Transforming growth factor β (TGFβ) binder and its use

Abstract

This invention provides a TGFβRII-ECD-based trap with tuned isoform specificity. [Solution] A tetravalent TGFβ receptor-extracellular domain-based trap having a tuned isoform specificity profile for neutralizing TGFβ ligands, and methods for using the same in the treatment of diseases and conditions related to TGFβ, particularly TGFβ1 and TGFβ3, are provided. In particular, a TGFβ conjugate is provided that is designed to tune TGFβ isoform specificity in order to maximize therapeutic effect in specific disease indications while minimizing adverse effects. The TGFβ conjugate comprises two polypeptides assembled via a multimerizing domain, each polypeptide having two TGFβII receptor (TGFβR) ligand-binding domains linked as a doublet, where the linker is selected to tune isoform specificity.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 038,290, filed on 12 June 2020, the contents of which Provisional Patent Application are incorporated herein by reference in their entirety.

[0002] Reference to electronically submitted sequence listings This application incorporates by reference the entire computer-readable format (CRF) of the sequence listing filed with this specification. The sequence listing text file filed with this specification, named "14247-632-228_SEQ_LISTING.txt", was created on June 7, 2021, and is 247,915 bytes in size.

[0003] This disclosure relates to TGFβ conjugates containing a fusion molecule derived from the extracellular domain of the TGFβ receptor (TGFβR-ECD) and to the use thereof for binding to and neutralizing TGFβ ligands, particularly for the treatment of TGFβ-related diseases or conditions. [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. Fluctuations in their levels and / or signaling can lead to significant pathological effects. TGFβ has been implicated in the development of many human disorders (Non-Patent Literature 1; Non-Patent Literature 2). For example, TGFβ and its activin ligand play significant pathogenic roles in many diseases, including fibrosis and cancer. Examples of TGFβ-related disorders include hematological malignancies, solid tumors, bone marrow failure states, and a wide variety of disorders characterized by unregulated fibrosis such as pulmonary, hepatic, renal, and vascular fibrosis, pulmonary hypertension, and systemic sclerosis (SSc; also known as scleroderma) (Non-Patent Literature 3; Non-Patent Literature 4).

[0005] Sustained activation of TGFβ signaling plays a central role in the development of fibrosis (Non-Patent Document 5). Standard TGFβ signaling stimulates the migration of fibroblasts to 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), TGFβ increases collagen deposition in the skin and / or lungs and stimulates fibroblast activation in myofibroblasts in the skin (Non-Patent Document 8; Non-Patent Document 10; Non-Patent Document 11). Furthermore, non-standard TGFβ pathways also contribute to the maintenance of the fibrotic phenotype (Non-Patent Document 12). Therefore, the TGFβ 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] TGFβ is also considered a key regulator of tumor progression and is overexpressed by most tumor types. It works favorably for tumorigenesis, for one thing, by inducing epithelial-mesenchymal transition (EMT) in epithelial tumor cells, leading to aggressive metastasis. TGFβ also promotes tumorigenesis by acting as a potent suppressor of the immune response in the tumor microenvironment. In fact, TGFβ is recognized as one of the most potent immunosuppressive factors present in the tumor microenvironment. Because TGFβ interferes with 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 TGFβ in the tumor microenvironment is highlighted by evidence showing that elevated levels of TGFβ 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 expended in devising antitumor therapies that involve TGFβ inhibition. These methods include the use of polypeptide fusions based on the extracellular domain of the TGFβ receptor that binds to or "captures" a TGFβ ligand (see, for example, Patent Document 1; Patent Document 2; Patent Document 3; Patent Document 4; Patent Document 5; Patent Document 6; Patent Document 7; Patent Document 8; Patent Document 9; Patent Document 10; Patent Document 11; Patent Document 12; Patent Document 13; Patent Document 14; Patent Document 15; Patent Document 16; Patent Document 17; and Patent Document 18).

[0007] One approach to developing therapeutic agents that inhibit TGFβ function involves using antibodies or soluble decoy receptors (also known as receptor extracellular domain (ECD)-based ligand traps) to bind to and sequester ligands, thereby blocking the ligand's access to its cell surface receptor. Generally, receptor ECD-based traps are a type of therapeutic agent that can selectively sequester ligands and can be optimized using protein engineering techniques.

[0008] Previously, it has been shown that a single-chain, bivalent TGFβ trap having two TGFβ receptor type II (TGFβRII) extracellular domains linked as a doublet can neutralize members of the TGFβ superfamily of ligands (Patent Document 3, Patent Document 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 domain. Further, it has been shown that when such bivalent doublets are bound in tandem to multimerization domains such as Fc components at the N-terminus or C-terminus, the potency increases (Patent Document 19, Patent Document 10).

[0009] To date, most therapeutic approaches to neutralize TGFβ have focused particularly on the TGFβ1 isoform, especially in cancer immunology. This is because TGFβ1 is the isoform predominantly expressed in the immune system (Non-Patent Document 15) as well as in many types of human tumors (Non-Patent Document 16). The target of interest was usually the TGFβ1 isoform, but most therapeutic agents under development generally inhibit other TGFβ isoforms with varying potencies. For example, fresolimumab is a monoclonal antibody that is a pan-inhibitor of all three TGFβ isoforms. It neutralizes all isoforms, but 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 is said to be involved 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 be spared from neutralization as it plays a beneficial role.

[0011] Therefore, it would be useful to provide a TGFβRII-ECD-based trap with adjusted isoform specificity to maximize the therapeutic effect in specific disease indications while minimizing adverse effects. In particular, it may be useful to provide a trap that neutralizes TGFβ3 with an efficacy similar to that of TGFβ1.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

[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 [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 having a tuned isoform specificity profile for neutralizing TGFβ ligands, and methods for using them in the treatment of TGFβ-related diseases and conditions. The tetravalent TGFβ conjugates provided herein comprise two polypeptides assembled via a multimerizing domain, each polypeptide having two TGFβII receptor (TGFβR) ligand-binding domains linked as a doublet. The TGFβ conjugates provided herein are designed to tune TGFβ isoform specificity to maximize therapeutic efficacy in specific disease indications while minimizing adverse effects.

[0015] The technology of the present invention is at least in part based on the inventors' recognition that a TGFβ ligand trap having isoform specificity that distinguishes it from other known drugs under development may be advantageous for the treatment of certain TGFβ-related diseases and conditions. Recent reports have shown the important role of TGFβ3 isoforms 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 on TGFβ3 production by immune cells (Komai, I. and Okamura, T., 2018). Regarding SSc, a genome-wide association study in African American patients identified TGFβ3 as a novel SSc-susceptible gene (Gourh, P. et al., 2017).

[0016] Regarding TGFβ2, the involvement of this isoform in cardiac homeostasis (Roberts, AB 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) suggests that it would be desirable to avoid neutralizing this isoform.

[0017] In summary, these findings suggest that neutralizing TGFβ1 and TGFβ3 to similar degrees may be beneficial in treating certain disorders, particularly those involving TGFβ3. Achieving nearly equivalent 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 when one of these isoforms is preferentially neutralized, and / or maximizing efficacy. It is also desirable to similarly inhibit TGFβ1 and TGFβ3 without neutralizing TGFβ2 signaling (as avoiding neutralization of this isoform may be beneficial).

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

[0019] While we do not wish to be limited by theory, the present invention is at least in part based on the finding that modifying one or more linkers in such TGFβ ligand traps (e.g., a linker that binds two TGFβR-LBDs together and / or a linker that binds TGFβR-LBDs to the polymerization domain) specifically affects the inhibitory efficacy of the binder against different TGFβ isoforms. In some cases, modifying one or both linkers does not increase undesirable inhibition of TGFβ2 and does not significantly reduce the overall potency (e.g., IC2). 50 (remains within a low picomolar range), TGFβ3:TGFβ1 IC 50 The ratio can be reduced or made equal (exhibiting similar or equal inhibitory efficacy for both isoforms), as is shown herein.

[0020] The TGFβ binding agents provided herein generally comprise a first polypeptide and a second polypeptide bound together via a multimerizing domain, each polypeptide comprising, in an N-terminus-C-terminus orientation: N-terminal region; first TGFβ receptor ligand-binding domain ((TGFβR-LBD); first linker; second TGFβR-LBD; second linker; and multimerizing domain. One embodiment of the TGFβ binding agent is schematically shown in Figure 1 (which shows one 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 may be bound to each other via their respective multimerizing domains, for example, by disulfide bonds (cysteine ​​crosslinks), by coil-coil interactions, etc.

[0021] The TGFβ binding agent of the present invention is TGFβ3:TGFβ1 IC2 50They are designed to reduce or equalize the ratio; that is, they are designed to exhibit reduced preferential inhibition of TGFβ1 compared to other known TGFβ traps. Thus, the TGFβ conjugates provided herein are characterized by their specificity profile for isoform inhibition: specifically, their relative inhibitory efficacy for TGFβ1 and TGFβ3 isoforms (TGFβ3:TGFβ1 IC2 in this specification). 50 The ratio (expressed as a ratio) is approximately 2.5:1 or less, and the activity of both 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 binders of the present invention may offer several advantages in addition to the adjusted isoform specificity. For example, and not limited to, the polypeptides and TGFβ binders may offer improved manufacturability, such as reduced glycosylation, increased homogeneity, and ease of expression. Thus, in certain embodiments, the polypeptides and TGFβ binders of the present invention offer one or more of the following advantages compared to previous TGFβ binders: improved therapeutic effect on specific disease indications, e.g., TGFβ3-mediated pathologies; reduced glycosylation; increased homogeneity; improved manufacturability; and increased production.

[0023] In one embodiment of the polypeptide and TGFβ binder of the present invention, the first and second linkers are designed to provide desired relative isoform specificity of inhibition. For example, in one embodiment, the lengths of the first and second linkers are such that TGFβ3:TGFβ1 IC 50Selected such that the ratio is about 2.5:1 or less, and both TGFβ3 and TGFβ1 isoform activities are inhibited with much higher potency (e.g., in the picomolar range for TGFβ3 and TGFβ1, and in the nanomolar range for TGFβ2) than TGFβ2 isoform activity.

[0024] In certain embodiments of the polypeptides and TGFβ binders of the technology of the present invention, the first linker and the second linker have a TGFβ3:TGFβ1 IC 50 ratio that is selected to be about 2.5:1 or less. In certain embodiments, the TGFβ3:TGFβ1 IC 50 ratio 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. In certain embodiments, the TGFβ3:TGFβ1 IC for the TGFβ binder 50 ratio is from about 1:1 to about 2:1, or is 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. In certain embodiments, the TGFβ3:TGFβ1 IC for the TGFβ binder 50 ratio is from about 1:1 to about 1.5:1 or from about 1.4:1 to about 1.6:1, or is 1.4:1, 1.5:1, or 1.6:1. In certain such embodiments, the TGFβ binder inhibits both TGFβ1 isoform activity and TGFβ3 isoform activity with a potency 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 higher than TGFβ2 isoform activity.

[0025] In one embodiment, the first linker is 33 amino acid lengths or less. In one embodiment, the first linker is about 10 to 33 amino acid lengths. In an embodiment, the first linker may be about 15 to 33 amino acid lengths, or about 18 to about 30 amino acid lengths. In one embodiment, the first linker is 16, 18, 30, or 32 amino acid lengths. In a particular embodiment, the first linker is 16 amino acid lengths. In some other embodiments, the first linker is 18 amino acid lengths. In some other embodiments, the first linker is 30 amino acid lengths. In some other embodiments, the first linker is 32 amino acid lengths.

[0026] In one embodiment, the second linker is 10 amino acids or longer. In one embodiment, the second linker is about 10 to about 35 amino acids long. In an embodiment, the second linker may be about 10 to about 34 or about 15 to about 34 amino acids long. In one embodiment, the second linker is 16, 30, 32, or 34 amino acids long. In a particular embodiment, the second linker is 30 amino acids long. In some other embodiments, the second linker is 16 amino acids long. In some other embodiments, the second linker is 32 amino acids long. In some other embodiments, the second linker is 34 amino acids long.

[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 yet another embodiment, the first linker is 32 amino acids and the second linker is 32 amino acids. In yet another embodiment, the first linker is 32 amino acids and the second linker is 34 amino acids. It should be understood that many other substitutions are possible, as long as the desired isoform specificity of inhibition is achieved.

[0028] In some embodiments, one or more of the first and second linkers include or consist of an IDR linker, IDR linker variant, hybrid linker, hybrid linker variant, cleavage linker, cleavage linker variant, or extension linker as disclosed herein. For example, one or more of the first and second linkers may independently include or consist of an amino acid sequence described in SEQ ID NOs. 4 or any one of 8-26, 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. In some embodiments, the first linker includes or consists of an amino acid sequence described 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 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 includes or comprises an amino acid sequence described 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 a typical embodiment, the first linker contains or comprises the amino acid sequence described in SEQ ID NO: 12, and / or the second linker contains or comprises the amino acid sequence described in SEQ ID NO: 11. In another typical embodiment, the first linker contains or comprises the amino acid sequence described in SEQ ID NO: 8, and / or the second linker contains or comprises the amino acid sequence described in SEQ ID NO: 9. It should be understood that other embodiments using combinations of linkers provided herein are encompassed, as long as the desired isoform specificity of inhibition is achieved.

