting examples of such embodiments include linkers having the amino acid sequences set forth in SEQ ID NOS: 8-16, 19, 22, 23, and 26.

[0121] In one embodiment, the IDR linker comprises or consists of the sequence set forth in SEQ ID NO:4.

[0122] In one embodiment, the IDR linker does not consist of the sequence set forth in SEQ ID NO: 7. In such embodiments, polypeptides and TGFβ binding agents comprising the sequence set forth in SEQ ID NO: 7 are excluded from the present invention. In certain embodiments, the desired isoform specificity (e.g., a desired TGFβ3:TGFβ1 IC 50 IDR linkers that do not provide a specific IDR sequence are excluded from the present invention, as are polypeptides and TGFβ binding agents that contain such sequences.

[0123] The IDR linker variants, hybrid linkers, hybrid linker variants, truncated linkers, truncated linker variants and extended linkers are derived from the sequences of the IDR linkers disclosed herein.

[0124] As used herein, the term "non-IDR linker" refers to a linker that does not share substantial homology or identity with the intrinsically disordered regions (IDRs) adjacent to the structured ligand-binding domain of the TGFβRII extracellular domain. In aspects and embodiments described herein, the non-IDR linker may be, for example, a flexible linker, including, but not limited to, glycine and glycine-serine (GS) linkers. When producing fusion constructs, it is common practice to introduce artificial, highly flexible glycine or glycine-serine linkers, such as GGGGS or [GS]n (where n is 1, 2, 3, 4, or 5 or more, e.g., 10, 25, or 50), between various regions of the construct. However, such artificial linkers may also be disadvantageous due to the potential for undesirable immunogenicity and their additional molecular weight. Entropy factors are also a potential disadvantage of glycine and GS linkers, which are highly flexible and may become partially restricted upon target binding, resulting in a loss of entropy that is detrimental to binding. Thus, in certain embodiments, the polypeptides and TGFβ binding agents of the present disclosure do not include a non-IDR linker, or include at least one IDR linker or IDR linker variant in addition to a non-IDR linker. Non-limiting examples of non-IDR linkers according to the present disclosure include SEQ ID NOs: 17, 20, 21, and 24.

[0125] In one embodiment, the linker comprises or consists of a mixture of IDR and GS linkers, e.g., the amino acid sequences set forth in SEQ ID NOs: 18 and 25. Such linkers are referred to herein as hybrid linkers. In one embodiment, linkers are provided in which 3 to 7 or 3 to 14 amino acid residues in any one of SEQ ID NOs: 4, 8 to 16, 19, 22, 23, and 26 are substituted with an amino acid sequence containing glycine and / or serine residues (glycine or GS linker). These linkers are referred to herein as hybrid linkers. Hybrid linker variants are also encompassed; these are functionally equivalent variants of hybrid linkers containing one or more insertions, deletions, or amino acid substitutions, optionally conservative amino acid substitutions. Variants are further described below.

[0126] In exemplary embodiments of hybrid linkers and hybrid linker variants, at least three consecutive amino acids of the IDR linker or IDR linker variant are substituted with glycine and / or serine residues. In further exemplary embodiments of hybrid linkers or hybrid linker variants, at least seven consecutive amino acids of the IDR linker or IDR linker variant are substituted with glycine and / or serine residues. In yet other exemplary embodiments of hybrid linkers or hybrid linker variants, two sets of three to seven consecutive amino acids of the IDR linker or IDR linker variant are substituted with glycine and / or serine residues. The two sets of three to seven consecutive amino acids may be spaced apart or consecutive within the linker sequence.

[0127] Examples of glycine and GS sequences for use in hybrid linkers and hybrid linker variants include, but are not limited to, GSG and any one of SEQ ID NOs: 17, 18, 20, 21, 24, and 25.

[0128] In certain embodiments, the linker is a truncated linker or truncated linker mutant. Such linkers have a truncation (deletion) of, for example, 1 to about 20 consecutive amino acids (and any range contained within 1 and about 20, e.g., 1 to about 10, 1 to about 5, etc.) at either or both the N-terminus or C-terminus of an IDR linker provided herein. In exemplary embodiments, the amino acid truncation can be at the N-terminus of any one of SEQ ID NOs: 3 or 8-26. In exemplary embodiments, the truncation can result in the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues at the N-terminus. In another exemplary embodiment, the truncation can be at the C-terminus of any one of SEQ ID NOs: 4 and 8-26. In exemplary embodiments, the truncation can result in the removal of 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues at the C-terminus. In another embodiment, the truncation may result in the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues at the C-terminus.

[0129] In another embodiment, the truncation can be an internal deletion, for example, a deletion starting at amino acid number 10 or 11 of SEQ ID NO: 7. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues are deleted from SEQ ID NO: 7, including amino acid number 10 and / or 11. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues are deleted internally from any one of SEQ ID NOs: 4 and 8-26.

[0130] In exemplary embodiments, the amino acid truncation may result in the removal of 1 to 10 amino acids encompassing the region defined by amino acid residue numbers 11 to 20 of any one of SEQ ID NOs: 7-26.

[0131] Other exemplary and non-limiting embodiments of cleavable linkers are provided in SEQ ID NOs: 8-16, 18, 19, 22, 23, and 26.

[0132] The present disclosure further provides truncation linker mutants, which may contain amino acid substitutions (conservative or non-conservative) compared to the truncation linkers disclosed herein.

[0133] In one embodiment, the first linker has (a) a deletion of at least one N-terminal amino acid residue compared to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:12, or SEQ ID NO:8; (b) a deletion of at least one C-terminal amino acid residue compared to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:12, or SEQ ID NO:8; (c) a deletion of at least one internal amino acid residue compared to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:12, or SEQ ID NO:8; or (d) one or more substitutions in the amino acid sequence compared to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:12, or SEQ ID NO:8, or comprises or consists of the amino acid sequence of any one of (a) to (c). In one embodiment thereof, the amino acid deletion is a deletion of 16 amino acids in SEQ ID NO:3.

[0134] In one embodiment, the second linker has (a) a deletion of at least one N-terminal amino acid residue compared to SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11; (b) a deletion of at least one C-terminal amino acid residue compared to SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11; (c) a deletion of at least one internal amino acid residue compared to SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11; or (d) one or more substitutions in the amino acid sequence compared to SEQ ID NO:4, 7, 9 or 11, or comprises or consists of the amino acid sequence of any one of (a) to (c).

[0135] In certain embodiments, the linker is an extended linker. Such linkers have an addition (extension) of 1 to 10 amino acids (and any range contained within 1 and 10, e.g., 1 to 7, 1 to 5, 1 to 3, 1, 2, 3, etc.) at either or both of the N-terminus or C-terminus of any IDR linker, IDR linker mutant, hybrid linker, hybrid linker mutant, truncated linker, or truncated linker mutant as disclosed herein. Each of these additional amino acids can be independently selected from any amino acid residue.

[0136] In exemplary embodiments, the linkers disclosed herein may comprise 1 to 5 additional amino acid residues at their N-terminus. In another exemplary embodiment, the linkers disclosed herein may comprise 1 to 5 additional amino acid residues at their C-terminus. In further exemplary embodiments, the linkers disclosed herein may comprise 1 to 5 additional amino acid residues at both their N-terminus and C-terminus. Such additional amino acid residues may be selected from any amino acid residue and may be the same or different. Other exemplary and non-limiting embodiments of extended linkers include the addition of 1 to 10 amino acids (and any range included within 1 and 10, e.g., 1 to 7, 1 to 5, 1 to 3, 1, 2, 3, etc.) at either or both the N-terminus or C-terminus of any one of SEQ ID NOs: 4 and 8-26. The added sequence may comprise any amino acid residues.

[0137] Exemplary embodiments of extended linkers also include those that include a non-IDR linker moiety at either or both of their N-terminus or C-terminus. For example, an IDR linker, IDR linker mutant, hybrid linker, hybrid linker mutant, truncated linker, or truncated linker mutant can be flanked at either or both of its N-terminus and C-terminus by at least one non-IDR linker. Alternatively, a non-IDR linker can be flanked at either or both of its N-terminus and C-terminus by at least an IDR linker, IDR linker mutant, hybrid linker, hybrid linker mutant, truncated linker, or truncated linker mutant.

[0138] In certain embodiments of the polypeptide constructs and TGFβ binding agents of the present disclosure, the N-terminal region comprises or consists of the N-terminal IDR (SEQ ID NO:3) in TGFβRII-ECD (SEQ ID NO:1), or a sequence substantially identical thereto, including, but not limited to, truncated or substituted variants thereof. It should be understood that the N-terminal region can be truncated and / or substituted and otherwise modified so long as the desired inhibitory potency and specificity are not adversely affected.

[0139] The present disclosure also encompasses variants of the polypeptides and TGFβ-binding agents described herein. Variants encompassed by the present disclosure include those having an alteration in the amino acid sequence of any one of the polypeptide or TGFβ-binding agent elements (such as the first and second TGFβ receptor ligand-binding domains (TGFβR-LBDs), first linker, second linker, N-terminal region, multimerization domain, etc.). Variants of polypeptides or TGFβ-binding agents include those having similar or improved binding affinity, avidity, isoform specificity, inhibitory potency, stability, manufacturability, and / or reduced aggregation, for example, compared to the polypeptides and TGFβ-binding agents disclosed herein.

[0140] Sites of interest for substitutional mutagenesis include the multimerization domain of the polypeptide or TGFβ-binding agent. Exemplary embodiments of polypeptide or TGFβ-binding agent variants of the present disclosure can include those with modified IgG1, IgG2, IgG3, or IgG4 constant regions or portions thereof. TGFβ-binding agents that can include an IgG1 constant region (modified or unmodified) are encompassed herein. TGFβ-binding agents that can include an IgG4 constant region (modified or unmodified) are also encompassed herein.

[0141] Variants encompassed by the present disclosure include those that may contain insertions, deletions, or amino acid substitutions (conservative or non-conservative). These variants may have at least one amino acid residue in the amino acid sequence removed and a different residue inserted in its original place.

[0142] Generally, a conservative amino acid substitution is the substitution of an amino acid residue for another amino acid residue having similar chemical properties (e.g., size, charge, or polarity). Conservative substitutions can be made by exchanging an amino acid from one of the groups listed below (Groups 1-6) for another amino acid of the same group.

[0143] Other exemplary embodiments of conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." If such substitutions result in undesirable properties, more substantial changes, designated in Table 1 as "exemplary substitutions" or described further below with reference to amino acid classes, can be introduced and the products screened.

[0144] It is known in the art that variants can be generated by substitution mutagenesis and retain the biological activity (i.e., functional equivalence) of the polypeptides of the present disclosure. These variants have, for example, one or more conservative amino acid substitutions in at least one amino acid residue in the amino acid sequence that has been removed and a different residue inserted in its original position. Examples of substitutions identified as "conservative substitutions" are shown in Table 1. If such a substitution results in an undesirable change, other types of substitutions designated as "exemplary substitutions" in Table 1 or further described herein with reference to amino acid classes are introduced and the products screened.

[0145] Amino acid residues can be divided into groups based on common side chain properties as follows: (Group 1) hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (Group 2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (Group 3) Acidic: Aspartic acid (Asp), Glutamic acid (Glu); (Group 4) Basic: histidine (His), lysine (Lys), arginine (Arg); (Group 5) residues that influence chain orientation: glycine (Gly), proline (Pro); and (Group 6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0146] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another. [Table 1]

[0147] In general, the degree of similarity and identity between variable chains is determined herein using the Blast2 sequence program (Tatusova, TA and Madden, TL, 1999) with default settings, i.e., the blastp program, BLOSUM62 matrix (open gap 11 and extension gap penalty 1; gap dropoff 50, expect 10.0, word size 3) and activation filter.

[0148] However, the level of identity can also be determined over the entire length of a given sequence. Thus, percent identity will indicate the amino acids that are identical and that may occupy the same or similar positions compared to the original peptide. Percent similarity will indicate the amino acids that are identical and those that are substituted with conservative amino acid substitutions compared to the original peptide in the same or similar positions.

[0149] Thus, in certain embodiments, a variant of the present disclosure comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the original sequence or portion of the original sequence.

[0150] In certain embodiments, the amino acid sequence changes occur in the TGFβ receptor ligand binding domain (TGFβR-LBD) of the polypeptide or TGFβ binding agent. In other embodiments, the changes can occur outside the TGFβ receptor ligand binding domain (TGFβR-LBD) of the TGFβ binding agent. Variants encompassed by the present disclosure can have a TGFβR-LBD that is identical or substantially identical to a structured ligand binding domain found in the extracellular domain (ECD) of a TGFβ receptor (including in humans, animals, etc.). In further embodiments, the amino acid sequence changes occur in the multimerization domain. In yet other embodiments, the amino acid sequence changes occur in the first and / or second linker. It should be understood that changes can occur in multiple regions of a polypeptide or TGFβ binding agent so long as the desired function is maintained.

[0151] In certain embodiments, a polypeptide or TGFβ binding agent of the disclosure can be conjugated to, for example, a targeting agent, a therapeutic moiety (for therapeutic purposes), or a detectable moiety (i.e., for detection or diagnostic purposes).

[0152] In exemplary embodiments, a polypeptide or TGFβ binding agent of the present disclosure is conjugated to a therapeutic moiety, such as, but not limited to, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an anti-fibrotic agent, an anti-cancer agent (e.g., a small molecule), a single chain antibody, or the like.

[0153] In another exemplary embodiment, a polypeptide or TGFβ binding agent of the present disclosure is conjugated to a detectable moiety, including, for example and without limitation, a moiety detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, and / or other physical means. The detectable moiety can be attached to the TGFβ binding agent directly or indirectly (for example, but not limited to, via a linkage such as, but not limited to, a DOTA or NHS linkage) using methods well known in the art. A wide variety of detectable moieties can be used, with the choice being made depending on the required sensitivity, ease of conjugation, stability requirements, and available instrumentation. Suitable detectable moieties include, but are not limited to, fluorescent labels, radioactive labels (for example, but not limited to, 125 I, In 111 , Tc 99 , I 131 and positron emitting isotopes for PET scanners and the like), nuclear magnetic resonance active labels, luminescent labels, chemiluminescent labels, chromophore labels, enzyme labels (for example and without limitation, horseradish peroxidase, alkaline phosphatase, etc.), quantum dots and / or nanoparticles. The detectable moiety may produce and / or generate a detectable signal, thereby allowing the signal from the detectable moiety to be detected.