[0030] In some embodiments of the polypeptides and TGFβ binders of the present invention, the N-terminal region includes or comprises an IDR linker, an IDR linker variant, a hybrid linker, a hybrid linker variant, a cleavage linker, a cleavage linker variant, or an elongation linker. For example, the N-terminal region may include or comprise the amino acid sequence described in SEQ ID NO: 3, 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.

[0031] In one embodiment of the polypeptide and TGFβ binder of the present invention, the first TGFβR-LBD and / or the second TGFβR-LBD comprises or consists of the amino acid sequence described in SEQ ID NO: 2, 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. In one embodiment, the first TGFβR-LBD and the second TGFβR-LBD are the same or substantially the same. In another embodiment, the first TGFβR-LBD and the second TGFβR-LBD may have different amino acid sequences.

[0032] In some embodiments of the polypeptide and TGFβ binder of the present invention, the polymerizing domain enables the dimerization of the two polypeptides according to the present disclosure in a non-covalent manner, for example, by a coil-coil interaction.

[0033] In other embodiments, the polymerizing domain enables the dimerization of the two polypeptides relating to this disclosure by a covalent method, for example, by disulfide crosslinking.

[0034] In one embodiment, the polymerizing domain is one or more constant regions of the antibody, for example, the second constant domain (C) of the antibody heavy chain. H 2) and / or a third constant domain (C H3) or comprising the Fc region of the antibody heavy chain. The antibody may be an IgG antibody, such as, but is not limited to, IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a human antibody, and for example, the multimerization domain comprises the constant region of the heavy chain of human IgG1, IgG2, IgG3, or IgG4. In some 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 constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the multimerization domain comprises or consists of the Fc region of a human IgG1 antibody. In some other embodiments, the multimerization domain comprises or consists of the Fc region of a human IgG4 antibody.

[0035] In one embodiment, the multimerizing domain includes one or more cysteine ​​residues for crosslinking the first polypeptide construct with the second polypeptide construct. For example, the multimerizing domain may include at least two cysteine ​​residues for forming a disulfide crosslink between the two polypeptide constructs, thereby forming a dimer.

[0036] In some embodiments, the multimerizing domain is manipulated to reduce aggregation or to modulate the stability of the dimer or multimer of the polypeptide construct. For example, the Fc region may contain one or more amino acid substitutions that reduce aggregation and / or enhance the stability of the TGFβ-binding agent compared to the native Fc sequence. In some embodiments, the multimerizing domain is selected to provide one or more effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement activation (complement-dependent cell-mediated cytotoxicity or CDC), or opsonization.

[0037] In one embodiment, the multimerization domain includes or comprises an amino acid sequence described in any one of SEQ ID NOs: 49-80, 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. In a particular embodiment, the multimerization domain includes or comprises an amino acid sequence described in SEQ ID NO: 49, 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. In several other embodiments, the multimerization domain includes or comprises an amino acid sequence described in SEQ ID NO: 50, 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.

[0038] In some embodiments of the polypeptide and TGFβ-binding agent of the present invention, the TGFβ-binding region (including the N-terminal domain, two LBDs, and two linkers) includes or comprises a sequence described in any one of SEQ ID NOs: 27-48, 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. In certain embodiments, the TGFβ-binding region includes or comprises the amino acid sequence described in SEQ ID NO: 27. In some other embodiments, the TGFβ-binding region includes or comprises the amino acid sequence described in SEQ ID NO: 29. In some other embodiments, the TGFβ-binding region includes or comprises the amino acid sequence described in SEQ ID NO: 32. In some other embodiments, the TGFβ-binding region includes or comprises the amino acid sequence described in SEQ ID NO: 41.

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

[0040] In some embodiments of the polypeptides and TGFβ binders of the present invention, the polypeptide construct comprises or consists of an amino acid sequence described in any one of SEQ ID NOs: 81-103 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. In a particular embodiment, the polypeptide construct comprises or consists of the amino acid sequence described in SEQ ID NO: 81. In some other embodiments, the polypeptide construct comprises or consists of the amino acid sequence described in SEQ ID NO: 84. In some other embodiments, the polypeptide construct comprises or consists of the amino acid sequence described in SEQ ID NO: 87. In some other embodiments, the polypeptide construct comprises or consists of the amino acid sequence described in SEQ ID NO: 96.

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

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

[0043] In one embodiment of the technology of the present invention, 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 sequences of the first linker, the second linker, LBD, the multimerization domain, and combinations thereof. Thus, each of the following may be the same or different in the two polypeptides independently: 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 one embodiment of the TGFβ binder, the first polypeptide construct and the second polypeptide construct contain or consist of the sequence described 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.

[0045] In one embodiment of the TGFβ binding agent, the different TGFβ binding regions include or consist of the amino acid sequence described 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 one embodiment of the TGFβ binder, the TGFβ binder is 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 polypeptide construct comprising the first Fc region of human IgG1, and 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 polypeptide construct containing the second Fc region of human IgG1. Includes; Here, the first polypeptide construct and the second polypeptide construct are linked together via the first and second Fc regions of human IgG1.

[0047] In one embodiment, the inhibitory efficacy of the TGFβ binder on both TGFβ1 isoform activity and TGFβ3 isoform activity is higher than that on TGFβ2 isoform activity; where the relative inhibitory efficacy of the TGFβ binder on TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC) 50 The ratio is approximately 2.5:1 or less.

[0048] In one embodiment, the TGFβ-binding agent provided herein is a homodimer of the polypeptide construct provided herein.

[0049] In alternative embodiments, the TGFβ binder is a homodimer, i.e., the first and second polypeptides are the same or substantially the same.

[0050] In one embodiment, the polypeptide or TGFβ conjugate may be conjugated with a targeting agent, a therapeutic portion, a detectable portion, and / or a diagnostic portion.

[0051] In another broader embodiment, nucleic acids encoding polypeptides and TGFβ-binding agents of the present invention are provided. Vectors and plasmids for the expression of such nucleic acids and / or polypeptides and TGFβ-binding agents are also provided. For example, in one embodiment, nucleic acids having the sequences described 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 with SEQ ID NOs: 106-109, 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, or capable of hybridizing to them under conditions of high stringency, are provided. Cells expressing polypeptides and TGFβ-binding agents of the present invention are also provided.

[0052] In a further embodiment, a method is provided for producing polypeptides and TGFβ-binding agents of the present invention, comprising expressing one or more polypeptides provided herein in a cell, followed by isolation and / or purification thereof. In one embodiment, the polypeptide construct and TGFβ-binding agent are expressed in a form that can be secreted by cells, for example, using a signal peptide at the N-terminus, to allow for the recovery of the polypeptide or TGFβ-binding agent from the culture medium.

[0053] In another broader embodiment, pharmaceutical compositions comprising polypeptide constructs or TGFβ binders relating to the present disclosure and pharmaceutically acceptable carriers, diluents, or excipients are provided. In some embodiments, the pharmaceutical composition is formulated for administration by injection or infusion, for example, intravenous, subcutaneous, intraperitoneal, or intramuscular administration. In some embodiments, the pharmaceutical composition is provided in unit dosage forms.

[0054] In yet another broader embodiment, a method is provided for preventing or treating a TGFβ-related disease or condition, comprising administering a therapeutically effective amount of a polypeptide, TGFβ-binding agent or pharmaceutical composition of the present invention to a subject such that the TGFβ-related disease or condition is prevented or treated.

[0055] Examples of TGFβ-related diseases or conditions that can be prevented or treated in accordance with this disclosure include, but are not limited to, fibrosis (e.g., fibrosis, fibrous scarring, fibroproliferative disorders); cancer (e.g., malignant tumors, solid tumors, metastases); and bone marrow failure (e.g., Schwachmann-Bodian-Diamond syndrome, Fanconi anemia). In some embodiments, the polypeptides or TGFβ conjugates described herein are used to treat or prevent fibrosis of tissues and / or organs, and fibrous scarring, including, but are not limited to, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), renal fibrosis, hepatic fibrosis (e.g., cirrhosis), systemic sclerosis, scleroderma, cutaneous fibrosis, cardiac fibrosis, myelofibrosis, etc.

[0056] In one embodiment, a method is provided for preventing or treating a disease or condition mediated by TGFβ1 and / or TGFβ3, comprising administering a therapeutically effective amount of a polypeptide, TGFβ binder, or pharmaceutical composition of the present invention to a subject so as to treat a disease or condition mediated by TGFβ1 and / or TGFβ3. In one embodiment, a method is provided for preventing or treating a disease or condition mediated by TGFβ3 in a subject requiring attention, comprising administering a therapeutically effective amount of a polypeptide, TGFβ binder, or pharmaceutical composition of the present invention to a subject so as to prevent or treat a disease or condition mediated by TGFβ3.

[0057] In one embodiment, a method is provided for preventing or treating fibrosis in a subject in need, comprising administering a therapeutically effective amount of a polypeptide, TGFβ-binding agent or pharmaceutical composition of the present invention to the subject so as to prevent or treat fibrosis.

[0058] In a broader embodiment, kits and packages are provided for treating TGFβ-related diseases or conditions in a subject requiring treatment, comprising the polypeptide, TGFβ binder or pharmaceutical composition relating to the Disclosure; optionally comprising one or more further components such as acids, bases, buffers, inorganic salts, solvents, antioxidants, preservatives or metal chelators, and / or tools for administration thereof, such as syringes or needles. Instructions for administration or use may also be included.

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

[0060] In yet another embodiment, polypeptide constructs or TGFβ binders produced by the manufacturing methods provided herein are provided herein.

[0061] Further scope, applicability, and merits of this disclosure will become apparent from the non-restrictive detailed description set forth herein. However, it should be understood that this detailed description illustrates exemplary embodiments of this disclosure, and refers to the accompanying drawings only as examples.

[0062] The patent or application file shall include at least one drawing made in color. A copy of this patent or patent application publication, accompanied by the color drawing, will be provided by the Patent Office upon request and payment of the necessary fees.

[0063] For a better understanding of the technology and to more clearly illustrate how it can be implemented, accompanying drawings illustrating, as an example, aspects and features of non-limiting embodiments of the technology of the present invention are referred to herein. [Brief explanation of the drawing]

[0064] [Figure 1] A schematic structure of the domain organization of a tetravalent TGFβ binder according to a specific embodiment is shown. The embodiment shown herein is a homodimer of a first polypeptide (left) and a second polypeptide (right) linked by a disulfide crosslink in a multimerization domain (shown as two lines). The ligand-binding domain (LBD) is shown as a circle, the multimerization domain as an ellipse, and the N-terminal region and linker as rectangles. In embodiments where the binder is a heterodimer, the first and second polypeptides differ in one or more regions or parts (not shown).

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

[0066] Figure 2B shows overlays of TGFβ1 dimers (blue) and TGFβ3 dimers (green). The corresponding monomers in the regions are superimposed to show the difference between monomers at the dimer angle.

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

[0068] [Figure 3] Figure 3A shows polyacrylamide gel electrophoresis analysis of representative TGFβ-binding agents under non-reducing conditions. After expression and purification, 2 μg of each protein was packed into the gel as shown: 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 for typical TGFβ-binding agents. After expression and purification, 2 ug of each protein was packed into the gel as shown: 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 from A549 / IL-11 cell-based assays for TGFβ1 inhibition for proteins 61, 96, 101, 107, and 112, as well as controls, as shown. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error (SEM).

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

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

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

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

[0075] Figure 6B shows representative results from A549 / IL-11 cell-based assays for TGFβ3 inhibition for proteins 113, 115, and 116, as well as for controls, as shown. The table shows the calculated ICs calculated in Graphpad Prism. 50 The values ​​are listed. Error bars indicate the standard error (SEM).

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

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

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

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

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

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

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

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

[0084] [Figure 11] Figure 11 shows representative results from A549 / IL-11 cell-based assays for TGFβ2 inhibition for proteins 61, 96, and 101, as well as controls, as shown. The table lists the calculated IC50 values ​​calculated in Graphpad Prism. Error bars indicate the standard error (SEM). [Modes for carrying out the invention]

[0085] The technology of the present invention is described in more detail below. This description is not intended to be a detailed list of all the different ways in which the technology can be carried out, or all the features that can be added to the technology of the present invention. For example, features shown in relation to one embodiment may be incorporated into other embodiments, and features shown in relation to a particular embodiment may be omitted from that embodiment. Furthermore, many variations and additions to the various embodiments suggested herein will be obvious to those skilled in the art in light of this disclosure, and such variations and additions will not depart from the technology of the present invention. Accordingly, the following description is intended to illustrate certain specific embodiments of the technology and is not intended to exhaustively describe all substitutions, combinations and variations thereof.

[0086] definition 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 those skilled in the art to which the invention relates.

[0087] The terms “a,” “an,” and “the,” used in the claims and / or herein with the term “including,” may mean “one,” but this also coincides with the meanings of “one or more,” “at least one,” and “one or two or more.” Similarly, the term “another” may mean at least a second or more. Unless otherwise specifically indicated herein or unless the context clearly contradicts this, these terms should be interpreted as encompassing both singular and plural.

[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 further, undescribed elements or process steps. The term “consisting of” should be interpreted as closed-ended.

[0089] The term “approximately” is used to indicate that a value or quantity refers to an actual given value and an approximation of such a given value that can be reasonably inferred by a person skilled in the art (including equivalents and approximations of such a given value under experimental and / or measurement conditions). For example, with respect to a given value or range, the term “approximately” 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] As used herein, the expression “and / or” should be interpreted 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 interpreted as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were individually described herein. Unless otherwise stated or evident from the context, the term “or” as used herein is understood to be inclusive and encompasses both “or” and “and.” For example, “one embodiment of a composition comprising A or B” would typically represent one aspect using a composition comprising both A and B. However, “or” should be interpreted as excluding aspects that cannot be combined in a consistent manner (e.g., a composition pH of 9-10 or 7-8).

[0091] It should be understood herein that terms such as “1-20” include any individual values ​​that are contained within and include 1 and 20. Therefore, it should be understood herein that the term “1-20” includes 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 “1-20” also include individual subranges that are contained within and include 1-20. Therefore, the term “1-20” also includes subranges such as “1-9”, “2-9”, “3-5”, “5-9”, “5-20”, and “8-20”. The same applies to, but is not limited to, similar expressions such as “1-19”, “1-18”, “1-10”, “1-9”, and “5-15”.

[0092] It should be understood herein that terms such as “approximately 15 to approximately 35” include and encompass 15 and 35. Accordingly, terms such as “approximately 15 to approximately 35” include any number from 15 to 35 and any number including 15 to 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 “approximately 15 to approximately 35” also include any individual subranges that include and encompass 15 to 35, “approximately 16 to approximately 34,” “approximately 16 to approximately 24,” “approximately 24 to approximately 34,” etc. With respect to the number of amino acids, the term “approximately” means that a given number of amino acids are specifically included, allowing for a variation of + / - 2 in the number of amino acid residues. Therefore, terms such as "approximately 15-35" also include "13-37," "13-35," "17-37," and "17-35." The same applies to, but is not limited to, similar expressions such as "approximately 16-34," "approximately 16-24," and "approximately 24-34."

[0093] It should be understood herein that terms such as “at least 80% identical” include and encompass any individual values ​​that fall within and include the range of 80% to 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 subranges that fall within and include the range of 80% to 100%, such as “85% to 99%”, “97% to 100%”, “90% to 100%”, etc. The same applies to, but is not limited to, similar expressions such as “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 specific biological or biochemical function, such as the binding of a protein receptor to its ligand, or the activation of a cell receptor by that ligand. In some embodiments, the inhibitory potency is determined by measuring the IC50 of the inhibitor against a particular ligand or substrate. In this case, the relative inhibitory potency against various inhibitors and / or ligands is expressed as IC50. 50 It can be evaluated by comparing values. For example, a relative inhibitory effect of 3:1 is equivalent to IC 50 This means the ratio of the values ​​is 3:1. The terms "inhibition potency," "inhibitory potency," "inhibitory potency," and "neutralizing potency" are used synonymously herein.

[0095] When used herein, "IC" 50 The term "IC" refers to the median inhibitory concentration (i.e., 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 particular biological or biochemical function. 50 The value is typically expressed as molar concentration. IC of inhibitors 50 This can be determined by constructing dose-response curves and examining the effects of various concentrations of the inhibitor on the specific biological or biochemical function in question.

[0096] As used herein, the term “avidity” refers to the overall strength of the binding interaction between a protein receptor and its ligand. Binding strength generally refers to the accumulated strength of multiple individual non-covalent interactions between a protein receptor and its ligand, and differs from “affinity,” which represents the strength of a single binding interaction. It should be understood that binding strength is rarely simply the sum of the affinities of its components, as many factors (such as local concentration or proximity, polymerization, 3D structure or conformation) can influence biomolecular interactions.

[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, for example, without any 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 the original or reference sequence and has a high degree of sequence identity thereto. 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, for example, with 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 pursuant to this disclosure are intended to be included.

[0098] As used herein, the term “multimerizing domain” refers to an amino acid sequence that enables a polypeptide chain to be assembled into a polymer. The term “polymer” refers to a molecule made up of multiple monomers. The term “polymer” is not limited to dimers, trimers, tetramers, pentamers, hexamers, octamers, decamers, and so on.

[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 within 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 binders Novel polypeptide constructs comprising a TGFβ-binding domain and a polymerizing domain, as well as TGFβ-binding agents comprising two such polypeptide constructs assembled via the polymerizing domain, are provided herein. The TGFβ-binding domain comprises two TGFβRII-LBDs linked side-by-side by a first linker, which are linked to the polymerizing domain by a second linker. The polypeptide constructs and TGFβ-binding agents of this disclosure are optimized by improving their binding to TGFβ. Specifically, the linkers are optimized without increasing undesirable inhibition of TGFβ2 and without significantly reducing the overall potency (e.g., IC2). 50 (While remaining within the picomolar range), TGFβ3:TGFβ1 IC 50 The TGFβ isoform specificity is optimized so that the ratio is approximately 2.5:1 or less (showing similar inhibitory efficacy for both isoforms).

[0103] In exemplary embodiments, the polypeptide constructs and TGFβ conjugates of the present disclosure comprise two polypeptide chains conjugated via the Fc region of an antibody, or via a constant CH2 domain, a constant CH3 domain, and / or a combination of CH2 and CH3. The constant region of the antibody may be derived from or substantially identical to that of a human IgG1, IgG2, IgG3, or IgG4 antibody. The conjugation of both polypeptide chains generally occurs, for example, in mammalian cells, for protein expression and secretion. In one exemplary embodiment, the TGFβ conjugate may comprise a homodimer, i.e., a dimer of a polypeptide construct having a sequence described in any one of SEQ ID NOs: 81-103 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. In other embodiments, the TGFβ binder comprises a heterodimer, i.e., a dimer of two different polypeptide constructs, at least one of which has a sequence described in any one of SEQ ID NOs. 81-103 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.

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

[0105] In exemplary embodiments, the multimerizing domain allows for the covalent assembly of two or more polypeptide chains, for example, by disulfide bonds between cysteine ​​residues. Alternatively, the multimerizing domain may allow the polypeptide chains to be assembled non-covalently, for example and not limited to, by a coil-like structure (De Crescenzo, G. et al., 2004).

[0106] In one embodiment, the multimerizing domain is a dimerizing domain, i.e., it enables the assembly of two polypeptide chains that form a dimer. According to this disclosure, such a dimer generally comprises two polypeptides, each polypeptide comprising two TGFβR-LBDs linked together and linked to the dimerizing domain described herein, thereby forming a tetravalent TGFβ binder. Homodimers and heterodimers of polypeptide constructs provided herein are included.

[0107] In some embodiments, the polypeptide multimerization or dimerization domain includes, for example, a constant region of an immunoglobulin heavy chain containing CH2 and / or CH3 domains. The Fc moiety of immunoglobulins is typically used. However, coiled structures have also been found to be suitable for dimerization. Exemplary embodiments of the Fc moiety include, for example and not limited to, those that have lost the ability to interact with specific Fc receptors. In further embodiments, the multimerization domain may include an IgG-like dimerization domain, e.g., an IgG1, IgG2, IgG3, or IgG4 dimerization domain. In some embodiments, the multimerization domain may provide one or more effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement activation (complement-dependent cell-mediated cytotoxicity, CDC), or opsonization.

[0108] In one embodiment, the multimerizing or dimerizing domain comprises CH2, CH3, or CH2 and CH3 from an antibody heavy chain of human origin. For example, though not intended to be limiting, the antibody heavy chain may be selected from the group consisting of human IgG1, IgG2, IgG3, or IgG4. In an embodiment, the constant domain in the construct is CH2 itself, or CH3 itself, or CH2-CH3. The antibody heavy chain components typically provide disulfide crosslinks between the same or different single-stranded polypeptide constructs. In one embodiment, the multimerizing domain provides at least one disulfide bond between single-stranded polypeptide constructs. In another embodiment, the multimerizing domain provides at least two disulfide bonds between single-stranded polypeptide constructs. In some cases, the antibody heavy chain also provides isolation of a dimerized polypeptide, e.g., a protein A-based molecule after production in a host cell.

[0109] Therefore, in some embodiments, the multimerizing or dimerizing domain is an antibody constant domain that provides crosslinking between two polypeptide constructs of the present invention. This is achieved, for example, when expressed polypeptide constructs are secreted from their expression host. Thus, the production of single-chain polypeptides may provide a dimeric construct in which two polypeptide chains are crosslinked via disulfide crosslinks containing one or more cysteine ​​residues in each of the antibody constant domains present in each polypeptide. In some embodiments, the multimerizing domain (e.g., constant domain) has no particular activity other than acting as a structure through which a multimer (e.g., dimer) can be formed. Such minimal constant domains may also be modified to provide some benefit by incorporating a corresponding hinge domain and optionally altering the cysteine ​​residue composition. For example, some or all of the cysteine ​​residues involved in crosslinking two Fc fragments or naturally used for crosslinking between the heavy and light chains of a complete antibody may 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 keeping in mind that the stability of Fc dimers may depend on the number of intermolecular disulfide bridges, it should be noted that not all native interhinge disulfide bonds need to be formed for Fc dimerization to occur.

[0110] In this specification, the terms “antibody” and “immunoglobulin (Ig)” are used synonymously to refer to proteins constructed from paired heavy and light polypeptide chains. The structures of antibodies and domains are well-established and well-known to those skilled in the art, and are only briefly summarized herein. When antibodies fold properly, each chain folds into several different globular domains linked by a more linear polypeptide sequence. In particular, the Ig light chain folds into variable (VL) and constant (CL) domains, while the heavy chain folds into 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 a Fab (antigen-binding fragment) containing a binding region (Fv); the interaction of the two heavy chains results in the pairing of the CH2 and CH3 domains, resulting in the formation of an Fc (crystallizable fragment). The properties described herein for the CH2 and CH3 domains also apply to Fc.

[0111] In certain embodiments and aspects of this disclosure, the polymerizing or dimerizing 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 the IgG1, IgG2, IgG3, or IgG4 domains. IgG1, IgG2, IgG3, or IgG4 may be derived from humans. In certain embodiments, the TGFβ binders described herein include those having a dimerizing domain of IgG1. In several other embodiments, the TGFβ binders described herein include those having a dimerizing domain of IgG4.

[0112] The multimerizing or dimerizing domains may be manipulated to reduce aggregation or to modulate the stability of the TGFβ-binding agent formed by the assembly of two or more polypeptides disclosed herein. Thus, for example, an Fc moiety having a mutation in the hinge region is encompassed by this disclosure. Exemplary embodiments of Fc variants and modified hinge regions are provided, for example, in patent applications published under the brochures International Publication No. 2018 / 158727 and International Publication No. 2017 / 037634. ​​Where the hinge portion of a multimerizing or dimerizing domain is referred to, it should be understood that the hinge is a portion of the multimerizing domain and not a portion of the second linker.

[0113] In exemplary embodiments, the multimerization or dimerization domain has sequences described in SEQ ID NOs: 49-80, or functionally equivalent variants thereof, or sequences that are 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 specific embodiments, the multimerization domain may include SEQ ID NOs: 49 or sequences that are 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 other embodiments, the multimerization domain may include SEQ ID NOs: 50 or sequences that are 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 this disclosure, the first and second polymerizing or dimerizing domains of the TGFβ-binding agent may have the same or substantially the same amino acid sequence in a particular embodiment. Alternatively, in a given embodiment, the polymerizing or dimerizing domains may be different, as long as this does not adversely affect polymerization.

[0115] It should be understood that the multimerization domain is not intended to be particularly limited. Any amino acid sequence that enables the binding of polypeptide chains forming the tetravalent TGFβ binder according to this disclosure may be used, as long as the desired function and isoform specificity are maintained.

[0116] According to this disclosure, linkers in polypeptide constructs and TGFβ binders include or may consist of IDR linkers, IDR linker variants, hybrid linkers, hybrid linker variants, cleavage linkers, cleavage linker variants, or extension linkers, 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 essentially 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 “essentially disordered region (IDR) linker” refers to a linker that includes, or comprises, at least a portion of one or both of the essentially disordered regions (IDRs) adjacent to the structured ligand-binding domain of the TGFβRII extracellular domain. An IDR linker generally has substantial sequence identity with at least one sequence of the essentially disordered region 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 a portion thereof. It should be understood that an IDR linker may include all or only a portion of the IDRs of TGFβR, or multiple portions linked together.