[0154] Therapeutic moieties include, for example and without limitation, yttrium-90, scandium-47, rhenium-186, iodine-131, iodine-125, and many others recognized by those skilled in the art (e.g., lutetium (e.g., Lu 177 ), bismuth (e.g., Bi 213 ), copper (e.g., Cu 67 )), 5-fluorouracil, adriamycin, irinotecan, taxanes, pseudomonas endotoxin, ricin, auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F), maytansinoids (e.g., mertansine), and other toxins.

[0155] In another embodiment, the therapeutic moiety may include another therapeutic agent for a TGFβ-related disease or condition. For example, and without limitation, one or more polypeptide constructs or TGFβ-binding agents may be linked to a cytotoxic agent to generate an antibody-drug conjugate (ADC).

[0156] A targeting agent can include, for example, an amino acid sequence for delivering a polypeptide or TGFβ binding agent to a desired tissue, organ, or location in a subject's body. For example, and without limitation, a targeting agent can include a polyaspartic acid sequence motif for bone targeting, or an antibody or antigen-binding fragment.

[0157] In other exemplary embodiments, the targeting agent, therapeutic moiety, or diagnostic moiety may include, for example and without limitation, an antibody or antigen-binding fragment thereof (e.g., a single chain antibody), a binding agent with affinity for another member of the TGFβ family or another therapeutic target, a radiotherapeutic agent, an imaging agent, a fluorescent moiety, a cytotoxic agent, a cytostatic agent, a nanoparticle-based carrier, a polymer conjugated to a drug, a nanocarrier, an imaging agent, a stabilizer, a drug, a nanocarrier, and / or a dendrimer.

[0158] It should be understood that the site for conjugation is not particularly limited, so long as there is no adverse effect on the function of the polypeptide or TGFβ-binding agent. For example, and without limitation, a targeting agent, therapeutic moiety, or detectable moiety may be conjugated at the linker portion of the polypeptide or TGFβ-binding agent (e.g., in a non-IDR linker), or at any other suitable site, such as at its N-terminus or multimerization domain.

[0159] Production of polypeptides and TGFβ binding agents The polypeptides or TGFβ binding agents disclosed herein can be made by a variety of methods known to those skilled in the art, including recombinant DNA methods.

[0160] To express a polypeptide or TGFβ binding agent, a nucleotide sequence capable of encoding a polypeptide chain described herein can be inserted into an expression vector, i.e., a vector containing elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements can include regulatory sequences, e.g., enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions. Methods well known to those of skill in the art can be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo genetic recombination, etc.

[0161] Various expression vectors and host cell systems known to those skilled in the art can be used to express the polypeptide chains described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus vectors; plant cell systems transformed with viral or bacterial expression vectors; and animal cell systems. For long-term production of recombinant proteins in mammalian systems, stable expression in mammalian cell lines can be used. For example, a nucleotide sequence capable of encoding any one of the polypeptide chains described herein can be transformed into a cell line using an expression vector that may contain a viral origin of replication and / or endogenous expression elements and a selectable or visible marker gene in the same or a separate vector. The present disclosure should not be limited by the vector or host cell used. In certain embodiments disclosed herein, nucleic acids capable of encoding the polypeptide chains described herein can be ligated into an expression vector. When the TGFβ binding agent is composed of different polypeptide chains (i.e., the first polypeptide and the second polypeptide are not identical), each of such polypeptide chains can be ligated into a separate vector or the same vector. According to the present disclosure, the polypeptide chains of a TGFβ binding agent can be encoded by a single vector or by separate vectors (e.g., a vector set). A cell is transformed with the desired vector or vector set.

[0162] Alternatively, the polypeptide chains can be expressed from an in vitro transcription system or a combined in vitro transcription / translation system, respectively, or any such cell-free system.

[0163] Host cells containing the nucleotide sequences can be cultured under conditions for transcription of the corresponding RNA (such as mRNA) and / or expression and secretion of the polypeptide from the cell culture. In exemplary embodiments, expression vectors containing nucleotide sequences capable of encoding the polypeptide chains described herein can be designed to include a signal sequence that directs secretion of the polypeptide through a prokaryotic or eukaryotic cell membrane.

[0164] Due to the inherent degeneracy of the genetic code, DNA sequences encoding the same, substantially the same, or functionally equivalent amino acid sequences can be generated and used. The nucleotide sequences of the present disclosure can be manipulated using methods generally known in the art to modify nucleotide sequences for various purposes, including, but not limited to, modifying the cloning, processing, and / or expression of gene products. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to manipulate nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations that generate new restriction sites, modify glycosylation patterns, change codon preferences, generate splice variants, and the like. Codon-optimized nucleic acids encoding the polypeptide chains described herein are encompassed by the present disclosure.

[0165] Additionally, a host cell line may be selected for its ability to modulate expression of the inserted sequences or process the expressed polypeptide in the desired manner. A variety of host cells (e.g., CHO, HeLa, MDCK, HEK293, and W138) with characteristic mechanisms for particular cellular and post-translational activities are commercially available and available from the American Type Culture Collection (ATCC) and can be selected to ensure proper modification and processing of the expressed polypeptide.

[0166] Those skilled in the art will also readily recognize that nucleic acid and polypeptide sequences can be synthesized, in whole or in part, using chemical or enzymatic methods well known in the art. For example, peptide synthesis can be performed using various solid-phase techniques, and machines such as the ABI 431A peptide synthesizer (PE Biosystems) can be used to automate synthesis. If desired, the amino acid sequence can be modified during synthesis and / or combined with sequences from other proteins to produce variant proteins.

[0167] Pharmaceutical Composition Pharmaceutical compositions comprising the polypeptides or TGFβ binding agents disclosed herein are also encompassed by the present disclosure. Pharmaceutical compositions generally comprise a polypeptide or TGFβ binding agent disclosed herein and a pharmaceutically acceptable carrier.

[0168] Pharmaceutical compositions can be prepared as known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition, 2000). For example, a therapeutic compound and / or composition, together with one or more solid or liquid pharmaceutical carrier materials and / or additives (or auxiliary substances), and optionally in combination with other pharmaceutically active compounds having a therapeutic or prophylactic effect, can be formulated into a suitable dosage form, which can then be used as a pharmaceutical in human or veterinary medicine. Pharmaceuticals can also contain additives, many of which are known in the art, such as fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersing agents, preservatives, sweeteners, colorants, flavorings, perfumes, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for changing osmotic pressure, coating agents, or antioxidants.

[0169] The term "pharmaceutical composition" refers to a composition comprising a polypeptide or TGFβ binding agent described herein and, depending on the method of administration and the nature of the dosage form, at least one component including a pharmaceutically acceptable carrier, diluent, adjuvant, excipient, or vehicle, such as preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, and dispersing agents.

[0170] The term "pharmaceutically acceptable carrier" is used to mean any carrier, diluent, adjuvant, excipient, or vehicle as described herein or known in the art. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin. Non-limiting examples of suitable carriers, diluents, solvents, or vehicles include water, salt solutions, phosphate buffered saline (PBS), gelatin, oils, alcohols, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic acid esters, such as ethyl oleate. Non-limiting examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Non-limiting examples of disintegrants include starch, alginic acid, and some complex silicates. Non-limiting examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol.

[0171] The term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with the cells of subjects, e.g., humans and animals, without undue toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit-risk ratio.

[0172] Pharmaceutically acceptable carriers may include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In one embodiment, the carrier is suitable for parenteral administration. The carrier may be suitable for intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. In other embodiments, the carrier is suitable for topical administration or administration by inhalation. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions provided herein is contemplated. Supplementary active compounds may also be incorporated into the compositions. For example, the pharmaceutical compositions provided herein may further comprise at least one additional therapeutic agent, as further described below.

[0173] In certain embodiments, the pharmaceutical compositions provided herein can be administered orally, for example, in the form of pills, tablets, lacquered tablets, dragees, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example, in the form of suppositories.

[0174] In other embodiments, the pharmaceutical compositions provided herein can be administered parenterally, for example subcutaneously, intramuscularly, or intravenously, in the form of a solution for injection or infusion. Other suitable administration forms are, for example, in the form of an ointment, cream, tincture, spray, or transdermal therapeutic system, for example, transdermal or topical administration, or inhalation administration in the form of a nasal spray or aerosol mixture, or, for example, microcapsules, implants, or wafers.

[0175] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Furthermore, the compounds may be administered in sustained-release formulations, for example, in compositions containing sustained-release polymers. The compounds may be prepared with carriers that will protect against rapid release, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polyglycolic acid copolymers (PLG).

[0176] Many methods for preparing such preparations are generally known to those skilled in the art.Sterile injectable solution can be prepared by incorporating the required amount of the active compound, such as the polypeptide or TGFβ binding agent provided herein, in a suitable solvent with one or a combination of the above-listed components as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other components required from the above-listed components.For the preparation of sterile injectable powder, the general preparation method is vacuum drying and freeze-drying, which produces a powder of active ingredient and any other desired components from the solution that has been previously sterilized and filtered.Compounds can also be formulated with one or more additional compounds that enhance their solubility.

[0177] It is often advantageous to formulate compositions (such as parenteral compositions) in dosage unit form for ease of administration and uniformity of dosage. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other animals, each unit containing a predetermined quantity of active material calculated to produce a desired therapeutic effect, together with a suitable pharmaceutical carrier. The specifications for the dosage unit forms of the present invention may vary and are determined by and directly respond to (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such therapeutic compounds for the prevention or treatment of TGFβ-related diseases or disorders. Dosage amounts are further described below.

[0178] In certain embodiments, pharmaceutical compositions are provided comprising an effective amount of a polypeptide and / or TGFβ binding agent described herein and a pharmaceutically acceptable carrier. In one embodiment, pharmaceutical compositions for treating or preventing fibrosis are provided, comprising a polypeptide or TGFβ binding agent described herein and a pharmaceutically acceptable carrier. In another embodiment, pharmaceutical compositions are provided for delaying cancer progression, inhibiting cancer invasion, e.g., malignant glial cell (MGC) infiltration, inhibiting cancer stem cell growth, survival, spheroid formation and / or proliferation, inhibiting metastasis, inhibiting cancer recurrence, and / or overcoming cancer chemoresistance, comprising a polypeptide and / or TGFβ binding agent described herein and a pharmaceutically acceptable carrier. In another embodiment, pharmaceutical compositions are provided for treating or preventing bone marrow failure conditions.

[0179] As used herein, "pharmaceutically acceptable carriers" or "pharmaceutical carriers" are known in the art and include, but are not limited to, 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. Furthermore, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic acid esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions (including saline and buffered media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, collating agents, and inert gases and the like.

[0180] For any compound, the therapeutically effective dose can be initially estimated in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. Animal models can also be used to determine concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. These techniques are well known to those skilled in the art, and a therapeutically effective dose refers to that amount of active ingredient that ameliorates symptoms or pathological conditions. Therapeutic efficacy and toxicity can be assessed by ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 The lethal dose can be determined by standard pharmaceutical procedures in cell culture or in experimental animals, such as by calculating and comparing the dose (lethal to 50% of the population) with the dose required for the treatment of a given disease. Any of the pharmaceutical compositions described herein can be applied to any subject in need of treatment, including, but not limited to, mammals, such as dogs, cats, cows, horses, rabbits, monkeys, and especially humans.

[0181] The pharmaceutical compositions described herein can be administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0182] How to use The polypeptides or TGFβ-binding agents described herein, and pharmaceutical compositions thereof, are useful for preventing or treating TGFβ-related diseases or conditions. Accordingly, provided are methods for preventing or treating TGFβ-related diseases or conditions in a subject, comprising administering a therapeutically effective amount of a polypeptide, TGFβ-binding agent, or pharmaceutical composition described herein. The polypeptides and TGFβ-binding agents are generally administered in the form of a pharmaceutical composition. The subject may be in need of such treatment, i.e., suffering from, suspected of suffering from, or at risk of suffering from a disease or condition associated with TGFβ (e.g., TGFβ1 and / or TGFβ3).

[0183] As used herein, the term "TGFβ-related disease or condition" refers to a disease or condition that can be ameliorated by inhibition of TGFβ activity, particularly TGFβ1 and / or TGFβ3 activity. TGFβ-related diseases or conditions include, but are not limited to, diseases or conditions associated with overexpression or overactivation of TGFβ ligands, particularly TGFβ1 and / or TGFβ3. In certain embodiments, the TGFβ-related disease or condition is mediated by TGFβ1 and / or TGFβ3. In one embodiment, the disease or condition being treated is mediated by TGFβ3. In another embodiment, the disease or condition being treated is mediated by a combination of TGFβ1 and TGFβ3. As used herein, the term "amelioration" means reducing, suppressing, alleviating, lowering, arresting, or stabilizing the onset or progression of a disease.

[0184] Examples of TGFβ-related diseases or conditions that can be prevented or treated in accordance with the present disclosure include, but are not limited to: fibrosis (e.g., fibrosis, fibrotic scarring, fibroproliferative disorders); cancer (e.g., malignant tumors, solid tumors, metastases); bone marrow failure (e.g., Shwachman-Bodian-Diamond syndrome, Fanconi anemia); eye diseases; and connective tissue genetic disorders.

[0185] In certain embodiments, the polypeptides or TGFβ binding agents described herein are used to treat or prevent fibrosis, including, for example and without limitation, fibrosis of tissues and / or organs, fibrotic scarring, and fibroproliferative disorders. Non-limiting examples of fibrosis or conditions that may be treated or prevented include pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), renal fibrosis, liver fibrosis (e.g., cirrhosis), systemic sclerosis, scleroderma, dermal fibrosis, cardiac fibrosis, myelofibrosis, and myelofibrosis. In a specific embodiment, systemic sclerosis (SSc) is treated or prevented. In other embodiments, scleroderma is treated or prevented. In other embodiments, myelofibrosis (MF) is treated or prevented.

[0186] Systemic sclerosis (SSc, also known as scleroderma) is a severe, debilitating fibrotic disease. TGFβ is a potent profibrotic cytokine that has been shown to be important for promoting several pathological processes in SSc, including increased collagen deposition in the skin and lungs (Varga, J. and Abraham, D., 2007; Varga, J. and Whitfield, M.L., 2009; Gabrielli, A. et al., 2009; Lafyatis, R., 2014; Allanore, Y. et al., 2015). SSc represents a significant unmet therapeutic challenge, with the average life expectancy of newly diagnosed SSc patients being approximately 11 years (Mayes, M.D. et al., 2003). A recent clinical study validated TGFβ as a driver of fibrosis in human SSc patients by demonstrating dramatic reversal of fibrosis after inhibition of TGFβ with the neutralizing antibody fresolimumab (Rice, LM et al., 2015). This clinical proof-of-principle study, together with extensive preclinical data demonstrating the importance of TGFβ in promoting fibrosis in SSc and other diseases, provides a compelling rationale for the use of TGFβ binding agents according to the present disclosure for the treatment of SSc patients.