[0119] In one embodiment, the IDR linker includes or consists of one or both portions 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 portions of the IDR may include the entire IDR sequence or its variants (e.g., substitutions, cleavages).

[0120] In one embodiment, the IDR linker includes or consists of portions of each IDR that are 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, for example, linkers having the amino acid sequences described in SEQ ID NOs: 8-16, 19, 22, 23, and 26.

[0121] In one embodiment, the IDR linker includes or consists of the sequence described in Sequence ID No. 4.

[0122] In one embodiment, the IDR linker does not consist of the sequence described in SEQ ID NO: 7. In such embodiments, polypeptides and TGFβ binders containing the sequence described in SEQ ID NO: 7 are excluded from the present invention. In some embodiments, desired isoform specificity (e.g., desired TGFβ3:TGFβ1 IC) is used. 50 IDR linkers that do not provide a ratio are excluded from the present invention, as are polypeptides and TGFβ binders containing such sequences.

[0123] IDR linker variants, hybrid linkers, hybrid linker variants, cleavage linkers, cleavage linker variants, and elongation linkers are derived from the IDR linker sequences disclosed herein.

[0124] As used herein, the term “non-IDR linker” means a linker that does not share substantial homology or identity with the essentially disordered region (IDR) adjacent to the structured ligand-binding domain of the TGFβRII extracellular domain. In the embodiments and examples described herein, non-IDR linkers may be flexible linkers, including, for example, glycine and glycine-serine (GS) linkers, but are not limited to these. When producing fusion constructs, it is common practice to introduce artificial, highly flexible glycine or glycine-serine linkers, such as GGGGS or [G4S]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 can also be inconvenient due to undesirable immunogenicity and the potential for their added molecular weight. Entropy factors are also a potential disadvantage of glycine and GS linkers, as glycine and GS linkers are highly flexible and can be partially restricted at target binding, potentially resulting in an unfavorable loss of entropy for binding. Therefore, in some embodiments, the polypeptides and TGFβ binders of this disclosure either do not contain a non-IDR linker or contain at least one IDR linker or IDR linker variant in addition to a non-IDR linker. Non-limiting examples of non-IDR linkers relating to this 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, for example, the amino acid sequences described 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, 8-16, 19, 22, 23, and 26 are replaced with amino acid sequences containing glycine and / or serine residues (glycine or GS linkers). These linkers are referred to herein as hybrid linkers. Hybrid linker variants are also included; these are functionally equivalent variants of a hybrid linker that include one or more insertions, deletions, or amino acid substitutions, optionally including conservative amino acid substitutions. Variants are described further below.

[0126] In exemplary embodiments of the hybrid linker and hybrid linker variant, 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 the hybrid linker or hybrid linker variant, at least seven consecutive amino acids of the IDR linker or IDR linker variant are substituted with glycine and / or serine residues. In yet another exemplary embodiment of the hybrid linker or hybrid linker variant, two sets of 3 to 7 consecutive amino acids of the IDR linker or IDR linker variant are substituted with glycine and / or serine residues. The two sets of 3 to 7 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 sequence numbers 17, 18, 20, 21, 24, and 25.

[0128] In one embodiment, the linker is a cleavage linker or a cleavage linker variant. Such a linker has a cleavage (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 the N-terminus or C-terminus or both of the IDR linker provided herein. In an exemplary embodiment, the amino acid cleavage may be at the N-terminus of either SEQ ID NO: 3 or 8-26. In an exemplary embodiment, the cleavage may result in the removal of residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 at the N-terminus. In another exemplary embodiment, the cleavage may be at the C-terminus of either SEQ ID NO: 4 or 8-26. In an exemplary embodiment, the cleavage may result in the removal of residues 1, 2, 3, 4, 5, 6, 7, 8, or 9 at the C-terminus. In another embodiment, the cleavage 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 cleavage may be an internal deletion, for example, a deletion starting from amino acid number 10 or 11 of SEQ ID NO: 7. In one embodiment, residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 are deleted from SEQ ID NO: 7, including amino acid numbers 10 and / or 11. In another embodiment, residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 are deleted internally from any one of SEQ ID NOs: 4 and 8-26.

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

[0131] Other exemplary and non-limiting embodiments of the cutting linker are provided in Sequence IDs 8-16, 18, 19, 22, 23, and 26.

[0132] This disclosure further provides cleavage linker variants, which may include amino acid substitutions (conservative or non-conservative) compared to the cleavage linkers disclosed herein.

[0133] In one embodiment, the first linker has (a) the 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) the 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) the 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 contains or consists of any one of the amino acid sequences of (a) to (c). In one embodiment, the amino acid deletion is the deletion of 16 amino acids in SEQ ID NO: 3.

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

[0135] In one embodiment, the linker is an elongated linker. Such a linker has the addition (elongation) 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 the N-terminus or C-terminus or both of any IDR linker, IDR linker variant, hybrid linker, hybrid linker variant, cleavage linker, or cleavage linker variant as disclosed herein. Each of these further amino acids can be independently selected from any amino acid residue.

[0136] In exemplary embodiments, the linkers disclosed herein may include 1 to 5 additional amino acid residues at their N-terminus. In another exemplary embodiment, the linkers disclosed herein may include 1 to 5 additional amino acid residues at their C-terminus. In yet another exemplary embodiment, the linkers disclosed herein may include 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 residues and may be the same or different. Other exemplary and non-limiting embodiments of the elongation linkers include the addition 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 the N-terminus or C-terminus or both of any one of SEQ ID NOs: 4 and 8-26. The added sequence may contain any amino acid residues.

[0137] Exemplary embodiments of the extension linker also include those that include a non-IDR linker portion at either or both of its N-terminus and / or C-terminus. For example, an IDR linker, IDR linker variant, hybrid linker, hybrid linker variant, cleavage linker, or cleavage linker variant may be flanked by at least one non-IDR linker at either or both of its N-terminus and / or C-terminus. Alternatively, a non-IDR linker may be flanked at least by an IDR linker, IDR linker variant, hybrid linker, hybrid linker variant, cleavage linker, or cleavage linker variant at either or both of its N-terminus and / or C-terminus.

[0138] In some embodiments of the polypeptide constructs and TGFβ binders of this disclosure, the N-terminal region comprises or consists of a sequence substantially identical to the N-terminal IDR (SEQ ID NO: 3) in TGFβRII-ECD (SEQ ID NO: 1), or, but not limited to, a cleaved or substituted variant thereof. It should be understood that the N-terminal region may be cleaved and / or substituted and otherwise modified, provided that this does not adversely affect the desired inhibitory efficacy and specificity.

[0139] This disclosure also includes variants of the polypeptides and TGFβ binders described herein. The variants included herein include those having a change in the amino acid sequence of any one of the elements of the polypeptide or TGFβ binder (e.g., the first and second TGFβ receptor ligand-binding domains (TGFβR-LBD), the first linker, the second linker, the N-terminal region, the polymerization domain, etc.). The variants of the polypeptide or TGFβ binder include, for example, those having similar or improved binding affinity, binding strength, isoform specificity, inhibitory potency, stability, manufacturability, and / or reduced aggregation compared to the polypeptides and TGFβ binders disclosed herein.

[0140] The sites targeted for substitutional mutagenesis include the polymerization domain of the polypeptide or TGFβ-binding agent. Exemplary embodiments of the polypeptide or TGFβ-binding agent variants of this disclosure may include those having modified IgG1, IgG2, IgG3, or IgG4 constant regions or portions thereof. TGFβ-binding agents that may include the IgG1 constant region (modified or unmodified) are included herein. TGFβ-binding agents that may include the IgG4 constant region (modified or unmodified) are also included herein.

[0141] The variants included in this disclosure may include insertions, deletions, or amino acid substitutions (conservative or non-conservative). These variants may have at least one amino acid residue removed from their amino acid sequence and a different residue inserted in its original position.

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

[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 may be introduced, indicated as “Exemplary Substitutions” in Table 1 or further described below with reference to amino acid classes, and the product may be screened.

[0144] It is known in the art that mutants can be generated by substitutional mutagenesis and retain the biological activity (i.e., functional equivalence) of the polypeptides of this disclosure. These mutants have, for example, one or more conserved amino acid substitutions in at least one amino acid residue in the removed amino acid sequence and a different residue inserted in its original position. Examples of substitutions identified as “conservative substitutions” are shown in Table 1. If such substitutions result in undesirable changes, other types of substitutions, indicated as “exemplary substitutions” in Table 1 or further described herein by reference to amino acid classes, are introduced and the product is screened.

[0145] Based on their general side-chain properties, amino acid residues can be grouped 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 affect chain orientation: glycine (Gly), proline (Pro); and (Group 6) Aromatics: Tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0146] Non-conservative substitutions involve replacing one member of these classes with another member. [Table 1]

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

[0148] However, the level of identity can also be determined over the entire length of a given sequence. Therefore, percentage identity would indicate amino acids that are identical to the original peptide and can occupy the same or similar positions. Percent similarity would indicate amino acids that are identical to the original peptide at the same or similar positions, and those that are substituted with conserved amino acid substitutions.

[0149] Accordingly, in some embodiments, the variants of the present disclosure include 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 with the original sequence or a portion of the original sequence.

[0150] In one embodiment, the amino acid sequence change occurs in the TGFβ receptor ligand-binding domain (TGFβR-LBD) of the polypeptide or TGFβ binder. In another embodiment, the change may occur outside the TGFβ receptor ligand-binding domain (TGFβR-LBD) of the TGFβ binder. The variants encompassed by this disclosure may have a TGFβR-LBD that is identical or substantially identical to the structured ligand-binding domain found in the extracellular domain (ECD) of the TGFβ receptor (including those in humans, animals, etc.). In yet another embodiment, the amino acid sequence change occurs in the polymerization domain. In yet another embodiment, the amino acid sequence change occurs in the first and / or second linker. It should be understood that the change may occur in multiple regions of the polypeptide or TGFβ binder, as long as the desired function is maintained.

[0151] In some embodiments, the polypeptide or TGFβ-binding agent of the present disclosure may be conjugated with, 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, the polypeptides or TGFβ conjugates of the present disclosure are conjugated with therapeutic moieties such as, for example and not limited to, chemotherapeutic agents, cytokines, cytotoxic agents, antifibrotic agents, anticancer agents (e.g., small molecules), and single-chain antibodies.

[0153] In another exemplary embodiment, the polypeptide or TGFβ binder of the Disclosure is conjugated with a detectable portion, for example and not limited to, a portion detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical and / or other physical means. The detectable portion may be conjugated to the TGFβ binder directly or indirectly (for example, via binding such as DOTA or NHS binding, for example) using methods well known in the art. A wide variety of detectable portions may be used, and the selection will depend on the required sensitivity, ease of conjugation, stability requirements and available instruments. Suitable detectable portions include, but are not limited to, fluorescently labeled, radiolabeled (for example, but are not limited to, 125 I, In 111 , Tc 99 , I 131 The detectable portion may include nuclear magnetic resonance (including positron-emitting isotopes for PET scanners, etc.), nuclear magnetic resonance (CRL) labeling, luminescence labeling, chemiluminescence labeling, chromophore labeling, enzyme labeling (e.g., but not limited to horseradish peroxidase, alkaline phosphatase, etc.), quantum dots and / or nanoparticles. The detectable portion may produce and / or generate a detectable signal, thereby enabling the detection of a signal from the detectable portion.

[0154] The therapeutic portion includes, for example and not limited to, 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 (for example, Bi 213 ), copper (for example, Cu 67 )), may include 5-fluorouracil, adriamycin, irinotecan, taxanes, Pseudomonas endotoxin, lysine, auristatin (e.g., monomethyl auristatin E, monomethyl auristatin F), mytansinoids (e.g., meltansine), and other toxins.

[0155] In another embodiment, the therapeutic portion may include another therapeutic agent for TGFβ-related disease or condition. For example, and not limited to, one or more polypeptide constructs or TGFβ conjugates may be linked to a cytotoxic agent to generate an antibody-drug conjugate (ADC).

[0156] The targeting agent may include, for example, an amino acid sequence for delivering a polypeptide or TGFβ-binding agent to a desired tissue, organ, or location in the target body. For example, but not limited to, the targeting agent may include a polyaspartate sequence motif for bone targeting, or an antibody or antigen-binding fragment.

[0157] In other exemplary embodiments, the targeting agent, therapeutic portion, or diagnostic portion may include, for example and not limited to, an antibody or its antigen-binding fragment (e.g., a single-chain antibody), a conjugate having affinity for another member of the TGFβ family or another therapeutic target, a radiotherapy agent, a contrast agent, a fluorescent portion, a cytotoxic agent, a cell division inhibitor, a nanoparticle-based carrier, a polymer conjugated with a drug, a nanocarrier, a contrast 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, as long as it does not adversely affect the function of the polypeptide or TGFβ binder. For example, and not limited to, a targeting agent, therapeutic portion, or detectable portion may be conjugated at the linker portion of the polypeptide or TGFβ binder (e.g., in a non-IDR linker), or at any other preferred site therein, such as its N-terminus or multimerization domain.

[0159] Production of polypeptides and TGFβ binders The polypeptides or TGFβ binders disclosed herein can be prepared by various methods well known to those skilled in the art, including recombinant DNA methods.

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

[0161] Various expression vectors and host cell lines known to those skilled in the art may 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 lines infected with baculovirus vectors; plant cell lines transformed with viral or bacterial expression vectors; and animal cell lines. Stable expression in mammalian cell lines may be used for long-term production of recombinant proteins in mammalian systems. For example, a nucleotide sequence capable of encoding any one of the polypeptide chains described herein may be transformed in a cell line using an expression vector and / or the same or a different vector containing endogenous expression elements and a selectable or visible marker gene. This 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 may be ligated into an expression vector. If the TGFβ-binding agent consists of different polypeptide chains (i.e., the first polypeptide and the second polypeptide are not identical), each of these polypeptide chains may be ligated into a separate vector or the same vector. According to this disclosure, the polypeptide chain of the TGFβ-binding agent may be encoded by a single vector or by separate vectors (e.g., a set of vectors). Cells are transformed with the desired vector or set of vectors.

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

[0163] Host cells containing nucleotide sequences can be cultured under conditions for transcription of the corresponding RNA (such as mRNA) and / or expression and secretion of polypeptides from the cell culture. In exemplary embodiments, an expression vector containing a nucleotide sequence capable of encoding the polypeptide chain described herein may be designed to include a signal sequence directed toward the secretion of polypeptides across 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 this disclosure can be manipulated using methods commonly known in the art to modify nucleotide sequences for a variety of purposes, including cloning, processing, and / or modification of gene product expression, but are not limited to these. Random fragmentation of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly can be used to manipulate nucleotide sequences. For example, oligonucleotide-mediated site-specific mutagenesis can be used to introduce mutations such as generating new restriction sites, altering glycosylation patterns, changing codon selection, and generating splice mutations. Codon-optimized nucleic acids encoding polypeptide chains described herein are encompassed by this disclosure.

[0165] Furthermore, host cell lines may be selected for their ability to regulate the expression of inserted sequences or process expressed polypeptides in the desired manner. Various host cells with specific cellular mechanisms and characteristic mechanisms for post-translational activity (e.g., CHO, HeLa, MDCK, HEK293, and W138) are commercially available and can be obtained from the American Type Culture Collection (ATCC) and may be selected to ensure appropriate modification and processing of expressed polypeptides.

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

[0167] Pharmaceutical composition Pharmaceutical compositions comprising polypeptides or TGFβ binders disclosed herein are also included by this disclosure. Pharmaceutical compositions generally comprise polypeptides or TGFβ binders disclosed herein and pharmaceutically acceptable carriers.

[0168] The preparation of pharmaceutical compositions may be carried out as is 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 may be put into a preferred dosage form together with one or more solid or liquid pharmaceutical carriers and / or additives (or auxiliary substances), and, if necessary, in combination with other pharmaceutically active compounds having therapeutic or prophylactic effects, and then it may be used as a pharmaceutical in human or veterinary medicine. Pharmaceuticals may also contain additives, many of which are known in the art, such as fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, fragrances, thickeners, diluents, buffers, solvents, solubilizers, agents for achieving depot effects, salts for altering osmotic pressure, coatings, or antioxidants.

[0169] The term "pharmaceutical composition" means a composition comprising a polypeptide or TGFβ binder described herein, and, depending on the method of administration and the nature of the dosage form, at least one pharmaceutically acceptable carrier, diluent, auxiliary, excipient, or vehicle, such as a preservative, filler, disintegrant, wetting agent, emulsifier, suspending agent, sweetener, flavoring agent, fragrance agent, antibacterial agent, antifungal agent, lubricant, and dispersant.

[0170] The term “pharmaceutically acceptable carrier” is used to mean any carrier, diluent, auxiliary agent, 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 thereof. Prevention of microbial action can be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride. Sustained absorption of injectable drug forms can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin. Non-limiting examples of suitable carriers, diluents, solvents, or vehicles include water, saline solutions, phosphate-buffered saline (PBS), gelatin, oils, alcohols, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and organic acid esters for injection, such as ethyl oleate. Non-limiting examples of excipients include lactose, lactose, sodium citrate, calcium carbonate, and dicalcium phosphate. Non-limiting examples of disintegrants include starch, alginic acid, and several 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 that, within reasonable medical judgment, it is suitable for use in contact with subjects, such as human and animal cells, without excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit-risk ratio.

[0172] Pharmacopoeia-acceptable carriers may include any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. 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 or inhalation administration. Pharmacopoeia-acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent may be used in the pharmaceutical compositions provided herein unless it is incompatible with the active compound. Auxiliary active compounds may also be incorporated into the compositions. For example, the pharmaceutical compositions provided herein may further include at least one further therapeutic agent, as will be further described below.

[0173] In one embodiment, the pharmaceutical compositions provided herein may be administered orally in the form of, for example, pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous solutions, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally in the form of, for example, suppositories.

[0174] In other embodiments, the pharmaceutical compositions provided herein may be administered parenterally, for example subcutaneously, intramuscularly, or intravenously, in the form of a solution for injection or infusion. Other preferred forms of administration include, for example, ointments, creams, tinctures, sprays, or transdermal therapeutic systems, for example, transdermal or topical administration, or inhalation in the form of a nasal spray or aerosol mixture, or, for example, microcapsules, implants, or wafers.

[0175] Pharmaceutical compositions must typically be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerin, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions by maintaining the required particle size, and by the use of surfactants. Often, it is preferable to include isotonic agents in the composition, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride. Sustained absorption of injectable compositions can be achieved by including absorption-delaying agents in the composition, such as monostearate salts and gelatin. Furthermore, compounds may be administered in sustained-release formulations, for example, in compositions containing sustained-release polymers. Compounds may be prepared with carriers that protect against rapid release, such as controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, polylactic acid, and polylactic acid-polyglycolic acid copolymer (PLG) may be used.

[0176] Many methods for preparing such formulations are generally known to those skilled in the art. Sterile injectable solutions can be prepared by incorporating, as needed, one or a combination of the components listed above, in a suitable solvent, an active compound such as a polypeptide or TGFβ binder provided herein, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, common preparation methods are vacuum drying and lyophilization, which yield powders of the active ingredient and any further desired components from the solution that has been previously sterile filtered. Compounds can also be formulated with one or more further compounds that increase their solubility.

[0177] For ease of administration and uniformity of dosage, it is often advantageous to formulate compositions (such as parenteral compositions) in dosage unit forms. The term "dosage unit" refers to a physically distinct unit suitable as a unit dose for human subjects and other animals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, along with a suitable pharmaceutical carrier. The specifications of the dosage unit forms of the present invention may vary and are determined by (a) the unique properties of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations specific to the art for formulating such therapeutic compounds for the prevention or treatment of TGFβ-related diseases or disorders, and directly therein. Dosages are further described below.

[0178] In one embodiment, a pharmaceutical composition is provided comprising an effective amount of a polypeptide and / or TGFβ binder described herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition for the treatment or prevention of fibrosis is provided, comprising a polypeptide or TGFβ binder described herein and a pharmaceutically acceptable carrier. In yet another embodiment, a pharmaceutical composition is provided for delaying the progression of cancer, inhibiting cancer invasion, e.g., malignant glial cell (MGC) invasion, inhibiting the growth, survival, spheroid formation and / or proliferation of cancer stem cells, inhibiting metastasis, inhibiting cancer recurrence, and / or overcoming cancer chemical resistance, comprising a polypeptide and / or TGFβ binder described herein and a pharmaceutically acceptable carrier. In yet another embodiment, a pharmaceutical composition for the treatment or prevention of bone marrow failure is provided.

[0179] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical carrier” includes, but is not limited to, 0.01 to 0.1 M or 0.05 M phosphate buffer or 0.8% physiological saline. Furthermore, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, e.g., olive oil, and organic acid esters for injection, e.g., ethyl oleate. Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions, or suspensions (including physiological saline and buffering media). Parenteral vehicles include sodium chloride solutions, ringer's dextrose, dextrose and sodium chloride, lactoringer's solution, or fixative oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements, e.g., ringer's dextrose-based ones. Other additives may also be present, such as preservatives, antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0180] For any compound, the therapeutically effective dose can first be estimated in a cell culture assay or in an animal model such as a mouse, rat, rabbit, dog, or pig. Animal models can also be used to determine the concentration range and route of administration. Such information can then be used to determine a useful dose and route for administration in humans. These techniques are well known to those skilled in the art, and the therapeutically effective dose refers to the amount of active ingredient that improves the symptoms or condition. Therapeutic efficacy and toxicity are determined by the ED. 50 (Therapeutic dose effective in 50% of the population) and LD 50 The dose (lethal to 50% of the population) may be determined by standard pharmaceutical procedures in cell cultures or in experimental animals, for example, by calculating and comparing the dose. Any of the pharmaceutical compositions described herein may be applied to any subject requiring treatment, including, but not limited to, mammals such as dogs, cats, cattle, horses, rabbits, monkeys, and especially humans.

[0181] The pharmaceutical compositions described herein may be administered by various routes, including, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal means.

[0182] How to use The polypeptides or TGFβ conjugates and their pharmaceutical compositions described herein are useful for the prevention or treatment of TGFβ-related diseases or conditions. Therefore, a method for the prevention or treatment of TGFβ-related diseases or conditions in a subject is provided, comprising administering a therapeutically effective amount of the polypeptides, TGFβ conjugates, or pharmaceutical compositions described herein. Polypeptides and TGFβ conjugates are generally administered in the form of pharmaceutical compositions. A subject may require such treatment, i.e., is suffering from, suspected of suffering from, or at risk of suffering from, a disease or condition related to 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 improved 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 the overexpression or overactivation of TGFβ ligands, particularly TGFβ1 and / or TGFβ3. In one embodiment, the TGFβ-related disease or condition is mediated by TGFβ1 and / or TGFβ3. In one embodiment, the disease or condition to be treated is mediated by TGFβ3. In another embodiment, the disease or condition to be treated is mediated by a combination of TGFβ1 and TGFβ3. As used herein, the term “improvement” means reducing, suppressing, mitigating, reducing, inhibiting, 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 this disclosure include, but are not limited to,: fibrosis (e.g., fibrosis, fibrous scarring, fibroproliferative disorders); cancer (e.g., malignant tumors, solid tumors, metastases); bone marrow failure (e.g., Schwachmann-Bodian-Diamond syndrome, Fanconi anemia); eye diseases; and connective tissue genetic disorders.

[0185] In some embodiments, the polypeptides or TGFβ-binding agents described herein are used to treat or prevent fibrosis, including, but not limited to, tissue and / or organ fibrosis, fibrous 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, hepatic fibrosis (e.g., cirrhosis), systemic sclerosis, scleroderma, cutaneous fibrosis, cardiac fibrosis, myelofibrosis, and myelofibrosis. In one particular embodiment, systemic sclerosis (SSc) is treated or prevented. In several other embodiments, scleroderma is treated or prevented. In several other embodiments, myelofibrosis (MF) is treated or prevented.

[0186] Systemic sclerosis (SSc, also known as scleroderma) is a severe wasting fibrosis. TGFβ has been shown to be important for promoting several pathological processes in SSc, including increased collagen deposition in the skin and lungs, as a potent profibrotic cytokine (Varga, J. and Abraham, D., 2007; Varga, J. and Whitfield, ML, 2009; Gabrielli, A. et al., 2009; Lafyatis, R., 2014; Allanore, Y. et al., 2015). SSc presents a significant unmet therapeutic challenge, with a mean life expectancy of approximately 11 years for newly diagnosed SSc patients (Mayes, MD et al., 2003). Recent clinical studies have demonstrated the driving force of fibrosis in human SSc patients by showing a dramatic reversal of fibrosis after inhibition of TGFβ with the neutralizing antibody fresolimmab (Rice, L M et al., 2015). This proof-of-principle clinical trial, along with extensive preclinical data demonstrating the importance of TGFβ in promoting fibrosis in SSc and other diseases, provides compelling rationale for the use of the TGFβ conjugates described herein for the treatment of SSc patients.

[0187] In myelofibrosis (MF), myelofibrosis is a characteristic feature 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, A et al., 2005), supporting the dual pathological role of TGFβ in MF, namely, myelofibrosis and promotion of myeloproliferation. Increased intraplatelet, peripheral blood mononuclear, and megakaryocyte-associated TGFβ has 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 compelling rationale for the use of TGFβ conjugates as disclosed herein for the treatment of MF patients.