[0187] In myelofibrosis (MF), myelofibrosis is a hallmark of the disease, and its severity correlates with clinical features, including anemia. Administration of TGFβ blockers has been shown to reduce myelofibrosis in several preclinical studies (Wang, JC et al., 2006; Vannucchi, AM et al., 2005), supporting the dual pathological role of TGFβ in MF: promoting myelofibrosis and myeloproliferation. Increased intraplatelet, peripheral blood mononuclear cell, and megakaryocyte-associated TGFβ have been demonstrated in MF patients. Overexpression of TGFβ in clinical samples, along with extensive preclinical data on the effects of TGFβ neutralization in MF models, provides a compelling rationale for the use of the TGFβ binding agents disclosed herein for the treatment of MF patients.

[0188] Other exemplary embodiments of fibrosis that may be prevented or treated include, for example and without limitation: interstitial lung disease; human fibrotic lung diseases (e.g., bronchiolitis obliterans, idiopathic pulmonary fibrosis, pulmonary fibrosis of known etiology, tumor interstitial in pulmonary disease, systemic sclerosis affecting the lungs, Hermansky-Pudlak syndrome, coal workers' pneumoconiosis, asbestosis, silicosis, chronic pulmonary hypertension); treatable types of fibrosis associated with AIDS, including pulmonary fibrosis, cystic fibrosis, hepatic fibrosis, cardiac fibrosis, mediastinal fibrosis, peritoneal fibrosis, myelofibrosis, and dermal fibrosis; scleroderma; and systemic sclerosis. Specific forms of fibrosis that may be treated or prevented include those affecting any organ or tissue or cell of the body, such as human tendon fibroblasts, kidneys, lungs, intestines, liver, heart, bone marrow, reproductive organs, skin, and eyes. These diseases include, but are not limited to, cystic fibrosis, systemic sclerosis, chronic obstructive pulmonary disease (COPD), Dupuytren's contracture, glomerulonephritis, liver fibrosis, post-infarction myocardial fibrosis, restenosis, ophthalmic surgery-induced fibrosis, and scarring. Genetic diseases of connective tissue may also be treated, including, but not limited to, Marfan syndrome (MFS) and osteogenesis imperfecta.

[0189] In certain embodiments, the polypeptides or TGFβ binding agents described herein are used to inhibit the differentiation of fibroblasts into myofibroblasts.

[0190] In certain embodiments, the polypeptides or TGFβ binding agents described herein are used to treat or prevent fibroproliferative disorders characterized by proliferation of fibroblasts and the corresponding overexpression of extracellular matrix proteins such as fibronectin, laminin, and collagen.

[0191] In certain embodiments, the polypeptides or TGFβ binding agents described herein are used to treat or prevent cancer, including, for example and without limitation, lung cancer, head and neck cancer, melanoma, colon cancer, pancreatic cancer, colorectal cancer, liver cancer, breast cancer, epithelial cancer, cholangiocarcinoma, solid tumors, and the like. In certain embodiments, the term "prevention" in the context of cancer can include preventing primary tumor invasion or metastasis. In certain embodiments, the term "treatment" in the context of cancer can include inhibiting TGFβ-mediated suppression of immune responses in the tumor microenvironment. With respect to solid tumors, the immunosuppressive role of TGFβ in the tumor microenvironment has been clearly demonstrated preclinically. Furthermore, clinical studies have recently shown that the lack of response to immune checkpoint inhibitors in patients with bladder cancer is associated with TGFβ signaling in the tumor microenvironment, supporting the notion that TGFβ suppresses anti-tumor immunity and suggesting that TGFβ inhibitors may have single-agent activity in some tumor environments and act to enhance anti-tumor activity when combined with immune checkpoint inhibitors in other tumor environments.

[0192] In certain embodiments, the polypeptide constructs or TGFβ binding agents described herein are used to treat or prevent disorders of abnormal cell proliferation and / or dysregulated apoptosis, including, but not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, osteosarcoma, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal tract (gastric, colorectal, and / or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, testicular cancer, hepatocellular (liver and / or bile duct) carcinoma, and primary or secondary central nervous system tumors. , primary or secondary brain tumors, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, cancer of the kidney and / or ureter, renal cell carcinoma, cancer of the renal pelvis, tumors of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination thereof.

[0193] In certain embodiments, the polypeptide constructs or TGFβ binding agents described herein are used to treat or prevent a disease or disorder selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, and pancreatic cancer.

[0194] In certain embodiments, the polypeptide constructs or TGFβ binding agents described herein are used to treat or prevent a disease or disorder that is a blood cancer, such as leukemia, lymphoma, or myeloma. In certain embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center cell lymphoma, transformed lymphoma, moderately differentiated lymphocytic lymphoma, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-cell lymphoma, mantle zone lymphoma, or the like. The disease or disorder is selected from the group consisting of myeloma, low-grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T-cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B-cell acute lymphoblastic leukemia, precursor T-cell acute lymphoblastic leukemia, Burkitt's leukemia (Burkitt's lymphoma), acute mixed lineage leukemia, chronic myelogenous lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. In certain embodiments, the disease or disorder is myeloma. In certain embodiments, the disease or disorder is myelodysplastic syndrome (MDS). In another specific embodiment, the disease or disorder is acute myeloid leukemia (AML). In another particular embodiment, the disease or disorder is chronic lymphocytic leukemia (CLL). In yet another particular embodiment, the myeloma is multiple myeloma (MM).

[0195] In other embodiments, the polypeptide constructs or TGFβ binding agents described herein are used to treat or prevent a disease or disorder that is a solid malignancy. In certain embodiments, the solid malignancy is selected from the group consisting of carcinoma, adenocarcinoma, adrenocortical carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, colorectal carcinoma, ductal cell carcinoma, lung carcinoma, thyroid carcinoma, nasopharyngeal carcinoma, melanoma, non-melanoma skin cancer, and lung carcinoma.

[0196] In some embodiments, the solid malignancy is an advanced non-CNS primary solid tumor, hi some embodiments, the solid malignancy is selected from the group consisting of gastric / gastroesophageal junction (GEJ) cancer, bladder / urothelial carcinoma, and non-small cell lung cancer (NSCLC).

[0197] In certain embodiments, the immune checkpoint inhibitor administered in combination with a polypeptide or TGFβ-binding agent described herein can be any pharmaceutical agent that inhibits or blocks the activity of an inhibitory immune checkpoint molecule. In certain embodiments, the activity is binding to a natural binding partner of the immune checkpoint molecule. If the immune checkpoint molecule is a receptor, the activity can be ligand-binding activity. If the immune checkpoint molecule is a ligand, the activity can be receptor-binding activity.

[0198] In certain embodiments, the immune checkpoint inhibitor administered in combination with a polypeptide or TGFβ-binding agent described herein is a negative checkpoint regulator involved in T cell activation. In some more specific embodiments, such negative checkpoint regulators include cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD80, CD86, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2), lymphocyte activation gene-3 (LAG-3; also known as CD223), galectin-3, B and T lymphocyte attenuator (BTLA), T cell membrane protein 3 (TIM3), galectin-9 (GAL9), B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, B7-H8, B7-H9, B7-H1, B7-H1, B7-H2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, B7-H8, B7-H9, B7-H1, B7-H1, B7-H1, B7-H2 ...1, B7-H2, B7-H1, B7-H1, B7-H1, B7-H1, B7-H2, B7-H1, B7-H1, B7-H1, B7-H1, B7-H1, B7-H1, B7-H1, B7-H1, B7-H1, B7- -H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9), V-domain Ig suppressor of T cell activation (VISTA), glucocorticoid-inducible tumor necrosis factor receptor-related (GITR) protein, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, or CGEN-15092. A summary of such checkpoint regulators and the drugs that target them is listed in Table 1. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, or TDO.

[0199] In some embodiments, the immune checkpoint inhibitor can be an antibody, a small molecule, or an oligonucleotide (such as an aptamer, shRNA, miRNA, siRNA, or antisense DNA). In certain embodiments, the immune checkpoint inhibitor is approved in the United States by the Food and Drug Administration (FDA) or a foreign equivalent agency for the treatment of cancer or a disease caused by a pathogen.

[0200] In certain embodiments, immune checkpoint inhibitors are antibodies that bind to immune checkpoints and inhibit immune checkpoint activity. Antibodies that can be immune checkpoint inhibitors include, but are not limited to, monoclonal antibodies (including Fc-optimized monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that retain antigen-binding activity, such as Fv, Fab, Fab', F(ab')2, bispecific antibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), multispecific antibodies formed from antibody fragments, and fusion proteins containing antibody fragments. In certain embodiments, the antibody is a monoclonal antibody. Preferably, the antibody is a humanized antibody.

[0201] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In certain embodiments, the immune checkpoint inhibitor is a monoclonal antibody that binds to PD-1 and inhibits an activity of PD-1 (e.g., ligand binding activity).

[0202] In some embodiments, the monoclonal antibody is selected from the group consisting of nivolumab, pidilizumab, MEDI0680, pembrolizumab, AMP-224, AMP-514, STI-A1110, TSR-042, AUR-012, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, and toripalimab.

[0203] In certain embodiments, the monoclonal antibody is nivolumab, pidilizumab, MEDI0680, or pembrolizumab. In a further specific embodiment, the monoclonal antibody is nivolumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is AMP-224. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is pidilizumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is pembrolizumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is MEDI0680. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is STI-A1110. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is TSR-042. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is AUR-012.

[0204] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In certain embodiments, the immune checkpoint inhibitor is a monoclonal antibody that binds to PD-L1 and inhibits PD-L1 activity (e.g., receptor binding activity).

[0205] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of mpdl3280A, durvalumab, avelumab, BMS-936559, atezolizumab, RG7446, and STI-A1010.

[0206] In certain embodiments, the monoclonal antibody is mpdl3280A, durvalumab, avelumab, BMS-936559, or atezolizumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-L1 is RG7446. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-L1 is STI-A1010.

[0207] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4 (e.g., ipilimumab).

[0208] In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3 (e.g., BMS-986016).

[0209] In some embodiments, immune checkpoint inhibitors administered in combination with a polypeptide or TGFβ binding agent described herein include, but are not limited to: OPDIVO® (nivolumab); YERVOY® (ipilimumab); leratolimab; linrodostat; EMPLICITI® (elotuzumab); BMS-986258; BMS 986315; BMS-986207; BMS-986249; and BMS-986218.

[0210] PD-1 inhibitors useful in the combinations described herein include any molecule capable of inhibiting, blocking, eliminating, or preventing the activity or expression of PD-1. In particular, anti-PD-1 inhibitors can be small molecule compounds, nucleic acids, polypeptides, antibodies, peptibodies, diabodies, minibodies, single-domain antibodies or nanobodies, single-chain variable fragments (ScFv), or functional fragments or variants thereof. In one example, the PD-1 inhibitor is a small molecule compound (e.g., a compound having a molecular weight of less than about 1000 Da). In other embodiments, PD-1 inhibitors useful in the combinations described herein include nucleic acids and polypeptides.

[0211] In some embodiments, methods are provided for preventing or inhibiting cancer recurrence after treatment, e.g., after drug treatment or surgical resection. In some embodiments, methods are provided for slowing cancer progression, wherein cancer regrowth is delayed by more than 30%, or more than 50%, or more than 70%, and / or the survival time of an affected subject is increased. Further provided are methods for enhancing the effectiveness of a cancer therapy for the treatment of cancer selected from the group including resection, chemotherapy, radiation therapy, immunotherapy, and / or gene therapy, comprising administering a polypeptide or TGFβ binding agent described herein and administering the cancer therapy simultaneously, separately, or sequentially. As used herein, the term "enhancing the effectiveness of a cancer therapy" refers to an improvement in conventional cancer therapy, and includes reducing the amount of an anti-cancer composition applied during conventional cancer therapy, e.g., the amount of radiation in radiation therapy, the amount of a chemotherapeutic agent in chemotherapy, the amount of an immunotherapeutic agent in immunotherapy, or the amount of a vector in gene therapy, and / or increasing the effectiveness of conventional therapies and anti-cancer compositions when applied in conventional doses or amounts during conventional cancer therapy. In one embodiment, increasing the effectiveness of cancer therapy refers to increasing the survival rate of the subject receiving the treatment.

[0212] In certain embodiments, the polypeptides or TGFβ-binding agents described herein are used to treat or prevent bone marrow failure in a subject, e.g., a human who has or is at risk of developing bone marrow failure. Exemplary types of bone marrow failure include, but are not limited to, SDS (also known as Shwachman-Bodian-Diamond syndrome or SBDS), Fanconi anemia (FA), dyskeratosis congenita (DC), congenital amegakaryocytic thrombocytopenia (CAMT), Blackfan-Diamond anemia (BDA), and reticular dysplasia (RD). Patients with Shwachman-Diamond syndrome (SDS) suffer from bone marrow failure, exocrine pancreatic insufficiency, bone malformations, and an increased risk of acute myeloid leukemia. In certain embodiments, the polypeptides or TGFβ-binding agents described herein are used to treat or prevent Fanconi anemia (FA). In other embodiments, the polypeptides or TGFβ-binding agents described herein are used to treat or prevent Shwachman-Diamond syndrome (SDS) in a subject.

[0213] Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. Patients with FA develop bone marrow failure during the first decade of life due to a reduction in hematopoietic stem and progenitor cells (HSPCs). FA is caused by mutations in one of the 19 Fanconi anemia complementation group (FANC) genes, whose products cooperate in the FA / BRCA DNA repair pathway. Bone marrow failure in FA may, in part, be the direct or indirect result of overactivation of growth inhibitory pathways induced by genotoxic stress. Canonical TGFβ pathway-mediated growth inhibition of hematopoietic stem cells (HSCs) has recently been identified as the cause of bone marrow failure in FA (Rio, P. and Bueren, JA, 2016; Zhang, H. et al., 2016). Shwachman-Diamond syndrome (SDS) is another rare bone marrow failure syndrome caused by mutations in the SBDS gene (Boocock, GR et al., 2003; Rogers, ZR, 2018). The TGFβ pathway has been shown to be dysregulated in SDS cells. Taken together, these findings provide a compelling rationale for the use of TGFβ binding agents according to the present disclosure for the treatment of bone marrow failure syndromes.