[0188] Other exemplary embodiments of fibrosis that can be prevented or treated include, but are not limited to, interstitial lung disease; human fibrous lung disease (e.g., bronchiolitis obliterans, idiopathic pulmonary fibrosis, pulmonary fibrosis of known etiology, tumor interstitial in lung disease, systemic sclerosis affecting the lungs, Hermanski-Puddlak syndrome, coal miner's 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 cutaneous fibrosis; scleroderma; and systemic sclerosis. Specific forms of fibrosis that can be treated or prevented include those affecting any organ, tissue, or cell of the body, such as human tendon fibroblasts, kidneys, lungs, intestines, liver, heart, bone marrow, genitals, skin, and eyes. These conditions include, but are not limited to, cystic fibrosis, systemic sclerosis, chronic obstructive pulmonary disease (COPD), Dupuytren's contracture, glomerulonephritis, hepatic fibrosis, post-infarction myocardial fibrosis, restenosis, ophthalmic surgery-induced fibrosis, and scarring. Genetic disorders of connective tissue may also be treated, but are not limited to, Marfan syndrome (MFS) and osteogenesis imperfecta.

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

[0190] In one embodiment, the polypeptides or TGFβ-binding agents described herein are used for the treatment or prevention of fibroproliferative disorders. Fibroproliferative disorders are characterized by the proliferation of fibroblasts and the corresponding overexpression of extracellular matrix components such as fibronectin, laminin, and collagen.

[0191] In some embodiments, the polypeptides or TGFβ conjugates described herein are used to treat or prevent cancers including, but are not limited to, lung cancer, head and neck cancer, melanoma, colon cancer, pancreatic cancer, colorectal cancer, liver cancer, breast cancer, epithelial cancer, cholangiocarcinoma, and solid tumors. In some embodiments, the term “prevention” in relation to cancer may include preventing major tumor invasion or metastasis. In some embodiments, the term “treatment” in relation to cancer may include inhibiting TGFβ-mediated suppression of the immune response in the tumor microenvironment. With respect to solid tumors, the immunosuppressive role of TGFβ in the tumor microenvironment has been clearly demonstrated preclinically. Furthermore, recent clinical studies have shown that a lack of response to immune checkpoint inhibitors in bladder cancer patients is related to TGFβ signaling in the tumor microenvironment, supporting the idea that TGFβ suppresses antitumor immunity and suggesting that TGFβ inhibitors may have monotherapy activity in one tumor environment and act to enhance antitumor activity when combined with immune checkpoint inhibitors in other tumor environments.

[0192] In some embodiments, the polypeptide constructs or TGFβ binders described herein are used to treat or prevent diseases of abnormal cell proliferation and / or unregulated apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, head and neck cancer, skin 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, gastric cancer, gastrointestinal (stomach, colorectal and / or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, testicular cancer, hepatocellular carcinoma (liver and / or bile duct) cancer, primary or secondary central nervous system tumors. These include 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 lymphoma, spinal axial tumors, brainstem gliomas, pituitary adenomas, adrenocortical carcinomas, gallbladder cancer, spleen cancer, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or combinations thereof.

[0193] In one embodiment, the polypeptide constructs or TGFβ binders described herein are used to treat or prevent diseases or disorders selected from the group consisting of bladder cancer, brain tumors, 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 splenic cancer.

[0194] In one embodiment, the polypeptide constructs or TGFβ binders described herein are used to treat or prevent diseases or disorders that are hematological cancers such as leukemia, lymphoma, or myeloma. In one embodiment, cancers include Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular central cell lymphoma, transformed lymphoma, moderately differentiated lymphocytic lymphoma, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), central cell lymphoma, diffuse dissecting cell lymphoma (DSCCL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma, mantle zone lymphoma 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 lymphoma), acute mixed leukemia, chronic myeloid lymphoma, chronic myeloid leukemia (CML), and chronic monocytic leukemia. In a particular embodiment, the disease or disorder is myeloma. In a particular embodiment, the disease or disorder is myelodysplastic syndrome (MDS). In another particular embodiment, the disease or disorder is acute myeloid leukemia (AML). In another specific embodiment, the disease or disorder is chronic lymphocytic leukemia (CLL). In yet another specific 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 diseases or disorders that are solid malignancies. In some embodiments, the solid malignancies are selected from the group consisting of carcinoma, adenocarcinoma, adrenocortical carcinoma, colonic adenocarcinoma, colorectal adenocarcinoma, colorectal cancer, ductal cell carcinoma, lung cancer, thyroid cancer, nasopharyngeal cancer, melanoma, non-melanoma skin cancer, and lung cancer.

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

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

[0198] In certain embodiments, the immune checkpoint inhibitor administered in combination with the polypeptides or TGFβ conjugates 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, and B7 These include T cell immune receptors with H3, B7-H4, Ig, and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9), V-domain Ig inhibitors of T cell activation (VISTA), glucocorticoid-induced tumor necrosis factor receptor-associated (GITR) proteins, herpesvirus entry mediators (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, or CGEN-15092. A summary of these checkpoint regulators and 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 may 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 an equivalent foreign agency for the treatment of cancer or disease caused by a pathogen.

[0200] In certain embodiments, an immune checkpoint inhibitor is an antibody that binds to an immune checkpoint and inhibits its activity. Antibodies that may 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 (e.g., 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 its activity (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, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, and tripalimab.

[0203] In certain embodiments, the monoclonal antibody is nivolumab, pidilizumab, MEDI0680, or pembrolizumab. In further specific embodiments, the monoclonal antibody is nivolumab. In another specific embodiment, the immune checkpoint inhibitor that is a PD-1 inhibitor is AMP-224. In another specific embodiment, the immune checkpoint inhibitor that is a PD-1 inhibitor is pidilizumab. In another specific embodiment, the immune checkpoint inhibitor that is a PD-1 inhibitor is pembrolizumab. In another specific embodiment, the immune checkpoint inhibitor that is a PD-1 inhibitor is MEDI0680. In another specific embodiment, the immune checkpoint inhibitor that is a PD-1 inhibitor is STI-A1110. In another specific embodiment, the immune checkpoint inhibitor that is a PD-1 inhibitor is TSR-042. In another specific embodiment, the immune checkpoint inhibitor that is a PD-1 inhibitor 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 its 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 a PD-L1 inhibitor is RG7446. In yet another specific embodiment, the immune checkpoint inhibitor that is a PD-L1 inhibitor is STI-A1010.

[0207] In some embodiments, the immune checkpoint inhibitor is a CTLA4 inhibitor (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 the polypeptides or TGFβ conjugates described herein include, but are not limited to: OPDIVO® (nivolumab); YERVOY® (ipilimumab); relatrimab; linrhodostat; EMPLICITI® (erotuzumab); BMS-986258; BMS-986315; BMS-986207; BMS-986249; and BMS-986218.

[0210] The PD-1 inhibitors useful in the combinations described herein include any molecule capable of inhibiting, blocking, eliminating, or interfering with the activity or expression of PD-1. In particular, anti-PD-1 inhibitors may be small molecule compounds, nucleic acids, polypeptides, antibodies, peptide bodies, diabodies, minibodies, single-domain antibodies or nanobodies, single-stranded 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 with a molecular weight of less than about 1000 Da). In other embodiments, the PD-1 inhibitors useful in the combinations described herein include nucleic acids and polypeptides.

[0211] In one embodiment, a method is provided for preventing or inhibiting cancer recurrence after treatment, for example, after drug therapy or surgical resection. In one embodiment, a method is provided for slowing the progression of cancer, wherein cancer regrowth is delayed by more than 30%, more than 50%, or more than 70%, and / or the survival time of the affected subject is increased. Further provided is a method for enhancing the effectiveness of a cancer treatment for the treatment of cancer, selected from the group including resection, chemotherapy, radiotherapy, immunotherapy, and / or gene therapy, comprising administering a polypeptide or TGFβ conjugate described herein, and administering the cancer treatment simultaneously, separately, or sequentially. As used herein, the term “enhancing the effectiveness of a cancer treatment” refers to an improvement of conventional cancer treatment, including a reduction in the amount of anticancer composition applied in conventional cancer treatment, for example, the amount of radiation in radiotherapy, the amount of chemotherapeutic drug in chemotherapy, the amount of immunotherapy drug in immunotherapy, or the amount of vector in gene therapy, and / or an increase in the effectiveness of conventional treatments and anticancer compositions when applied in conventional doses or amounts in conventional cancer treatment. In one embodiment, improving the effectiveness of cancer treatment means increasing the survival rate of those receiving treatment.

[0212] In certain embodiments, the polypeptides or TGFβ conjugates described herein are used to treat or prevent bone marrow failure in subjects, for example, humans who have or are at risk of developing bone marrow failure. Exemplary types of bone marrow failure include, but are not limited to, SDS (also known as Schwachmann-Bodian-Diamond syndrome or SBDS), Fanconi anemia (FA), congenital dyskeratosis (DC), congenital anegakaryotic thrombocytopenia (CAMT), Blackfan-Diamond anemia (BDA), and reticular dysplasia (RD). Patients with Schwachmann-Diamond syndrome (SDS) have an increased risk of bone marrow failure, exocrine pancreatic insufficiency, bone malformations, and acute myeloid leukemia. In certain embodiments, the polypeptides or TGFβ conjugates described herein are used to treat or prevent Fanconi anemia (FA) in subjects. In several other embodiments, the polypeptides or TGFβ conjugates described herein are used to treat or prevent Schwachmann-Diamond syndrome (SDS) in subjects.

[0213] Fanconi anemia (FA) is the most common hereditary bone marrow failure syndrome. FA patients develop bone marrow failure within the first 10 years of life due to a reduction in hematopoietic stem and progenitor cells (HSPCs). FA is caused by a mutation in one of the 19 Fanconi anemia complementation (FANC) genes (whose products cooperate in the FA / BRCA DNA repair pathway). Bone marrow failure in FA may be, in part, a direct or indirect consequence of hyperactivation of growth inhibitory pathways induced by genotoxic stress. Standard TGFβ pathway-mediated growth inhibition of hematopoietic stem cells (HSCs) has recently been identified as a cause of bone marrow failure in FA (Rio, P. and Bueren, JA, 2016; Zhang, H. et al., 2016). Schwachmann-Diamond syndrome (SDS) is another rare bone marrow failure syndrome caused by a mutation in the SBDS gene (Boocock, G 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 the TGFβ-binding agents described herein for the treatment of myelodeficiency syndromes.

[0214] In one embodiment, a method is provided for treating or preventing bone marrow failure, such as SDS, comprising administering an effective amount of the polypeptide or TGFβ-binding agent according to the Disclosure to a subject in need. 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., showing an absolute neutrophil count <1500 / mL), anemia, and thrombocytopenia (e.g., 50,000 cells / mm³). 3 The group consists of the following: (showing a platelet count of less than 1), exocrine pancreatic insufficiency, growth retardation, chronic steatorrhea, metaphyseal dysplasia, myelodysplasia, megakaryocyte dysplasia, erythrodysplasia, acute myeloid leukemia (AML), and systemic osteopenia. For further information on bone marrow failure, see, for example, International Publication No. 2016 / 138300 and International Publication No. 2019 / 018662.

[0215] As used herein, the terms “effective dose” and “therapeutic effective dose” are used synonymously to refer to the amount or dose of a compound or composition that, upon single or multiple dose administration to a subject, provides a desired effect in the subject being treated (e.g., a desired biological or pharmacological response to improve, reduce, or prevent a disease, disorder, or condition). In some embodiments, the effective dose is the 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 some embodiments, the effective dose is the amount or dose of a compound or composition that inhibits the activity of one or more TGFβ (e.g., TGFβ1 and / or TGFβ3) in a subject, as described herein.

[0216] The terms “inhibit” or “to inhibit” are used herein in general to mean reducing, slowing, limiting, delaying, suppressing, blocking, neutralizing, interfering with, or preventing a process, for example, reducing or delaying the growth, metastasis, or survival of TGFβ-related diseases or conditions such as fibrosis, cancer or tumors, or bone marrow failure.

[0217] In the spirit of this disclosure, the terms “to treat” or “treatment” refer to both therapeutic actions and preventive or protective measures, the purpose of which is to improve the targeted disease or condition. Persons requiring treatment include those already suffering from the disorder, as well as those susceptible to the disorder or those for whom the disorder should be prevented. In certain embodiments, “to 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, “to treat” or “treatment” refers to inhibiting or improving a disease or condition, physically (e.g., stabilizing recognizable symptoms), physiologically (e.g., stabilizing physical parameters), or both. In some embodiments, “to treat” or “treatment” refers to delaying the onset (or recurrence) of a disease or condition. The terms “to treat” or “treatment” can mean any measure of success in treating or improving a disease or condition, including any objective or subjective parameters, such as reducing; alleviating; reducing symptoms or making the disease or condition more tolerable to the subject; improving the physical or mental health of the subject, such as reducing pain or discomfort experienced by the patient; and, in some circumstances, further improving at least one clinical parameter of the disease or condition.