[0214] In certain embodiments, therefore, there is provided a method for treating or preventing bone marrow failure, such as SDS, comprising administering to a subject in need thereof an effective amount of a polypeptide or TGFβ binding agent according to the present disclosure. In such embodiments, the polypeptide or TGFβ binding agent may reduce or inhibit symptoms or sequelae associated with SDS. Exemplary symptoms or sequelae associated with SDS include neutropenia (e.g., exhibiting an absolute neutrophil count <1500 / mL), anemia, thrombocytopenia (e.g., thrombocytopenia <50,000 / mm ), and / or thrombocytopenia (e.g., thrombocytopenia <50,000 / mm ). 3 The bone marrow failure is selected from the group consisting of: bone marrow failure (showing a platelet count of less than 0.05), exocrine pancreatic insufficiency, growth retardation, chronic steatorrhea, metaphyseal dysplasia, myelodysplasia, megakaryocytic dysplasia, erythroid dysplasia, acute myeloid leukemia (AML), and generalized osteopenia. For further description of bone marrow failure, see, e.g., WO 2016 / 138300 and WO 2019 / 018662.

[0215] As used herein, the terms "effective amount" and "therapeutically effective amount" are used interchangeably to refer to an amount or dose of a compound or composition that, upon administration to a subject in single or multiple doses, provides a desired effect in the treated subject (e.g., a desired biological or pharmacological response to ameliorate, reduce, or prevent a disease, disorder, or condition). In certain embodiments, an effective amount is an amount or dose of a compound or composition that prevents or treats a TGFβ-related disease or condition in a subject, as described herein. In certain embodiments, an effective amount is an amount or dose of a compound or composition that inhibits one or more activities of TGFβ (e.g., TGFβ1 and / or TGFβ3) in a subject, as described herein.

[0216] The terms "inhibition" or "inhibiting" are used herein generally to refer to decreasing, slowing, restricting, retarding, suppressing, blocking, neutralizing, impeding, or preventing a process, such as, for example, reducing or delaying the growth, metastasis, or survival of a TGFβ-related disease or condition, such as, but not limited to, fibrosis, cancer or tumor, or bone marrow failure.

[0217] The term "treat" or "treatment," for the purposes of this disclosure, refers to both therapeutic treatment and prophylactic or preventative measures, where the goal is to ameliorate the targeted disease or condition. Those in need of treatment include those already afflicted with the disorder as well as those susceptible to the disorder or those in whom the disorder is to be prevented. In certain embodiments, "treat" or "treatment" refers to improving at least one physical parameter, such as skin thickening, fibrous scarring, or tumor size, growth, or migration. In some embodiments, "treat" or "treatment" refers to inhibiting or ameliorating a disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In some embodiments, "treat" or "treatment" refers to delaying the onset (or recurrence) of a disease or condition. The term "treat" or "treatment" can refer to any indicator of successful treatment or amelioration of a disease or condition, including any objective or subjective parameter, such as alleviation; mitigation; diminishing symptoms or making a disease or condition more tolerable by the subject; improving the physical or mental well-being of a subject, such as reducing pain or discomfort experienced by a patient; and, in some circumstances, further improving at least one clinical parameter of the disease or condition.

[0218] In certain embodiments of the present disclosure, "treating" refers to neutralizing the biological activity of excess TGFβ, which may be determined by a suitable clinical parameter of improvement; by pathological assessment of, for example, fibrosis and / or effects on immunosuppression or prevention of fibrosis; by direct inhibition of TGFβ signaling; or by another measure appropriate to the disease or condition being treated.

[0219] As used herein, "prevent" or "prevention" is intended to refer to at least a reduction in the likelihood of, or risk of, acquiring a disease or disorder, or a reduction in susceptibility thereto (i.e., the failure to develop at least one clinical symptom of a disease in a patient who may be susceptible to, or predisposed to, the disease, but who has not yet developed or displayed symptoms of the disease). The terms "prevention" or "preventing" are also used to refer to the administration of a compound or composition described herein to a subject who is at risk (or susceptible) for such a disease or condition. Subjects suitable for treatment for the prevention of a disease or condition include individuals who are at risk for the disease or condition but do not display symptoms, as well as patients who currently display symptoms. In certain embodiments, "prevention" or "preventing" is used to refer to the administration of a compound or composition described herein to a subject who has been diagnosed with or treated for a disease or condition and is at risk of a recurrence of the disease or condition.

[0220] In certain embodiments, treatment or prevention is within the context of the present invention if there is a measurable difference between the performance of subjects treated with the TGFβ binding agents, compositions and methods provided herein compared to members of a placebo group, historical controls, or between subsequent tests given to the same subjects.

[0221] The term "subject" includes organisms having, or susceptible to, or at risk of, a TGFβ-related disease or condition. Examples of subjects include mammals, e.g., humans, monkeys, cows, rabbits, sheep, goats, pigs, dogs, cats, rats, mice, and transgenic species thereof. The term "subject" generally includes animals, e.g., mammals, e.g., primates, e.g., humans, that are susceptible to a condition characterized by a TGFβ-related disease or condition, such as fibrosis or cancer. The animal may also be an animal model for the disorder, e.g., a mouse model, a xenograft recipient, etc. In some embodiments, the subject is a human.

[0222] The dosage of each TGFβ-binding agent for use in the compositions provided herein is not particularly limited. Exemplary dosages include milligram or microgram amounts of compound per kilogram of subject or sample weight (e.g., about 50 micrograms / kilogram to about 500 milligrams / kilogram, about 1 milligram / kilogram to about 100 milligrams / kilogram, about 1 milligram / kilogram to about 50 milligrams / kilogram, about 1 milligram / kilogram to about 10 milligrams / kilogram, or about 3 milligrams / kilogram to about 5 milligrams / kilogram). Further exemplary dosages include doses of about 5 to about 500 mg, about 25 to about 300 mg, about 25 to about 200 mg, about 50 to about 150 mg, or about 50, about 100, about 150 mg, about 200 mg, or about 250 mg, and may be administered daily or twice daily, or in lesser or greater amounts.

[0223] In certain embodiments, the dosage range for adults is generally 0.005 mg to 10 g per day. Polypeptides, TGFβ binding agents, and compositions thereof may be provided in unit dosage form, e.g., as a unit or multiples thereof effective in such dosage amounts, e.g., units containing 5 mg to 500 mg, typically about 10 mg to 200 mg. Dosage units may be, for example, 1 to 30 mg, 1 to 40 mg, 1 to 100 mg, 1 to 300 mg, 1 to 500 mg, 2 to 500 mg, 3 to 100 mg, 5 to 20 mg, 5 to 100 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg) of a polypeptide, TGFβ binding agent, or composition described herein.

[0224] It should be understood that the effective amount of a polypeptide or TGFβ binding agent for therapeutic treatment of a disease or condition will vary depending on the method of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. It should be understood that the dosage or amount of a polypeptide or TGFβ binding agent, used alone or in combination with one or more administered active compounds, will depend on the individual case and, as is customary, should be adapted to the individual circumstances to obtain optimal efficacy. Dosage and administration regimens are within the skill of those skilled in the art, and the appropriate dose will depend on several factors within the knowledge of a physician, veterinarian, or researcher of ordinary skill (see, for example, Wells et al. eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000)). For example, the dosage and administration regimen will depend on the nature and severity of the disorder being treated, and further on the sex, age, weight and individual responsiveness of the human or animal being treated, the efficacy and duration of action of the compound used, whether the treatment is acute or chronic or prophylactic, and / or whether other active compounds are administered in addition to the therapeutic molecule.

[0225] The compounds and compositions provided herein can be administered using known procedures at dosages and for periods effective to achieve the desired purpose. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the circumstances of the therapeutic situation. In some embodiments, the compounds or compositions are administered at effective dosages sufficient to prevent or treat fibrosis in a subject.

[0226] The route of administration of each of the TGFβ-binding agents for use in the compositions provided herein is not particularly limited. A polypeptide, TGFβ-binding agent, or composition thereof can be administered using any suitable route or means, for example, but not limited to, oral, parenteral, intravenous, intraperitoneal, intramuscular, subcutaneous, sublingual, topical, or nasal administration, by inhalation, injection, infusion, or other such routes known in the art. In certain embodiments, a polypeptide, TGFβ-binding agent, or composition thereof is administered by injection or infusion, for example, but not limited to, intravenously, intraperitoneally, intramuscularly, or subcutaneously.

[0227] In certain embodiments of the methods of the present disclosure, one or more symptoms of the onset or progression of a TGFβ-related disease or condition are alleviated in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0228] In certain embodiments of the disclosed methods, a fibrotic condition is reduced in a subject. For example, the polypeptide, TGFβ binding agent, or composition may reduce fibrosis, fibrotic scarring, or skin thickening in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0229] In certain embodiments of the methods of the present disclosure, differentiation of fibroblasts into myofibroblasts is inhibited in a subject by, e.g., at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0230] In certain embodiments of the methods of the present disclosure, tumor growth and / or metastasis is inhibited in a subject by, e.g., at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0231] In certain embodiments of the methods of the present disclosure, hematopoietic colony formation and / or hematopoiesis in bone marrow hematopoietic stem or progenitor cells (HSPCs) is increased in the subject's bone marrow, e.g., by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0232] In certain embodiments of the methods of the present disclosure, a positive response in pulmonary fibrosis is manifested as a sustained slowing in the rate of decline in lung function as measured by forced vital capacity.

[0233] In some embodiments of the method of the present disclosure, the favorable response of systemic sclerosis-associated skin fibrosis is determined by the improvement of modified Rodnan skin score (MRSS).For example, the MRSS of the subject can be improved by at least 5%, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0234] In certain embodiments, in bone marrow failure diseases, including myelofibrosis, a favorable response is manifested by improvement in anemia (e.g., a transfusion-independent patient exhibiting an increase in hemoglobin levels, a transfusion-dependent patient becoming transfusion-independent).

[0235] In certain embodiments, the polypeptide or TGFβ binding agent may be conjugated to a therapeutic moiety, as described herein. A desirable therapeutic moiety may be selected for its ability to prevent or treat the same disease or condition targeted by the polypeptide or TGFβ binding agent.

[0236] In certain embodiments, methods are provided for preventing or treating a TGFβ-related disease or condition in a subject by administering an effective amount of a polypeptide or TGFβ-binding agent described herein, such that the TGFβ-related disease or condition is prevented or treated in the subject.

[0237] In certain embodiments, there are provided methods of inhibiting TGFβ in a subject by administering an effective amount of a polypeptide or TGFβ-binding agent described herein such that TGFβ is inhibited in the subject. In certain such embodiments, there are provided methods of inhibiting TGFβ3 in a subject by administering an effective amount of a polypeptide or TGFβ-binding agent described herein such that TGFβ3 is inhibited in the subject. In certain such embodiments, there are provided methods of inhibiting TGFβ3 and TGFβ1 in a subject by administering an effective amount of a polypeptide or TGFβ-binding agent described herein such that TGFβ3 and TGFβ3 are inhibited in the subject.

[0238] In certain embodiments, methods are provided for inhibiting the differentiation of fibroblasts into myofibroblasts, either in vitro, ex vivo, or in vivo.

[0239] In some embodiments of the therapeutic and prophylactic treatments provided herein, the polypeptide or TGFβ-binding agent is administered in combination with one or more additional therapies or therapeutic agents. The additional therapies or therapeutic agents may be administered before, after, or simultaneously with the administration of the polypeptide, TGFβ-binding agent, or composition described herein. In some embodiments, the additional therapies or therapeutic agents are formulated together with the polypeptide or TGFβ-binding agent in the same composition. In other embodiments, the additional therapies or therapeutic agents are administered separately. Examples of additional therapies and therapeutic agents include, but are not limited to, anti-fibrotic agents; anti-cancer agents; and other TGFβ-binding agents or inhibitors, such as antibodies, antibody fragments, antigen-binding fragments, soluble TGFβ-ligand traps, etc. In one embodiment, the additional therapeutic agent is nintedanib (sold under the trade names Ofev® and Vargatef®). In one embodiment, the additional therapeutic agent is pirfenidone. In one embodiment, the additional therapeutic agent is an immune checkpoint inhibitor.

[0240] Alternatively, in certain embodiments, the polypeptide or TGFβ-binding agent may be conjugated to a detectable or diagnostic moiety that is useful for tracking the polypeptide or TGFβ-binding agent, or cells or tissues that express TGFβ. In certain such embodiments, methods are provided for diagnosing a TGFβ-associated disease or condition comprising administering to a subject a polypeptide or TGFβ-binding agent of the disclosure conjugated to a detectable or diagnostic moiety, and detecting the polypeptide or TGFβ-binding agent such that a disease or condition associated with TGFβ (e.g., overexpression of TGFβ1 and / or TGFβ3) is diagnosed.

[0241] kit According to the present disclosure, the polypeptides, TGFβ-binding agents, and pharmaceutical compositions described herein can be assembled into kits or pharmaceutical systems for use in treating or preventing TGFβ-related diseases or conditions. The kits or pharmaceutical systems can include a container (e.g., a package, box, cardboard, vial, etc.) having one or more containers, such as vials, tubes, ampoules, or bottles, containing the polypeptide, TGFβ-binding agent, or pharmaceutical composition in its closed containment. Additional kit components can include acids, bases, buffering agents, inorganic salts, solvents, antioxidants, preservatives, or metal chelators. The additional kit components can be present as pure compositions or as aqueous or organic solutions incorporating one or more additional kit components. Any or all of the kit components can optionally further comprise a buffer. The kits can also include tools for administration, such as needles and syringes. The kits can be used according to the methods described herein and can include instructions for use in such methods. The kits can also include instructions for administering and using the polypeptide, TGFβ-binding agent, or pharmaceutical composition.

[0242] The sequences are shown in the sequence table in Table 2. In Table 2, the N-terminal IDRs in the human TGFβRII extracellular domain and sequences derived therefrom are underlined; the C-terminal IDRs in the human TGFβRII extracellular domain and sequences derived therefrom are double underlined; the Gly-Ser linker region is italicized and underlined; and the multimerization domain is italicized and bolded. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18] [Table 2-19] [Table 2-20] [Table 2-21] [Table 2-22] [Example]

[0243] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and should not be construed as limiting its scope in any way.