[0218] In some embodiments of this disclosure, “to treat” means to neutralize the biological activity of excess TGFβ. This may be determined by preferred clinical variables of improvement; for example, by pathological evaluation of the effect on immunosuppression or prophylaxis of fibrosis and / or fibrosis; by direct inhibition of TGFβ signaling; or by another measure preferred to the disease or condition being treated.

[0219] As used herein, “prevent” or “prevent” is intended to mean at least a reduction in the likelihood, risk, or susceptibility to the disease or disorder (i.e., preventing the development of at least one clinical symptom of the disease in a patient who is predisposed to or at risk of developing the disease but has not yet developed or shown any symptoms of the disease). The term “prevent” or “prevent” is also used to describe the administration of the compounds or compositions described herein to subjects at risk of (or susceptible to) such disease or condition. Subjects suitable for treatment for the prevention of disease or condition include individuals at risk of disease or condition but not showing symptoms, as well as patients currently showing symptoms. In some embodiments, “prevent” or “prevent” is used to describe the administration of the compounds or compositions described herein to subjects who have been diagnosed with or are being treated for disease or condition and are at risk of recurrence of disease or condition.

[0220] In some embodiments, treatment or prevention falls within the scope of the present invention if there is a measurable difference between the results of subjects treated with the TGFβ conjugates, compositions, and methods provided herein, compared to a placebo group, a member of a historical control, or between subsequent trials given to the same subjects.

[0221] The term "subject" includes organisms with TGFβ-related diseases or conditions, or organisms that are susceptible to or at risk of developing them. Examples of subjects include mammals, such as humans, monkeys, cattle, rabbits, sheep, goats, pigs, dogs, cats, rats, mice, and their transgenic species. The term "subject" generally includes animals susceptible to conditions characterized by TGFβ-related diseases or conditions, such as fibrosis or cancer, such as mammals, such as primates, such as humans. Animals may also be animal models for the disorder, such as mouse models or xenograft recipients. In some embodiments, the subject is human.

[0222] The respective doses of the TGFβ binder for use in the compositions provided herein are not particularly limited. Exemplary doses include amounts of the compound in milligrams or micrograms per kilogram of the weight of the subject or sample (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 doses 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 include, for example, daily or twice daily, or less or more.

[0223] In one embodiment, the adult dose range is generally 0.005 mg to 10 g / day. Polypeptides, TGFβ binders, and compositions thereof may be provided in unit dosage forms, for example, as units or a plurality of units effective at such doses, for example, in units containing 5 mg to 500 mg, usually about 10 mg to 200 mg. Dosage units include, 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). This may include polypeptides, TGFβ binders, or compositions described herein in amounts of 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.

[0224] It should be understood that the effective dose of polypeptides or TGFβ conjugates for the therapeutic treatment of a disease or condition varies 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 dose and dosage plan. It should be understood that the dosage or amount of polypeptides or TGFβ conjugates used alone or in combination with one or more active compounds administered should be adapted to the individual circumstances to obtain the optimal effect, as is customary and individualized. Dosage and dosage plans are within the scope of the skills of those skilled in the art, and the appropriate dose depends on several factors within the scope of 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 medication and administration plan depends on the nature and severity of the disorder being treated, as well as the sex, age, weight and individual responsiveness of the person or animal being treated, the efficacy and duration of action of the compounds 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 may be administered using known procedures in doses and over periods effective to achieve the desired objective. The dosage plan may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the circumstances of the treatment situation. In some embodiments, the compound or composition is administered in an effective dose sufficient to prevent or treat fibrosis in a subject.

[0226] The respective routes of administration for each TGFβ binder used in the compositions provided herein are not particularly limited. Polypeptides, TGFβ binders, or compositions thereof may be administered by any preferred route or means, for example, by inhalation, injection, infusion, or other such routes known in the art, for example, by oral, parenteral, intravenous, intraperitoneal, intramuscular, subcutaneous, sublingual, topical, or nasal administration. In certain embodiments, polypeptides, TGFβ binders, or compositions thereof are administered by injection or infusion, for example, but not limited to, intravenous, intraperitoneal, intramuscular, or subcutaneous.

[0227] In one embodiment relating to the method of the present disclosure, one or more symptoms of the onset or progression of a TGFβ-related disease or condition are reduced 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% in the subject.

[0228] In one embodiment relating to the method of the present disclosure, fibrous symptoms are mitigated in the subject. For example, a polypeptide, a TGFβ binder, or a composition can reduce fibrosis, fibrous scarring, or skin thickening in the subject 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%.

[0229] In certain embodiments of the methods of the present disclosure, differentiation of fibroblasts into myofibroblasts is inhibited in a subject, for example, 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%.

[0230] In certain embodiments of the methods of the present disclosure, tumor growth and / or metastasis is inhibited in a subject, for example, 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%.

[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 bone marrow of a subject, for example, 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%.

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

[0233] In certain embodiments of the methods of the present disclosure, a favorable response in dermal fibrosis associated with systemic sclerosis is determined by improvement in the modified Rodnan skin score (MRSS). For example, the MRSS can improve by at least 5%, such as 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% in a subject.

[0234] In certain embodiments, in myelodysplastic disorders including myelofibrosis, a favorable response is manifested by improvement in anemia (e.g., a transfusion-independent patient showing an increase in hemoglobin level, and a transfusion-dependent patient becomes transfusion-independent).

[0235] In certain embodiments, a polypeptide or TGFβ binder can be conjugated to a therapeutic moiety as described herein. Desirable therapeutic moieties can be selected for their ability to prevent or treat the same disease or condition targeted by the polypeptide or TGFβ binder.

[0236] In certain embodiments, a method for the prevention or treatment of a TGFβ-related disease or condition in a subject is provided by administering to the subject an effective amount of a polypeptide or TGFβ binder as described herein such that the TGFβ-related disease or condition is prevented or treated in the subject.

[0237] In one embodiment, a method is provided for inhibiting TGFβ in a subject by administering an effective amount of a polypeptide or TGFβ binder described herein so as to inhibit TGFβ in the subject. In one such embodiment, a method is provided for inhibiting TGFβ3 in a subject by administering an effective amount of a polypeptide or TGFβ binder described herein so as to inhibit TGFβ3 in the subject. In one such embodiment, a method is provided for inhibiting TGFβ3 and TGFβ1 in a subject by administering an effective amount of a polypeptide or TGFβ binder described herein so as to inhibit TGFβ3 and TGFβ1 in the subject.

[0238] In one embodiment, a method is 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 conjugate is administered in combination with one or more further therapies or therapeutic agents. These further therapies or therapeutic agents may be administered before, after, or concurrently with the administration of the polypeptide, TGFβ conjugate, or composition described herein. In some embodiments, the further therapies or therapeutic agents are formulated together with the polypeptide or TGFβ conjugate in the same composition. In other embodiments, the further therapies or therapeutic agents are administered separately. Examples of further therapies and therapeutic agents include, but are not limited to, antifibrotic agents; anticancer agents; and other TGFβ conjugates or inhibitors, such as antibodies, antibody fragments, antigen-binding fragments, and soluble TGFβ ligand traps. In one embodiment, the further therapeutic agent is nintedanib (marketed under the trademarks Ofev® and Vargatef®). In one embodiment, the further therapeutic agent is pirfenidone. In one embodiment, the further therapeutic agent is an immune checkpoint inhibitor.

[0240] Alternatively, in one embodiment, the polypeptide or TGFβ conjugate may be conjugated with a detectable or diagnostic portion useful for tracking cells or tissues expressing TGFβ. In one such embodiment, a method is provided for diagnosing a TGFβ-related disease or condition, comprising administering the polypeptide or TGFβ conjugate of the Disclosure conjugated with a detectable or diagnostic portion to a target, and detecting the polypeptide or TGFβ conjugate so as to diagnose a disease or condition associated with TGFβ (e.g., overexpression of TGFβ1 and / or TGFβ3).

[0241] kit According to this disclosure, the polypeptides, TGFβ binders, and pharmaceutical compositions described herein may be assembled into kits or pharmaceutical systems for use in treating or preventing TGFβ-related diseases or conditions. The kit or pharmaceutical system may include one or more containers, such as vials, tubes, ampoules, or bottles, containing the polypeptides, TGFβ binders, or pharmaceutical compositions (e.g., packaging, boxes, cardboard, vials, etc.). Further kit components may include acids, bases, buffers, inorganic salts, solvents, antioxidants, preservatives, or metal chelating agents. Further kit components may exist as pure compositions or as aqueous or organic solutions incorporating one or more further kit components. Any or all of the kit components may optionally further include buffers. The kit may also include tools for administration, such as needles or syringes. The kit may be used in accordance with the methods described herein, and in such a manner may include instructions for use. The kit may also include instructions for the administration and use of the polypeptides, TGFβ binders, or pharmaceutical compositions.

[0242] The array is shown in the table of arrays in Table 2. In Table 2, the N-terminal IDR in the human TGFβRII extracellular domain and the sequences derived therefrom are shown underlined; the C-terminal IDR in the human TGFβRII extracellular domain and the sequences derived therefrom are shown double-underlined; the Gly-Ser linker region is shown italicized and underlined; and the multimerization domain is shown italicized and in bold.

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

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

[0244] Unless otherwise defined or specifically indicated in the context, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. It should be understood that any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention.

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

[0246] Previously, TGFβRII extracellular domain fusion molecules were described (International Publication No. 2017 / 037634, International Publication No. 2018 / 158727). Such fusion molecules contain two structured ligand-binding domains (SEQ ID NO: 2) linked side-by-side (head-to-tail) by linkers derived from IDR and its variants. As reported in International Publication No. 2018 / 158727, such polypeptide constructs have demonstrated at least 600-fold higher inhibitory efficacy than similar constructs having only a single ligand-binding domain (see International Publication No. 2018 / 158727, pp. 2, pp. 27-31, where it is stated that "a [construct] comprising a (TβRII-ECD)-(TβRIIECD) doublet in which the first region is linked at its C-terminus by an antibody constant domain inhibits TGFβ activity at at least 600-fold higher efficacy than an equivalent construct having a single TβRII-ECD linked at its C-terminus by an antibody constant domain (i.e., in the absence of a second ECD, also referred to herein as a singlet)"). Furthermore, such “doublet” polypeptide constructs (referred to as “T22d35-Fc”) can 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, in the case of the T22d35-Fc-IgG1-v1(CC) mutant, the neutralizing efficacy against TGFβ1 and TGFβ3 was described as being very similar, but this efficacy 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 International Publication No. 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 binders, the inventors selected an exemplary fusion molecule, "T22d35-Fc-IgG1-v1(CC)," for further investigation. In this fusion molecule (SEQ ID NO: 6 in this specification; SEQ ID NO: 14 in International Publication No. 2018 / 158727), the linker between the two structured ligand-binding domains is a fusion of the C-terminal 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, the inventors used the A549 IL-11 release assay to determine the inhibitory efficacy (IC) of T22d35-Fc-IgG1-v1(CC) fusion against various isoforms of TGFβ, particularly TGFβ1 and TGFβ3. 50 A more detailed characterization of ) was performed. The A549 IL-11 release assay was carried out substantially as described (International Publication No. 2018 / 158727 brochure), and is described in more detail below in Example 2.

[0249] By analyzing the inhibitory effect in more detail, we can achieve higher accuracy in IC 50 To obtain the value, the inventors performed an IC in the A549 IL-11 release assay. 50 We tested higher concentrations of TGFβ (more points on the curve) that were close to the target value. Representative results are shown in Figures 4A and 4B, and the results from the average of six experiments are shown in Table 3. The inventors found an average IC of 2.89 pM ± 0.16 relative to TGFβ1. 50 The value was determined, and this is consistent with previous reports (International Publication No. 2018 / 158727 pamphlet). However, for TGFβ3, the average IC 50The value was 8.64 pM ± 0.43, which is considerably higher than expected (i.e., lower inhibitory efficacy than expected). The results showed approximately 3 to 3.5 times higher inhibitory efficacy against TGFβ1 compared to TGFβ3, indicating preferential inhibition or neutralization of the TGFβ1 ligand by T22d35-Fc-IgG1-v1(CC) fusion. It should be noted that, as previously reported (International Publication No. 2018 / 158727; Figure 11), the inhibitory efficacy against TGFβ2 was considerably lower than that against TGFβ1 and TGFβ3. [Table 3]

[0250] These results are consistent with the description in International Publication No. 2018 / 158727, which suggests higher inhibition or neutralization of TGFβ1 compared to TGFβ3 (see, for example, page 3, lines 26-28 of International Publication No. 2018 / 158727, where it is stated that "the Fc-doublet (T22d35-Fc) shows at least 970-fold higher potency enhancement against TGFβ1 and at least 240-fold higher potency enhancement against TGFβ3 compared to the non-Fc-fused ECD doublet").