[0244] Unless otherwise defined or indicated otherwise by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0245] Example 1. Characterization and structural analysis of known TGFβ binding agents. The TGFβRII extracellular domain (SEQ ID NO: 1) comprises a structured portion, which represents a ligand-binding domain (SEQ ID NO: 2) flanked at both the N- and C-termini by intrinsically disordered regions (IDRs; SEQ ID NOs: 3 and 4, respectively).

[0246] Previously, TGFβRII extracellular domain fusion molecules have been described (WO 2017 / 037634, WO 2018 / 158727). Such fusion molecules comprise two structured ligand-binding domains (SEQ ID NO: 2) linked together side-by-side (head-to-tail) by a linker derived from an IDR and its variants. As reported in WO 2018 / 158727, such polypeptide constructs demonstrated inhibitory potency at least 600-fold greater than similar constructs having only a single ligand-binding domain (see WO 2018 / 158727, pp. 27-31, which states that "constructs in which the first region comprises a (TβRII-ECD)-(TβRIIECD) doublet linked at its C-terminus by an antibody constant domain inhibit TGFβ activity with at least 600-fold greater potency than equivalent constructs having a single TβRII-ECD linked at its C-terminus by an antibody constant domain (i.e., in the absence of the second ECD, also referred to herein as singlets)"). Furthermore, such a "doublet" polypeptide construct (termed "T22d35-Fc") could bind to and neutralize all three isoforms of TGFβ (i.e., TGF-β1, β2, and β3) to varying degrees, although TGFβ2 was generally neutralized to a much lesser extent than TGFβ1 and TGFβ3. For example, the T22d35-Fc-IgG1-v1(CC) mutant was described to have very similar neutralizing potencies against TGFβ1 and TGFβ3, but this potency was much lower against TGFβ2 (IC50 = 17.33 nM for TGFβ2 compared to 0.003327 nM and 0.003251 nM for TGFβ1 and TGFβ3, respectively; see WO 2018 / 158727, p. 26, lines 24-27). Similar results have been reported for other mutants.

[0247] To further investigate the binding and neutralization properties of such TGFβ-binding agents, we selected an exemplary fusion molecule, "T22d35-Fc-IgG1-v1(CC)" for further investigation. In this fusion molecule (SEQ ID NO: 6 herein; SEQ ID NO: 14 in WO 2018 / 158727), the linker between the two structured ligand-binding domains is a fusion of the C- and N-terminal IDRs, in that order (the entire linker sequence is shown in SEQ ID NO: 7), and the linker between the second ligand-binding domain and the multimerization domain is the C-terminal IDR of TβRII-ECD (SEQ ID NO: 4). In this fusion molecule, the multimerization domain is the hIgG1Fc(CC) region (SEQ ID NO: 49).

[0248] First, we used the A549 IL-11 release assay to measure the inhibitory potency (IC) of T22d35-Fc-IgG1-v1(CC) fusion against various isoforms of TGFβ, particularly TGFβ1 and TGFβ3. 50 ) was subjected to more detailed characterization. The A549 IL-11 release assay was performed essentially as described (WO 2018 / 158727) and is described in further detail in Example 2 below.

[0249] Analyze inhibitory potency in more detail and obtain IC with higher accuracy 50 To obtain this value, we performed an IC50 assay in the A549 IL-11 release assay. 50 Higher concentrations of TGFβ (more points on the curve) closer to the IC50 were tested. Representative results are shown in Figures 4A and 4B, and results from an average of six experiments are shown in Table 3. We found an average IC50 of 2.89 pM ± 0.16 for TGFβ1. 50 The mean IC values ​​were determined, which are consistent with previous reports (WO 2018 / 158727). However, for TGFβ3, the mean IC 50The IgG1-v1(CC) fusion antibody (T22d35-Fc-IgG1-v1) showed a potency of approximately 3-3.5 times higher than expected (i.e., lower inhibitory potency than expected). The results showed approximately 3-3.5 times higher inhibitory potency against TGFβ1 compared to TGFβ3, indicating preferential inhibition or neutralization of the TGFβ1 ligand by the T22d35-Fc-IgG1-v1(CC) fusion. It is noted that the inhibitory potency against TGFβ2 was significantly lower than that against TGFβ1 and TGFβ3, as previously reported (WO 2018 / 158727; Figure 11). [Table 3]

[0250] These results are consistent with the statements in WO 2018 / 158727, which suggest greater inhibition or neutralization of TGFβ1 compared to TGFβ3 (see, e.g., WO 2018 / 158727, page 3, lines 26-28, which states that "the Fc-doublet (T22d35-Fc) exhibits at least 970-fold increased potency against TGFβ1 and at least 240-fold increased potency against TGFβ3 compared to the non-Fc-fused ECD doublet").

[0251] To better understand the potential mechanism underlying the preferential inhibition of TGFβ1 ligand over TGFβ3 ligand by the previous fusion construct, we next performed a structural comparison of the two ligand isoforms. It should be noted that the ligand-binding domains of the previous fusion construct interact with virtually identical epitopes in the TGFβ1 and TGFβ3 ligands (Baardsnes, J. et al., 2009). Therefore, differential affinity for monomers within the TGFβ dimer does not readily explain the preferential inhibition of the TGFβ1 ligand. An overlay of the monomer structures of TGFβ1 (blue) and TGFβ3 (green) is shown in Figure 2A. An overlay of the TGFβ1 and TGFβ3 dimers, as observed in the Protein Data Bank (PDB) IDs 3KFD and 1KTZ, respectively, is shown in Figure 2B (the Protein Data Bank (PDB) IDs for the structures are 3KFD and 1KTZ, respectively). Although TGFβ1 and TGFβ3 are very similar at the amino acid sequence level, and both monomers adopt a similar extended cysteine ​​knot fold (Figure 2A), the arrangement of the two monomers in the biologically active dimer is found to be quite different when comparing the structures of the two isoforms. It is clear that each ligand isoform has a specific range of dimerization angles. The range of dimerization angles affects the overall shape, spatial extent, and compactness of the dimeric molecule. This difference in the shape of TGFβ1 and TGFβ3 dimers may result in preferential neutralization of TGFβ1 over TGFβ3 by previous fusion constructs; i.e., the specific spacing of the ligand-binding domains in the fusion constructs may have resulted in preferential interaction (preferential avidity) with the TGFβ1 ligand by its isoform dimer with a different shape.

[0252] Figure 2C shows a representative model of the fusion construct (T22d35-Fc-IgG1-v1(CC), SEQ ID NO: 6) bound to a TGFβ ligand, showing the second ligand-binding domain, second linker, and Fc region. This model is shown herein to demonstrate the impact of the short (10 amino acid) second linker. The green line indicates that the linker / spacer length is at least 25 Å short to allow ligand binding between the linked binding domains. Specifically, the length of the 10 amino acid linker in T22d35-Fc-IgG1-v1(CC) is approximately 35 Å even in the extended conformation, which is approximately 20 Å shorter than the optimal linker length calculated using molecular modeling. Thus, in this fusion construct, the second ligand-binding domain is sterically restricted from accommodating the TGFβ dimer.

[0253] It should be noted that the structured ligand-binding domain is the portion of the fusion construct that contributes to the interaction interface with TGFβ ligands, e.g., TGFβ1 and TGFβ3, and the linker region does not directly interact with the bound ligand. However, given the differences in dimeric structure for TGFβ1 and TGFβ3 and the structural constraints imposed by the linker region, our analysis suggested that modifying the linker region could affect the binding properties of a TGFβ binder to alter its ligand-binding specificity. In particular, shortening the first linker region between the ligand-binding domains and lengthening the second linker region between the second ligand-binding domain and the multimerization domain would relax steric and structural constraints to alter the relative inhibitory potency for TGFβ1 and TGFβ3 ligands.

[0254] Example 2. Design and characterization of TGFβ binding agents with altered isoform specificity. The present inventors generated molecules with various linker sequences and lengths to investigate whether modifying the linker could specifically affect isoform specificity and inhibitory potency for TGFβ ligands, particularly TGFβ1 and TGFβ3. Based on structural analysis, the present inventors focused on molecules with a shortened linker portion between the two ligand-binding domains (first linker portion) and an extended linker portion between the second ligand-binding domain and the multimerization domain (second linker portion). Our goal was to develop binders with a less preferential inhibition of TGFβ1 over TGFβ3 (i.e., a lower TGFβ3:TGFβ1 IC ) while maintaining good overall inhibitory potency, in order to endow the binder with beneficial therapeutic properties for specific disease indications. 50 The objective of this study was to design a TGFβ binding agent with a high binding ratio.

[0255] A series of TGFβ-binding agents with linkers of various lengths and sequences were designed. The structures of representative fusion proteins are summarized in Table 4. The sequences are shown in Table 2.

[0256] The test binding agent was a homodimer, with each polypeptide in the homodimer containing an N-terminal region containing the N-terminal IDR of the TGFβRII extracellular domain (SEQ ID NO: 3); two TGFβ receptor type II (TGFβRII) ligand-binding domains (SEQ ID NO: 2); an 18-amino acid first linker portion between the two ligand-binding domains (SEQ ID NO: 8 or 12); a second ligand-binding domain and a multimerization domain (SEQ ID NO: 4, 9, 11, or 15); and a 10-, 16-, or 30-amino acid second linker portion between the hIgG1Fc(CC) multimerization domain (SEQ ID NO: 49). T22d35-Fc-IgG1-v1(CC) fusion (SEQ ID NO: 6; WO 2018 / 158727) was used as a positive control (CTL). The complete sequences of protein 61 (p61), protein 96 (p96), protein 101 (p101), protein 107 (p107), and protein 112 (p112) are shown in SEQ ID NOs: 81, 84, 87, 89, and 92, respectively (Table 2). [Table 4]

[0257] Generation and purification of recombinant fusion molecules. All constructs contained the secretory signal sequence MDWTWRILFLVAAATGTHA (SEQ ID NO: 104) at the N-terminus when expressed. Complementary (c)DNA encoding the constructs was prepared synthetically (GeneArt, ThermoFisher Scientific). cDNA was cloned into the EcoR1 (5' end) and BamH1 (3' end) sites of the pTT5 mammalian expression plasmid vector (Durocher et al., 2002). Representative cDNA sequences used for expression of fusion proteins are shown in Table 2 (SEQ ID NOs: 106-109, used for expression of p61, p96, p101, and p128, respectively). The signal peptide is cleaved intracellularly during expression and is not included in the purified fusion proteins.

[0258] Fusion proteins were expressed by transient transfection of Chinese hamster ovary (CHO) cells. Briefly, expression plasmids encoding the fusion proteins were transfected into 100 mL cultures of CHO-3E7 cells in Freestyle F17 medium (Invitrogen) containing 4 mM glutamine and 0.1% Kolliphor p-1 88 (Sigma).

[0259] All cell cultures were performed at 37°C and 5% CO2. The transfection conditions were as follows: the transfected DNA consisted of plasmid DNA for expressing the fusion protein and 30% salmon sperm DNA, mixed with polyethyleneimine-pro (Polyplus) at a ratio of 1:4. 24 hours after transfection, 1% Tryptone NI feed (TekniScience Inc.) was added. 0.5 mM VPA (Sigma) was added, and the incubator temperature was lowered to 32°C and 5% CO2. This was done to promote the production and secretion of the fusion protein. This was maintained for 4 days post-transfection (dpt), after which the culture was harvested. On day 4, the harvested supernatant was filtered (0.2 μm) and purified using an AKTA pure 25L (GE). The supernatant was loaded onto a MabSelect PrismA Protein A column and purified by affinity chromatography. The column was then washed with 8 column volumes of PBS and the protein was eluted with 5 column volumes of 0.1 M sodium citrate, pH 3.2. Fractions were then buffer exchanged into formulation buffer (20 mM L-histidine, 100 mM NaCl, pH 7) using a HiPrep 26 / 10 desalting column (GE).

[0260] Figures 3A and 3B show polyacrylamide gel electrophoresis analysis of purified proteins 61, 96, 101, 107, and 112 (see lanes designated as p61, p96, p101, p107, and p112, respectively) and samples from T22d35-Fc-IgG1-v1(CC)(Ctl) under both non-reducing (Figure 3A) and reducing (Figure 3B) conditions. Protein (P) was electrophoresed on a 12% Bis-Tris acrylamide gel (NuPAGE™ 12% Bis-Tris Protein Gel, Cat# NP0341BOX, Life Technologies) under both non-reducing and reducing conditions. These fusion proteins are tetravalent, homodimeric TGFβ-binding agents, each comprising two polypeptide chains (i.e., they are homodimers of two polypeptide chains, the first and second polypeptides being identical, and each polypeptide comprising two ligand-binding domains). The two polypeptide chains are dimerized via disulfide bridges involving one or more cysteine ​​residues in their multimerization domains, as determined by the difference in size under reducing versus non-reducing conditions.

[0261] Inhibition of TGFβ1 and TGFβ3 activity by fusion proteins. To determine the inhibitory potency of proteins 61, 96, 101, 107, and 112, TGFβ neutralization was assessed and the inhibitory potency was compared to that of a positive control (T22d35-Fc-IgG1-v1 (CC), two TGFβRII-ECD doublets linked via the Fc portion (SEQ ID NO: 6)). It should be noted that a single non-FC-fused TGFβRII extracellular domain (SEQ ID NO: 1) does not neutralize either TGFβ1, β2, or β3 (De Crescenzo et al., 2004). The terms "inhibitory potency" and "neutralization potency" are used interchangeably herein.

[0262] The TGFβ neutralization potency of the purified fusion proteins was determined using a cell-based signaling assay, specifically, an A549 cell / IL-11 release assay using a colorimetric ELISA. Briefly, human A549 lung cancer cells (ATCC-CCL-185, Cedarlane, Burlington, ON) were plated in 96-well plates (5 × 10 3 Cells were seeded at 1000 x 1000 cells / well and incubated at 37°C and 5% CO2 in a humidified atmosphere. The following day, 10 pM TGFβ in complete medium in the absence or presence of increasing concentrations of fusion protein was incubated for 30 minutes at room temperature (RT) before being added to the cells. After 24 hours (h) of incubation, conditioned medium was collected and stored at 4°C. The following day, IL-11 ELISA was performed according to the manufacturer's instructions (Human IL-11 Duoset ELISA Kit, Cat# DY218, R&D Systems, Inc.). This IL-11 release assay serves as a model of TGFβ-mediated signaling: relative IL-11 release after TGFβ treatment is a measure of TGFβ activity. A decrease in IL-11 release after addition of the test fusion protein indicates TGFβ activity. Data were plotted and analyzed using Prism8 (GraphPad, San Diego) to generate dose-response curves from absorbance values ​​using a four-parameter fit logistic model (absorbance vs. concentration). Values ​​were then normalized to the positive control (TGFβ treatment in the absence of any inhibitor).

[0263] Results from a representative set of experiments are shown in Figures 4A and 4B, comparing the inhibitory potency of proteins 61, 96, 101, 107, and 112 against TGFβ1 and TGFβ3 with a positive control (SEQ ID NO: 6). The highest potency was seen with the positive control. However, the positive control also had the highest IC for TGFβ3:TGFβ1. 50 In contrast, proteins 61, 96, 101, 107, and 112 had a TGFβ3:TGFβ1 IC ratio (3.41 in this experiment). 50 The ratios were 1.66, 1.72, 1.51, 1.24, and 1.95, respectively, in this experiment (Figures 4A-4B). [Table 5]

[0264] The results show that modifications of the linker regions, particularly shortening the first linker region and lengthening the second linker region, significantly improve the TGFβ3:TGFβ1 IC 50 The ratio was significantly reduced, indicating a reduction in preferential inhibition of TGFβ1 while still maintaining inhibitory potency in the picomolar range.

[0265] Another set of representative binders is shown in Figures 5A-5B, which show polyacrylamide gel electrophoresis analysis of samples from purified proteins 112, 111, 106, 105, 104, 101, 99, and 71, respectively, under non-reducing (Figure 5A) and reducing (Figure 5B) conditions.

[0266] In a representative set of experiments shown in Figures 6A-6B, the neutralization potencies of proteins 113, 115, and 116 compared to a positive control (SEQ ID NO: 6) are shown. The results are also shown in Table 5. The results show that the inhibitory potencies of these proteins were comparable to the control against TGFβ1, but significantly higher against TGFβ3, with significantly lower TGFβ3:TGFβ1 IC 50 This shows that the ratio

[0267] In a representative set of experiments shown in Figures 7A-7B, the neutralization potencies of proteins 101, 129, and 130 compared to a positive control (SEQ ID NO: 6) are shown. The results are also shown in Table 5. The results indicate that the inhibitory potencies of these proteins were lower against TGFβ1, similar (P101, P130) or lower (P129) against TGFβ3, and significantly lower TGFβ3:TGFβ1 IC 50 This shows that the ratio

[0268] In a representative set of experiments shown in Figures 8A-8B, the neutralization potencies of proteins 101, 131, 132, and 133 compared to a positive control (SEQ ID NO: 6) are shown. The results are also shown in Table 5. The results show that the inhibitory potencies of these proteins were lower against TGFβ1 and similar or higher against TGFβ3 compared to the control, with significantly lower TGFβ3:TGFβ1 IC 50 This shows that the ratio

[0269] In a representative set of experiments shown in Figures 9A-9B, the neutralization potencies of proteins 96, 134, and 135 compared to a positive control (SEQ ID NO: 6) are shown. The results are also shown in Table 5. The results indicate that the inhibitory potencies of these proteins were reduced more against TGFβ1 than against TGFβ3 compared to the control, thereby reducing the TGFβ3:TGFβ1 IC 50 Comparing proteins 134 and 135 with protein 96, the results show that replacing either the first or second linker with a Gly-Ser linker resulted in a lower TGFβ3:TGFβ1 IC 50 The results show that the inhibitory potency against both TGFβ1 and TGFβ3 was significantly reduced while maintaining the ratio.

[0270] The multimerization domain does not affect TGFβ isoform specificity. TGFβ binders with the same TGFβ binding region but differing only in the multimerization domain were tested to determine what effect, if any, the multimerization domain has on the relative inhibitory potency against TGFβ1 and TGFβ3 isoforms. The results are shown in Figures 10A-10B (which show the neutralization potency of proteins 101 and 128 compared to the positive control (SEQ ID NO: 6)) and Table 5. Figures 10A-10B show one representative assay; results averaged from several assays are shown in Table 5. The results show that changing the multimerization domain from IgG1 (protein 101) to IgG4 (protein 128) did not have a significant effect on inhibitory potency against TGFβ1 and TGFβ3, as expected. The same results were obtained for proteins 61 and 96, as well as proteins 113 and 115 (Table 5).

[0271] Neutralization of TGFβ2 isoforms. We also tested whether the relative inhibition of TGFβ2 was affected by the TGFβ binding agents provided herein compared to a control. A representative set of experiments is shown in FIG. 11. As shown in FIG. 11 for proteins 61, 96, and 101, the neutralization potency against the TGFβ2 isoform was over 1000-fold lower (in other words, IC ) than against the TGFβ1 and TGFβ3 isoforms. 50 (>1000-fold higher), which is the same as the control. The results demonstrate that leveling or changing the relative inhibitory potencies for TGFβ1 and TGFβ3 isoforms had no significant effect on the very low inhibition of the TGFβ2 isoform.

[0272] Taken together, the results reported herein demonstrate that shortening the first linker region to fewer than 34 amino acids and / or extending the second linker region to more than 10 amino acids significantly improves the TGFβ3:TGFβ1 IC for these binders. 50These results demonstrate that TGFβ3:TGFβ1 IC agonists have been effective in lowering the ratio, in some cases nearly equalizing the inhibitory potency against the two isoforms. 50 It should be noted that the ratio was decreased by increasing inhibitory potency against TGFβ3 without adversely affecting potency against TGFβ1 (e.g., proteins 113, 115, 116). In other cases, the ratio was decreased primarily by decreasing inhibitory potency against TGFβ1 without adversely affecting potency against TGFβ3 (e.g., proteins 61, 96, 101, 107, 128), although in some cases a slight decrease in TGFβ3 potency was also observed. Nevertheless, all binders maintained significantly higher inhibitory potency against both TGFβ1 and TGFβ3 than against TGFβ2, consistent with their potential use as therapeutic agents for the treatment of TGFβ-related disorders, particularly those mediated by TGFβ3.

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[0274] All publications and references cited herein are hereby incorporated by reference in their entirety.

[0275] While the present invention will be described in detail with reference to embodiments thereof, these embodiments are provided to illustrate the invention and not to limit it, other embodiments may be made which utilize the principles of the invention and are within its spirit and scope as defined by the appended claims. The present disclosure provides, for example: [Section 1] 1. A polypeptide construct useful for inhibiting the effects of transforming growth factor β (TGFβ) isoforms, said construct comprising: TGFβ binding region, and Multimerization domain Includes; wherein the N-terminus of the multimerization domain is linked to the C-terminus of the TGFβ binding region; the TGFβ binding region comprises, in N-terminal to C-terminal direction, an N-terminal region, a first TGFβ receptor ligand binding domain (TGFβR-LBD), a first linker, a second TGFβR ligand binding domain, and a second linker; wherein the inhibitory potency of said polypeptide construct against both TGFβ1 isoform activity and TGFβ3 isoform activity is higher than that against TGFβ2 isoform activity; wherein the first linker and the second linker are linked together to determine the relative inhibitory potency of the polypeptide construct against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1). 50 The polypeptide construct, wherein the ratio of the polypeptide to the polypeptide chain is selected to be about 2.5:1 or less. [Section 2] The relative inhibitory potency of the polypeptide constructs against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 Item 2. The polypeptide construct of Item 1, wherein the ratio (ratio) of the β-amino acid to the β-amino acid is less than about 2.5:1, about 2.3:1 or less, about 2:1 or less, about 1.8:1 or less, about 1.5:1 or less, about 1.3:1 or less, about 1:1 or less, about 1:1 or less, about 0.8:1 or less, or about 0.5:1 or less. [Section 3] The relative inhibitory potency of the polypeptide constructs against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 Item 3. The polypeptide construct according to Item 1 or 2, wherein the ratio of the total number of amino acids to the total number of amino acids is about 1:1 to about 2:1. [Section 4] The relative inhibitory potency of the polypeptide constructs against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 Item 4. The polypeptide construct according to Item 3, wherein the ratio of the total amino acid sequence to the total amino acid sequence is about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, or 1.9:1. [Section 5] The relative inhibitory potency of the polypeptide constructs against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 Item 5. The polypeptide construct according to Item 4, wherein the ratio of the total amino acid sequence to the total amino acid sequence is about 1.4:1 to about 1.6:1. [Section 6] The relative inhibitory potency of the polypeptide constructs against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 Item 6. The polypeptide construct according to Item 5, wherein the ratio of the total amino acid sequence to the total amino acid sequence is about 1.4:1, about 1.5:1, or about 1.6:1. [Section 7] Item 7. The polypeptide construct according to any one of Items 1 to 6, wherein the polypeptide construct inhibits both TGFβ1 isoform activity and TGFβ3 isoform activity with at least 20-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold greater potency than TGFβ2 isoform activity. [Section 8] Item 8. The polypeptide construct according to any one of Items 1 to 7, wherein the first linker is 33 amino acids or less in length. [Section 9] Item 9. The polypeptide construct according to any one of Items 1 to 8, wherein the second linker is 10 amino acids or more in length. [Section 10] Item 10. The polypeptide construct of any one of items 1 to 9, wherein one or more of the first linker and the second linker comprises or consists of an IDR linker, an IDR linker mutant, a hybrid linker, a hybrid linker mutant, a truncated linker, a truncated linker mutant, or an extended linker. [Section 11] Item 11. The polypeptide construct of item 10, wherein one of the first linker and the second linker comprises or consists of a non-IDR linker. [Section 12] Item 11. The polypeptide construct of any one of items 1 to 10, wherein both the first linker moiety and the second linker moiety comprise or consist of an IDR linker, an IDR linker mutant, a hybrid linker, a hybrid linker mutant, a truncated linker, a truncated linker mutant, or an extended linker. [Section 13] Item 13. The polypeptide construct according to any one of Items 1 to 12, wherein the first linker is 10 amino acids or more, 15 amino acids or more, or 18 amino acids or more in length. [Section 14] Item 14. The polypeptide construct according to any one of Items 1 to 13, wherein the first linker is about 15 to 33 amino acids in length, or about 18 to 30 amino acids in length. [Section 15] Item 15. The polypeptide construct according to any one of Items 1 to 14, wherein the first linker is about 16, about 18, about 30, or about 32 amino acids in length. [Section 16] 16. The polypeptide construct of paragraph 15, wherein the first linker is 18 amino acids in length. [Section 17] Item 16. The polypeptide construct of Item 15, wherein the first linker is 16 amino acids in length. [Section 18] 16. The polypeptide construct of paragraph 15, wherein the first linker is 30 amino acids in length. [Section 19] 16. The polypeptide construct of paragraph 15, wherein the first linker is 32 amino acids in length. [Section 20] Item 20. The polypeptide construct according to any one of Items 1 to 19, wherein the second linker is 35 amino acids or less in length, or 10 to 34 amino acids in length. [Section 21] 21. The polypeptide construct according to any one of Items 1 to 20, wherein the second linker is about 15 to about 35 amino acids in length. [Section 22] Item 22. The polypeptide construct of any one of Items 1 to 21, wherein the second linker is about 16, about 30, about 32, or about 34 amino acids in length. [Section 23] 23. The polypeptide construct of claim 22, wherein the second linker is 30 amino acids in length. [Section 24] 23. The polypeptide construct of paragraph 22, wherein the second linker is 16 amino acids in length. [Section 25] 23. The polypeptide construct of claim 22, wherein the second linker is 32 amino acids in length. [Section 26] 23. The polypeptide construct of claim 22, wherein the second linker is 34 amino acids in length. [Section 27] 27. The polypeptide construct according to any one of items 1 to 26, wherein one or more of the first linker and the second linker comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 4 and 8 to 26, or a 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 thereto. [Section 28] 28. The polypeptide construct of claim 27, wherein the first linker comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 16, 21, 22, 23, and 26, or a 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 thereto. [Section 29] 29. The polypeptide construct of paragraph 27 or 28, wherein the second linker comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 4, 9, 11, 15, 17, 18, 19, 20, 22, 23, 24, 25, and 26, or a 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 thereto. [Section 30] Item 30. The polypeptide construct according to any one of Items 1 to 29, wherein the first linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:12. [Section 31] Item 30. The polypeptide construct according to any one of Items 1 to 29, wherein the first linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:8. [Section 32] Item 32. The polypeptide construct according to any one of Items 1 to 31, wherein the second linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:11. [Section 33] Item 32. The polypeptide construct according to any one of Items 1 to 31, wherein the second linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:9. [Section 34] The first linker is (a) a deletion of at least one N-terminal amino acid residue compared to SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:8; (b) a deletion of at least one C-terminal amino acid residue compared to SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:8; (c) a deletion of at least one internal amino acid residue compared to SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:8; or (d) The polypeptide construct according to any one of items 1 to 29, which has one or more substitutions in the amino acid sequence compared to SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 8, or which comprises or consists of any one of the amino acid sequences of (a) to (c). [Section 35] 35. The polypeptide construct of claim 34, wherein the amino acid deletion is a deletion of 16 amino acids of SEQ ID NO:3. [Section 36] The second linker is (a) a deletion of at least one N-terminal amino acid residue compared to SEQ ID NO:9 or SEQ ID NO:11; (b) a deletion of at least one C-terminal amino acid residue compared to SEQ ID NO:9 or SEQ ID NO:11; (c) a deletion of at least one internal amino acid residue compared to SEQ ID NO:9 or SEQ ID NO:11; or (d) The polypeptide construct according to any one of items 1 to 35, having one or more substitutions in the amino acid sequence compared to SEQ ID NO: 4, 9 or 11, or comprising or consisting of the amino acid sequence of any one of (a) to (c). [Section 37] Item 37. The polypeptide construct of any one of Items 1 to 36, wherein the N-terminal region comprises or consists of an IDR linker, an IDR linker mutant, a hybrid linker, a hybrid linker mutant, a truncated linker, a truncated linker mutant, or an extended linker. [Section 38] Item 38. The polypeptide construct of any one of items 1 to 37, wherein the N-terminal region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, or a 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 thereto. [Section 39] 39. The polypeptide construct of any one of paragraphs 1 to 38, wherein one or more of the first TGFβR-LBD and the second TGFβR-LBD comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, or a sequence which 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 thereto. [Section 40] Item 40. The polypeptide construct of any one of Items 1 to 39, wherein the first TGFβR-LBD and the second TGFβR-LBD are the same or substantially the same. [Section 41] 41. The polypeptide construct of paragraph 40, wherein both the first TGFβR-LBD and the second TGFβR-LBD comprise or consist of the amino acid sequence set forth in SEQ ID NO: 2, or a sequence which 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 thereto. [Section 42] 42. The polypeptide construct of any one of paragraphs 1 to 41, wherein the multimerization domain allows dimerization of the polypeptide construct with a second polypeptide construct of any one of paragraphs 1 to 41 in a non-covalent manner. [Section 43] 42. The polypeptide construct of any one of claims 1 to 41, wherein the multimerization domain allows for dimerization of the polypeptide construct with a second polypeptide construct of any one of claims 1 to 41 in a covalent manner. [Section 44] Item 44. The polypeptide construct of any one of items 1 to 43, wherein the multimerization domain comprises one or more constant regions of an antibody. [Section 45] The multimerization domain is a second constant domain of an antibody heavy chain (C H 2) and / or a third constant domain (C H 45. The polypeptide construct of claim 44, comprising: [Section 46] Item 46. The polypeptide construct according to any one of Items 1 to 45, wherein the multimerization domain comprises an Fc region of an antibody heavy chain. [Section 47] 47. The polypeptide construct according to any one of Items 44 to 46, wherein the antibody is an IgG antibody. [Section 48] 48. The polypeptide construct of clause 47, wherein the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody, optionally a human antibody. [Section 49] 49. The polypeptide construct of clause 48, wherein the multimerization domain has 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% sequence identity to a human IgG1, IgG2, IgG3, or IgG4 constant region. [Section 50] 50. The polypeptide construct of any one of paragraphs 1 to 49, wherein the multimerization domain comprises a cysteine ​​residue for crosslinking the polypeptide construct with a second polypeptide construct of any one of paragraphs 1 to 49. [Section 51] 51. The polypeptide construct of paragraph 50, wherein the multimerization domain comprises at least two cysteine ​​residues for forming disulfide bridges with the second polypeptide construct. [Section 52] Item 52. The polypeptide construct of any one of items 1 to 51, wherein the multimerization domain is engineered to reduce aggregation or modulate the stability of dimers or multimers of the polypeptide construct. [Section 53] Item 53. The polypeptide construct according to any one of Items 1 to 52, wherein the multimerization domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:49. [Section 54] Item 53. The polypeptide construct according to any one of Items 1 to 52, wherein the multimerization domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:50. [Section 55] 53. The polypeptide construct of any one of items 1 to 52, wherein the multimerization domain comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 49 to 80, or a 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 thereto. [Section 56] 56. The polypeptide construct of any one of items 1 to 55, wherein the TGFβ binding region comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 27 to 48, or a 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 thereto. [Section 57] Item 57. The polypeptide construct according to any one of Items 1 to 56, wherein the TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO:27. [Section 58] Item 57. The polypeptide construct according to any one of Items 1 to 56, wherein the TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO:29. [Section 59] Item 57. The polypeptide construct according to any one of Items 1 to 56, wherein the TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO:32. [Section 60] Item 57. The polypeptide construct according to any one of Items 1 to 56, wherein the TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO:40. [Section 61] Item 57. The polypeptide construct according to any one of Items 1 to 56, wherein the TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO:41. [Section 62] 62. The polypeptide construct of any one of paragraphs 1 to 61, wherein the polypeptide construct comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 103 and 105, or a 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 thereto. [Section 63] 62. The polypeptide construct of any one of paragraphs 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 81, or a 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 thereto. [Section 64] 62. The polypeptide construct of any one of paragraphs 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 84, or a 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 thereto. [Section 65] 62. The polypeptide construct of any one of paragraphs 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87, or a 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 thereto. [Section 66] 62. The polypeptide construct of any one of paragraphs 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95, or a 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 thereto. [Section 67] 62. The polypeptide construct of any one of paragraphs 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96, or a 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 thereto. [Section 68] Item 68. The polypeptide construct of any one of items 1 to 67, wherein the polypeptide construct further comprises an amino acid sequence suitable for expression, detection and / or purification of the TGFβ binding agent. [Section 69] 69. The polypeptide construct of Paragraph 68, wherein the polypeptide construct further comprises a signal peptide having the sequence set forth in SEQ ID NO: 104, or a sequence substantially identical thereto. [Section 70] 70. The polypeptide construct of any one of paragraphs 1 to 69, wherein the polypeptide construct is a dimeric polypeptide comprising a first and a second polypeptide construct of any one of paragraphs 1 to 69 linked between their respective multimerization domains by at least one disulfide bridge. [Section 71] 71. The polypeptide construct of Paragraph 70, wherein the first and second polypeptide constructs comprise the same or substantially the same amino acid sequence. [Section 72] 71. The polypeptide construct of Paragraph 70, wherein the first and second polypeptide constructs comprise different amino acid sequences. [Section 73] 73. The polypeptide construct of claim 72, wherein the first and second polypeptide constructs comprise the same or substantially the same multimerization domain and different TGFβ binding regions. [Section 74] 74. The polypeptide construct of any one of items 70 to 73, wherein the first polypeptide and / or the second polypeptide construct further comprises a site for conjugation. [Section 75] 75. The polypeptide construct of paragraph 74, wherein the first polypeptide and / or the second polypeptide construct is conjugated to a targeting agent, therapeutic moiety, detectable moiety, or diagnostic moiety. [Section 76] 76. The polypeptide construct of paragraph 75, wherein the targeting agent, therapeutic moiety, detectable moiety, or diagnostic moiety comprises an antibody or antigen-binding fragment thereof, a binding agent with affinity for another member of the TGFβ family or another therapeutic target, a radiotherapeutic agent, an imaging agent, a fluorescent moiety, a cytotoxic agent, a cytostatic drug, a nanoparticle-based carrier, a drug conjugated to a polymer, a nanocarrier, an imaging agent, a stabilizer, a drug, a nanocarrier, or a dendrimer. [Section 77] A polypeptide construct comprising, from the N-terminus to the C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) an Fc region of human IgG1. [Section 78] 78. A nucleic acid molecule encoding the polypeptide construct of any one of items 1 to 77. [Section 79] 79. The nucleic acid molecule of Paragraph 78, wherein said nucleic acid molecule encodes said polypeptide construct in a form that is secretable by a selected expression host. [Section 80] A nucleic acid molecule encoding at least one polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 103 and 105, or a sequence substantially identical thereto. [Section 81] A nucleic acid molecule having a sequence set forth in any one of SEQ ID NOs: 106 to 109, or a sequence substantially identical thereto. [Section 82] 81. The nucleic acid molecule of Paragraph 80, further comprising at the 5' end the sequence set forth in SEQ ID NO: 110 or SEQ ID NO: 111, or a sequence substantially identical thereto. [Section 83] 83. A vector comprising the nucleic acid molecule according to any one of Items 78 to 82. [Section 84] A cellular host comprising the nucleic acid molecule of any one of Items 78 to 82 or the vector of Item 83. [Section 85] Item 78. The first polypeptide construct according to any one of items 1 to 77, and Item 78. The second polypeptide construct according to any one of items 1 to 77. 1. A TGFβ binding agent comprising: wherein said first polypeptide construct and said second polypeptide construct are linked together by their respective multimerization domains; wherein the inhibitory potency of said TGFβ binding agent against both TGFβ1 isoform activity and TGFβ3 isoform activity is higher than against TGFβ2 isoform activity; wherein the first linker and the second linker are linked to a TGFβ binding agent that is capable of inhibiting TGFβ3 isoform activity relative to TGFβ1 isoform activity (IC for TGFβ3 vs TGFβ1). 50 The TGFβ binding agent is selected so that the ratio of TGFβ to TGFβ is about 2.5:1 or less. [Section 86] The relative inhibitory potency of the TGFβ binding agents against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 86. The TGFβ binding agent of paragraph 85, wherein the ratio (ratio) of the TGFβ to the TGFβ is less than about 2.5:1, about 2.3:1 or less, about 2:1 or less, about 1.8:1 or less, about 1.5:1 or less, about 1.3:1 or less, about 1:1 or less, about 1:1 or less, about 0.8:1 or less, or about 0.5:1 or less. [Section 87] The relative inhibitory potency of the TGFβ binding agents against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1)50 Item 87. The TGFβ binding agent of Item 85 or 86, wherein the ratio of the TGFβ to the TGFβ is about 1:1 to about 2:1. [Section 88] The relative inhibitory potency of the TGFβ binding agents against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 88. The TGFβ binding agent of paragraph 87, wherein the ratio of β to β is about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, or 1.9:1. [Section 89] The relative inhibitory potency of the TGFβ binding agents against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 Item 89. The TGFβ binding agent of Item 88, wherein the ratio of the TGFβ to the TGFβ is from about 1.4:1 to about 1.6:1. [Section 90] The relative inhibitory potency of the TGFβ binding agents against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3:TGFβ1) 50 90. The TGFβ binding agent of paragraph 89, wherein the ratio of β to β is about 1.4:1, about 1.5:1, or about 1.6:1. [Section 91] 91. The TGFβ binding agent of any one of items 85 to 90, wherein the TGFβ binding agent inhibits both TGFβ1 and TGFβ3 isoform activity with at least 20, 100, 200, 300, 400, 500, 600, 700, 800, or 900 times greater potency than TGFβ2 isoform activity. [Section 92] 92. The TGFβ binding agent of any one of paragraphs 85 to 91, wherein the TGFβ binding agent is a dimer, wherein the first polypeptide construct and the second polypeptide construct are linked between their respective multimerization domains by at least one disulfide bridge. [Section 93] 93. The TGFβ binding agent of paragraph 92, wherein the TGFβ binding agent is a homodimer and the first polypeptide construct and the second polypeptide construct are the same or substantially the same. [Section 94] 94. The TGFβ binding agent of paragraph 93, wherein the first polypeptide construct and the second polypeptide construct comprise or consist of a sequence set forth in any one of SEQ ID NOs: 81, 84, 87, or 96, or a sequence which 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 thereto. [Section 95] 95. The TGFβ binding agent of paragraph 93 or 94, wherein the first polypeptide construct and the second polypeptide construct comprise or consist of the sequence set forth in SEQ ID NO:87. [Section 96] 95. The TGFβ binding agent of paragraph 93 or 94, wherein the first polypeptide construct and the second polypeptide construct comprise or consist of the sequence set forth in SEQ ID NO:96. [Section 97] 94. The TGFβ binding agent of paragraph 93, wherein the first polypeptide construct and the second polypeptide construct comprise or consist of a sequence set forth in SEQ ID NO: 95, or a sequence which 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 thereto. [Section 98] 98. The TGFβ binding agent of paragraph 97, wherein the first polypeptide construct and the second polypeptide construct comprise or consist of the sequence set forth in SEQ ID NO:95. [Section 99] 93. The TGFβ binding agent of Paragraph 92, wherein the TGFβ binding agent is a heterodimer and the first polypeptide construct and the second polypeptide construct comprise different amino acid sequences. [Section 100] 100. The TGFβ binding agent of Paragraph 99, wherein the first polypeptide construct and the second polypeptide construct comprise the same or substantially the same multimerization domain and different TGFβ binding regions. [Section 101] 101. The TGFβ binding agent of paragraph 100, wherein the different TGFβ binding region comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 87 and 96, or a sequence 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 thereto. [Section 102] 101. The TGFβ binding agent of paragraph 100, wherein the different TGFβ binding region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95, or a sequence 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 thereto. [Section 103] A first polypeptide construct comprising, from the N-terminus to the C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) a first Fc region of human IgG1, and A second polypeptide construct comprising, from the N-terminus to the C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) a second Fc region of human IgG1. 1. A TGFβ binding agent comprising: wherein said first polypeptide construct and said second polypeptide construct are linked together via said first and second Fc regions of human IgG1. [Section 104] The inhibitory potency of the TGFβ binding agent against both TGFβ1 and TGFβ3 isoform activity is higher than that against TGFβ2 isoform activity; the relative inhibitory potency of the TGFβ binding agent against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC for TGFβ3 vs TGFβ1) 50 104. The TGFβ binding agent of paragraph 103, wherein the ratio of [Section 105] 78. A TGFβ binding agent which is a homodimer of the polypeptide construct of any one of items 1 to 77. [Section 106] A pharmaceutical composition comprising the polypeptide construct of any one of items 1 to 77 or the TGFβ binding agent of any one of items 85 to 105 and a pharmaceutically acceptable carrier, diluent or excipient. [Section 107] 107. The pharmaceutical composition of paragraph 106, wherein the composition comprises the polypeptide construct of paragraph 64 or 67 or the TGFβ binding agent of paragraph 95 or 96, or a combination thereof. [Section 108] 107. The pharmaceutical composition of Clause 106, wherein the composition comprises the polypeptide construct of Clause 66 or the TGFβ binding agent of Clause 98, or a combination thereof. [Section 109] 107. The pharmaceutical composition of Clause 106, wherein the composition comprises the polypeptide construct of Clause 66 or the TGFβ binding agent of Clause 103, or a combination thereof. [Section 110] Item 110. The pharmaceutical composition according to any one of items 106 to 109, wherein the composition is formulated for administration by injection or infusion. [Section 111] 111. The pharmaceutical composition of claim 110, wherein the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration. [Section 112] A method for producing the polypeptide construct of any one of items 1 to 77 or the TGFβ binding agent of any one of items 85 to 105, the method comprising expressing the first polypeptide construct and / or the second polypeptide construct in a cell. [Section 113] 113. The method of paragraph 112, further comprising culturing said cells and isolating and / or purifying said polypeptide construct or said TGFβ binding agent expressed in said cells. [Section 114] 114. The method of paragraph 113, wherein the polypeptide construct and / or the TGFβ binding agent are secreted by the cells and the polypeptide construct and / or the TGFβ binding agent are obtained from the medium in which the cells are cultured. [Section 115] 106. A method of treating or preventing a TGFβ-related disease or condition in a subject in need thereof, comprising administering to said subject the polypeptide construct of any one of paragraphs 1 to 77 or the TGFβ-binding agent of any one of paragraphs 85 to 105, such that said TGFβ-related disease or condition is treated or prevented in said subject. [Section 116] 116. The method of paragraph 115, wherein the subject is a mammal. [Section 117] 117. The method of paragraph 116, wherein the mammal is a human. [Section 118] 118. The method of any one of items 115 to 117, wherein the subject is suffering from or suspected of suffering from a disease or condition mediated by TGFβ1 and / or TGFβ3. [Section 119] 119. The method of any one of items 115 to 118, wherein the subject is suffering from or suspected of suffering from a disease or condition mediated by TGFβ3. [Section 120] 1. A method of treating or preventing a disease or condition mediated by TGFβ1 and / or TGFβ3 in a subject, comprising administering to said subject a compound or compound(s) comprising administering to said subject a compound or compound(s) mediated by TGFβ1 and / or TGFβ3, such that said disease or condition mediated by TGFβ1 and / or TGFβ3 is treated or prevented in said subject, A method comprising administering to said subject the polypeptide construct of any one of paragraphs 1 to 77 or the TGFβ binding agent of any one of paragraphs 85 to 105. [Section 121] The method of paragraph 120, wherein the disease is mediated by TGFβ3. [Section 122] Item 122. The method according to any one of Items 115 to 121, wherein the disease or condition is characterized by overexpression or overactivation of TGFβ1 and / or TGFβ3. [Section 123] Item 123. The method according to any one of Items 115 to 122, wherein the disease or condition is fibrosis. [Section 124] Item 124. The method of item 123, wherein the fibrosis is pulmonary fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, renal fibrosis, myelofibrosis, systemic sclerosis, dermal fibrosis, cardiac fibrosis, myelofibrosis, a fibroproliferative disease or a connective tissue disorder. [Section 125] Item 123. The method according to any one of Items 115 to 122, wherein the disease or condition is a bone marrow failure disease. [Section 126] 126. The method of claim 125, wherein the disease or condition is Shwachman-Bodian-Diamond syndrome or Fanconi anemia. [Section 127] A method of producing the polypeptide construct of any one of paragraphs 1 to 77 or the TGFβ binding agent of any one of paragraphs 85 to 105, the method comprising culturing the host cell of paragraph 84 under conditions suitable for protein expression; and harvesting the polypeptide construct of any one of paragraphs 1 to 77 or the TGFβ binding agent of any one of paragraphs 85 to 105. [Section 128] 128. A polypeptide construct or TGFβ binding agent produced by the method of paragraph 127.

Claims

1. 1. A polypeptide construct useful for inhibiting the effects of transforming growth factor beta (TGFβ) isoforms, said construct comprising: TGFβ binding region, and Multimerization domain Including; wherein the N-terminus of the multimerization domain is linked to the C-terminus of the TGFβ binding region; the TGFβ binding region comprises, in N-terminal to C-terminal direction, an N-terminal region, a first TGFβ receptor ligand binding domain (TGFβR-LBD), a first linker, a second TGFβR ligand binding domain, and a second linker; wherein the first linker is a minimum of 15 and a maximum of 33 amino acids in length and links the first and second TGFβR ligand binding domains; wherein the second linker is a minimum of 10 and a maximum of 34 amino acids in length and connects the multimerization domain and the TGFβ binding region; wherein said multimerization domain comprises the second constant domain (C H 2) and / or the third constant domain (C H 3) of an antibody heavy chain; wherein the inhibitory potency of said polypeptide construct against each of TGFβ1 isoform activity and TGFβ3 isoform activity is higher than that against TGFβ2 isoform activity; wherein the first linker and the second linker have a ratio of IC 50 for inhibition of TGFβ3 compared to IC 50 for inhibition of TGFβ1 (TGFβ3:TGFβ1 IC ５０ The polypeptide construct is selected so that the ratio of the polypeptides to the polypeptides is 2.5:1 or less.

2. 2. The polypeptide construct of claim 1, wherein the first linker and the second linker are selected such that the ratio of IC50 for inhibiting TGFβ3 compared to IC50 for inhibiting TGFβ1 is 2:1 or less.

3. 2. The polypeptide construct of claim 1, wherein the first linker and the second linker are selected such that the ratio of IC50 for inhibiting TGFβ3 compared to IC50 for inhibiting TGFβ1 is 1.8:1 or less.

4. 4. The polypeptide construct of any one of claims 1 to 3, wherein said polypeptide construct inhibits both TGFβ1 and TGFβ3 isoform activity with at least 1000 times greater potency than TGFβ2 isoform activity.

5. 5. The polypeptide construct of claim 1, wherein the first linker is 16, 18, 30, or 32 amino acids in length and / or the second linker is 16, 30, 32, or 34 amino acids in length.

6. 6. The polypeptide construct of any one of claims 1 to 5, wherein the first linker and / or the second linker each independently comprise or consist of an intrinsically disordered region (IDR) linker, a hybrid linker, a truncated linker, or an extended linker.

7. A polypeptide construct described in any one of claims 1 to 6, wherein one of the first linker and the second linker comprises or consists of a non-IDR linker.

8. 8. The polypeptide construct of any one of claims 1 to 7, wherein one or more of the first linker and the second linker comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 4 and 8 to 26, or a sequence that is at least 90% identical thereto.

9. (a) the first linker comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 16, 21, 22, 23, and 26, or a sequence at least 90% identical thereto; and / or (b) The polypeptide construct of claim 8, wherein the second linker comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 4, 9, 11, 15, 17, 18, 19, 20, 22, 23, 24, 25, and 26, or a sequence that is at least 90% identical thereto.

10. The polypeptide construct of claim 9, wherein the first linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 8; and / or the second linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO:

9.

11. (a) the first linker is (i) a deletion of 1 to 20 N-terminal amino acid residues compared to SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:8; (ii) a deletion of 1 to 20 C-terminal amino acid residues compared to SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:8; (iii) a deletion of 1 to 20 internal amino acid residues compared to SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:8; or (iv) one or more conservative amino acid substitutions in the amino acid sequence compared to SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 8, or any one of (i) to (iii). and / or comprising or consisting of an amino acid sequence having the amino acid sequence (b) the second linker is (i) a deletion of 1 to 20 N-terminal amino acid residues compared to SEQ ID NO:9 or SEQ ID NO:11; (ii) a deletion of 1 to 20 C-terminal amino acid residues compared to SEQ ID NO:9 or SEQ ID NO:11; (iii) a deletion of 1 to 20 internal amino acid residues compared to SEQ ID NO:9 or SEQ ID NO:11; or (iv) one or more conservative amino acid substitutions in the amino acid sequence compared to SEQ ID NO: 4, 9, or 11, or any one of (i) to (iii). A polypeptide construct according to any one of claims 1 to 10, comprising or consisting of an amino acid sequence having the following structure:

12. 12. The polypeptide construct of claim 11, wherein the amino acid deletion in the first linker is a deletion of 16 amino acids of SEQ ID NO:

3.

13. The N-terminal region is (a) an IDR linker, hybrid linker, cleavage linker or extension linker; and / or (b) the amino acid sequence set forth in SEQ ID NO: 3, or a sequence at least 90% identical thereto; A polypeptide construct according to any one of claims 1 to 12, comprising or consisting of:

14. (a) one or more of the first TGFβR-LBD and the second TGFβR-LBD comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, or a sequence at least 90% identical thereto; and / or (b) the first TGFβR-LBD has an amino acid sequence that is the same as or at least 90% identical to the amino acid sequence of the second TGFβR-LBD; and / or (c) the multimerization domain allows the dimerization of said polypeptide construct with a second polypeptide construct, in a covalent or non-covalent manner, wherein the second polypeptide construct is a polypeptide construct according to any one of claims 1 to 13; and / or (d) the multimerization domain comprises an Fc region of an antibody heavy chain; 15. The polypeptide construct of claim 14, wherein both the first TGFβR-LBD and the second TGFβR-LBD comprise or consist of the amino acid sequence set forth in SEQ ID NO: 2, or a sequence that is at least 90% identical thereto.

16. A polypeptide construct described in any one of claims 1 to 15, wherein the multimerization domain comprises an Fc region of an antibody heavy chain, and the antibody is an IgG antibody.

17. 17. The polypeptide construct of claim 16, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

18. 17. The polypeptide construct of claim 16, wherein the multimerization domain has at least 90% sequence identity with a human IgG1, IgG2, IgG3 or IgG4 constant region.

19. (a) the multimerization domain comprises a cysteine ​​residue for bridging said polypeptide construct with a second polypeptide construct, said second polypeptide construct being a polypeptide construct according to any one of claims 1 to 18; and / or (b) the multimerization domain comprises at least two cysteine ​​residues for forming disulfide bridges with the second polypeptide construct, the second polypeptide construct being a polypeptide construct according to any one of claims 1 to 18; and / or (c) the multimerization domain is engineered to reduce aggregation or modulate the dimeric or multimeric stability of the polypeptide construct; and / or (d) the multimerization domain comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 49 to 80 or a sequence at least 90% identical thereto. A polypeptide construct according to any one of claims 1 to 18.

20. 20. The polypeptide construct according to any one of claims 1 to 19, wherein said multimerization domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO:

50.

21. 21. A polypeptide construct according to any one of claims 1 to 20, wherein the TGFβ binding region comprises or consists of the amino acid sequence set out in SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 40 or SEQ ID NO:

41.

22. 22. The polypeptide construct of any one of claims 1 to 21, wherein the polypeptide construct comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 103 and 105, or a sequence that is at least 90% identical thereto.

23. 23. The polypeptide construct of claim 22, wherein the polypeptide construct comprises or consists of an amino acid sequence set forth in SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:95 or SEQ ID NO:96, or a sequence that is at least 90% identical to SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:95 or SEQ ID NO:

96.

24. 24. The polypeptide construct of claim 22 or 23, wherein the polypeptide construct further comprises a signal peptide having the sequence set forth in SEQ ID NO: 104, or a sequence that is at least 90% identical thereto.

25. A dimeric polypeptide comprising a first polypeptide construct and a second polypeptide construct, wherein the first polypeptide construct and the second polypeptide construct are each independently a polypeptide construct described in any one of claims 1 to 24, and the first polypeptide construct and the second polypeptide construct are linked between their respective multimerization domains by at least one disulfide bridge.

26. A dimeric polypeptide described in claim 25, wherein the amino acid sequence of the first polypeptide construct is the same as or at least 90% identical to the amino acid sequence of the second polypeptide construct.

27. 27. The dimeric polypeptide of claim 26, wherein the first polypeptide construct and the second polypeptide construct each independently comprise, from N-terminus to C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) an Fc region of human IgG1.

28. 26. The dimeric polypeptide of claim 25, wherein the first polypeptide construct and the second polypeptide construct comprise different amino acid sequences, and / or the first polypeptide construct and the second polypeptide construct each comprise a multimerization domain having the same amino acid sequence or an amino acid sequence that is at least 90% identical.

29. 29. The dimeric polypeptide according to any one of claims 25 to 28, wherein the first polypeptide construct and / or the second polypeptide construct further comprises a site for conjugation.

30. 30. The dimeric polypeptide of claim 29, wherein the first polypeptide construct and / or the second polypeptide construct is conjugated to a targeting agent, a therapeutic moiety, a detectable moiety, or a diagnostic moiety.

31. 31. The dimeric polypeptide of claim 30, wherein the targeting agent, therapeutic moiety, detectable moiety, or diagnostic moiety comprises an antibody or antigen-binding fragment thereof, a binding agent with affinity for another member of the TGFβ family or another therapeutic target, a radiotherapeutic agent, an imaging agent, a fluorescent moiety, a cytotoxic drug, a cytostatic drug, a nanoparticle-based carrier, a drug conjugated to a polymer, a nanocarrier, an imaging agent, a stabilizer, a drug, a nanocarrier, or a dendrimer.

32. A nucleic acid molecule encoding a polypeptide construct according to any one of claims 1 to 24 or a dimeric polypeptide according to any one of claims 25 to 31.

33. 33. The nucleic acid molecule of claim 32, which encodes the polypeptide construct or dimeric polypeptide in a form that is secretable by a selected expression host.

34. A vector comprising the nucleic acid molecule of claim 32 or 33.

35. A cellular host comprising a nucleic acid molecule according to claim 32 or 33 or a vector according to claim 34.

36. a first polypeptide construct, which is a polypeptide construct according to any one of claims 1 to 24; and A second polypeptide construct, which is a polypeptide construct according to any one of claims 1 to 24.

1. A TGFβ binding agent comprising: wherein said first polypeptide construct and said second polypeptide construct are linked together by their respective multimerization domains; wherein the inhibitory potency of the TGFβ binding agent against each of the TGFβ1 isoform activity and the TGFβ3 isoform activity is higher than that against the TGFβ2 isoform activity; wherein the first linker and the second linker are linked together such that the ratio of IC 50 for inhibition of TGFβ3 compared to IC 50 for inhibition of TGFβ1 (TGFβ3:TGFβ1 IC ５０ The TGFβ binding agent is selected so that the ratio of TGFβ to TGFβ is 2.5:1 or less.

37. 37. The TGFβ binding agent of claim 36, wherein the first linker and the second linker are selected such that the ratio of IC50 for inhibition of TGFβ3 compared to IC50 for inhibition of TGFβ1 is 2:1 or less.

38. 37. The TGFβ binding agent of claim 36, wherein the first linker and the second linker are selected such that the ratio of IC50 for inhibition of TGFβ3 compared to IC50 for inhibition of TGFβ1 is 1.8:1 or less.

39. 39. The TGFβ binding agent of claims 36-38, wherein the first polypeptide construct comprises, from N-terminus to C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) a first Fc region of human IgG1, and the second polypeptide construct comprises, from N-terminus to C-terminus: (i) an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 40; and (ii) a second Fc region of human IgG1; and wherein the first polypeptide construct and the second polypeptide construct are linked together via the first and second Fc regions of human IgG1.

40. 40. A pharmaceutical composition comprising a polypeptide construct according to any one of claims 1 to 24, a dimeric polypeptide according to any one of claims 25 to 31, or a TGFβ binding agent according to any one of claims 36 to 39, and a pharmaceutically acceptable carrier, diluent or excipient.

41. 41. The pharmaceutical composition of claim 40, wherein the composition is formulated for administration by injection or infusion.

42. 42. The pharmaceutical composition of claim 41, wherein the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

43. 43. A polypeptide construct according to any one of claims 1 to 24, a dimeric polypeptide according to any one of claims 25 to 31, a TGFβ-binding agent according to any one of claims 36 to 39, or a pharmaceutical composition according to any one of claims 40 to 42 for use in a method for treating or preventing a TGFβ-related disease or condition in a subject in need thereof, wherein the method comprises administering to the subject the polypeptide construct, dimeric polypeptide, TGFβ-binding agent or pharmaceutical composition such that the TGFβ-related disease or condition is treated or prevented in the subject.

44. 44. The polypeptide construct, dimeric polypeptide, TGFβ binding agent or pharmaceutical composition for use according to claim 43, wherein the subject is a human.

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