[0251] To better understand the underlying mechanism of the preferential inhibition of TGFβ1 ligand compared to TGFβ3 ligand by the previous fusion construct, we then performed a structural comparison of the two ligand isoforms. It should be noted that the ligand-binding domain of the previous fusion construct interacts with substantially the same epitope in 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 TGFβ1 ligand. Overlays of the monomer structures of TGFβ1 (blue) and TGFβ3 (green) are shown in Figure 2A. Overlays of the TGFβ1 and TGFβ3 dimers are shown in Figure 2B, as observed in Protein Databank (PDB) IDs 3KFD and 1KTZ, respectively (Protein Databank (PDB) IDs for the structures are 3KFD and 1KTZ, respectively). TGFβ1 and TGFβ3 are remarkably similar at the amino acid sequence level, and both monomers adopt similar elongated cysteine ​​knot folding (Figure 2A). However, 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 evident that each ligand isoform has a specific range of dimerization angles. The range of dimerization angles affects the overall shape, spatial extent, and density of the dimer molecule. This difference in the shapes of the TGFβ1 and TGFβ3 dimers may result in preferential neutralization of TGFβ1 over TGFβ3 by the previous fusion construct; that is, a specific spacing of the ligand-binding domains in the fusion construct may have resulted in preferential interaction (preferential binding force) 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), Sequence ID No. 6) bound to a TGFβ ligand exhibiting a second ligand-binding domain, a second linker, and an Fc region. This model is shown herein to illustrate the effect of a short (10 amino acid) second linker. The green line indicates that the linker / spacer length is at least 25 angstroms shorter to allow ligand binding between the bound 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 structure, which is about 20 Å shorter than the optimal linker length calculated using molecular modeling. Therefore, in this fusion construct, the second ligand-binding domain is sterically constrained from accommodating the TGFβ dimer.

[0253] It should be noted that the structured ligand-binding domain is a fusion construct that contributes to the interaction interface with TGFβ ligands, such as TGFβ1 and TGFβ3, and that the linker region does not directly interact with the binding ligand. However, considering the differences in dimer structures with respect to TGFβ1 and TGFβ3 and the structural constraints imposed by the linker region, our analysis suggests that modifying the linker region may affect the binding properties of the TGFβ binder in order to alter its ligand-binding specificity. In particular, shortening of the first linker region between ligand-binding domains and lengthening of the second linker region between the second ligand-binding domain and the polymerization domain would relax steric and structural constraints in order to alter the relative inhibitory efficacy against TGFβ1 and TGFβ3 ligands.

[0254] Example 2. Design and characterization of a modified TGFβ binder with isoform specificity. The inventors generated molecules with various linker sequences and lengths to investigate whether modifying the linker can specifically affect the isoform specificity and inhibitory efficacy of TGFβ ligands, particularly TGFβ1 and TGFβ3. Based on structural analysis, the inventors focused on molecules having a shortened linker moiety (first linker moiety) between two ligand-binding domains and an elongated linker moiety (second linker moiety) between the second ligand-binding domain and the polymerization domain. The inventors' objective was to provide the binder with beneficial therapeutic properties for specific disease indications, while maintaining good overall inhibitory efficacy, and exhibiting lower-priority inhibition of TGFβ1 than TGFβ3 (i.e., lower TGFβ3:TGFβ1 IC2). 50 The objective was to design a TGFβ-binding agent with a specific ratio.

[0255] A series of TGFβ-binding agents with linkers of varying lengths and sequences were designed. Representative fusion protein structures are summarized in Table 4. Their sequences are shown in Table 2.

[0256] The test conjugate is a homodimer, and each polypeptide in the homodimer contains 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 moiety between the two ligand-binding domains (SEQ ID NO: 8 or 12); a second ligand-binding domain and multimerization domain (SEQ ID NO: 4, 9, 11, or 15); and a 10, 16, or 30-amino acid second linker moiety between the hIgG1Fc(CC) multimerization domain (SEQ ID NO: 49). The T22d35-Fc-IgG1-v1(CC) fusion (SEQ ID NO: 6; International Publication No. 2018 / 158727 brochure) 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 sequence numbers 81, 84, 87, 89, and 92, respectively (Table 2). [Table 4]

[0257] Recombinant fusion molecule generation and purification. All constructs contained the secretion 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). The cDNA was cloned into EcoR1 (5' end) and BamH1 (3' end) of the pTT5 mammalian expression plasmid vector (Durocher et al., 2002). Representative cDNA sequences used for the expression of the fusion proteins are shown in Table 2 (SEQ ID NOs: 106-109, used for the expression of p61, p96, p101, and p128, respectively). The signal peptide was cleaved intracellularly during expression and was not included in the purified fusion protein.

[0258] The fusion protein was expressed by transient transfection of Chinese hamster ovary (CHO). Briefly, each expression plasmid encoding the fusion protein was transfected into 100 mL of CHO-3E7 cell culture 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, which was mixed with polyethyleneimine-pro (Polyplus) in a ratio of 1:4. 24 hours after transfection, 1% Tryptone Nl feed (TekniScience Inc.) and 0.5 mM VPA (Sigma) were added, and the incubator temperature was reduced to 32°C and 5% CO2. This was done to promote the production and secretion of the fusion protein and was maintained for 4 days post-transfection (dpt), after which the culture was collected. On day 4, the collected supernatant was filtered (0.2 μm) and purified using AKTA pure 25L (GE). The supernatant was packed into a MabSelect PrismA protein A column and purified by affinity chromatography. Next, the column was 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. Then, the fraction was buffered using a HiPrep 26 / 10 desalting column (GE) with formulation buffer (20 mM L-histidine, 100 mM NaCl, pH 7).

[0260] Figures 3A and 3B show polyacrylamide gel electrophoresis analysis of purified proteins 61, 96, 101, 107, and 112 (see lanes labeled 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. Proteins (P) were electrophoresed on a 12% bis-trisacrylamide gel (NuPAGE® 12% bis-trisprotein gel, Cat# NP0341BOX, Life Technologies) under both non-reducing and reducing conditions. These fusion proteins are tetravalent, homodimeric TGFβ-binding agents, each containing two polypeptide chains (i.e., they are homodimers of two polypeptide chains, with the first and second polypeptides being identical, and each polypeptide containing two ligand-binding domains). The two polypeptide chains are dimerized via disulfide crosslinks containing one or more cysteine ​​residues in their multimerization domains, as evidenced by the size difference under reducing versus non-reducing conditions.

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

[0262] The TGFβ neutralizing efficacy of the purified fusion protein was determined using cell-based signaling assays, particularly an A549 cell / IL-11 release assay using colorimetric ELISA. In short, human A549 lung cancer cells (ATCC-CCL-185, Cedarlane Burlington ON) were used in a 96-well plate (5 × 10⁶). 3 Cells were seeded in individual cells / well and incubated in a humidified atmosphere at 37°C with 5% CO2. The following day, 10 pM TGFβ in complete medium was incubated at room temperature (RT) for 30 minutes in the absence or presence of increasing concentrations of the fusion protein before being added to the cells. After 24 hours (h) of incubation, the prepared 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 for TGFβ-mediated signaling: relative IL-11 release after TGFβ treatment is a measure of TGFβ activity. A decrease in IL-11 release after the addition of the test fusion protein indicates TGFβ activity. The data were plotted and analyzed using Prism8 (GraphPad, San Diego), and a dose-response curve was generated from absorbance values ​​using a 4-parameter fit logistic model (absorbance vs. concentration). The values ​​were then normalized relative to a positive control (TGFβ treatment in the absence of any inhibitor).

[0263] Results from representative experimental sets are shown in Figures 4A and 4B, comparing the inhibitory efficacy of proteins 61, 96, 101, 107, and 112 against TGFβ1 and TGFβ3 with that of a positive control (SEQ ID NO: 6). The highest efficacy was observed in the positive control. However, the positive control also showed the highest IC for TGFβ3:TGFβ1. 50 The ratio was 3.41 in this experiment. In contrast, TGFβ3:TGFβ1 IC2 of proteins 61, 96, 101, 107, and 112 50 The ratios in this experiment were 1.66, 1.72, 1.51, 1.24, and 1.95, respectively (Figures 4A-4B). [Table 5]

[0264] The results showed that modifications to the linker region, particularly shortening of the first linker region and lengthening of the second linker region, were associated with TGFβ3:TGFβ1 IC 50 This shows a significant reduction in the ratio, indicating a decrease in preferential inhibition of TGFβ1 while still maintaining inhibitory efficacy within the picomolar range.

[0265] Representative binders for another set are 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] The neutralizing efficacy of proteins 113, 115, and 116 compared to the positive control (SEQ ID NO: 6) is shown in representative experimental sets, as presented in Figures 6A-6B. The results are also shown in Table 5. The results indicate that the inhibitory efficacy of these proteins was equivalent to that of the control against TGFβ1, but significantly higher against TGFβ3, and significantly lower against TGFβ3:TGFβ1 IC. 50 This indicates that a ratio was brought about.

[0267] In representative experimental sets shown in Figures 7A-7B, the neutralizing efficacy of proteins 101, 129, and 130 compared to the positive control (SEQ ID NO: 6) is demonstrated. The results are also shown in Table 5. The results show that the inhibitory efficacy of these proteins was lower against TGFβ1 compared to the control, and approximately the same (P101, P130) or lower (P129) against TGFβ3, with a significantly lower TGFβ3:TGFβ1 IC2. 50 This indicates that a ratio was brought about.

[0268] In representative experimental sets shown in Figures 8A-8B, the neutralizing efficacy of proteins 101, 131, 132, and 133 compared to the positive control (SEQ ID NO: 6) is demonstrated. The results are also shown in Table 5. The results show that the inhibitory efficacy of these proteins was lower against TGFβ1 compared to the control, and approximately the same as or higher against TGFβ3, with a significantly lower TGFβ3:TGFβ1 IC2. 50 This indicates that a ratio was brought about.

[0269] In representative experimental sets shown in Figures 9A-9B, the neutralizing efficacy of proteins 96, 134, and 135 compared to the positive control (SEQ ID NO: 6) is demonstrated. The results are also shown in Table 5. The results show that the inhibitory efficacy of these proteins was reduced more against TGFβ1 than against TGFβ3 compared to the control, thereby reducing the TGFβ3:TGFβ1 IC2 50 The ratio was significantly lower than that of the control. Comparing proteins 134 and 135 with protein 96, the results showed that replacing either the first or second linker with a Gly-Ser linker resulted in a lower TGFβ3:TGFβ1 IC2. 50 This demonstrates a significant reduction in inhibitory activity against both TGFβ1 and TGFβ3 while maintaining the ratio.

[0270] The multimerization domain does not affect TGFβ isoform specificity. If TGFβ binders have the same TGFβ binding region but differ only in the multimerization domain, they were tested to investigate how the multimerization domain affects the relative inhibitory efficacy against TGFβ1 and TGFβ3 isoforms. The results are shown in Figures 10A–10B (showing the neutralizing efficacy of proteins 101 and 128 compared to a positive control (SEQ ID NO: 6)) and Table 5. Figures 10A–10B show one representative assay; the averaged results from several assays are shown in Table 5. The results indicate that, as expected, the change in the multimerization domain from IgG1 (protein 101) to IgG4 (protein 128) did not have a significant effect on the inhibitory efficacy against TGFβ1 and TGFβ3. The same results were obtained for proteins 61 and 96, and proteins 113 and 115 (Table 5).

[0271] Neutralization of TGFβ2 isoforms. The inventors also tested whether the relative inhibition of TGFβ2 was affected by the TGFβ binding agents provided herein compared to controls. A representative set of experiments is shown in Figure 11. As shown in Figure 11 for proteins 61, 96, and 101, the neutralizing potency for TGFβ2 isoforms was more than 1000 times lower than for TGFβ1 and TGFβ3 isoforms (in other words, IC). 50 However, it was more than 1000 times higher), which is the same as the control. The results demonstrate that homogenization or changes in relative inhibitory efficacy against TGFβ1 and TGFβ3 isoforms did not have a significant effect on the very low inhibition of the TGFβ2 isoform.

[0272] In summary, the results reported herein indicate 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 is effective for TGFβ3:TGFβ1 IC2 for these binders. 50It was shown to be effective in reducing the ratio and, in some cases, nearly homogenizing the inhibitory efficacy against the two isoforms. In some cases, TGFβ3:TGFβ1 IC 50 It should be noted that the ratio was reduced by increasing the inhibitory efficacy against TGFβ3 without adversely affecting the efficacy against TGFβ1 (e.g., proteins 113, 115, 116). In other cases, this ratio was reduced primarily by decreasing the inhibitory efficacy against TGFβ1 without adversely affecting the efficacy against TGFβ3 (e.g., proteins 61, 96, 101, 107, 128), although in some cases a slight decrease in TGFβ3 efficacy was also observed. Nevertheless, all binders maintained significantly higher inhibitory efficacy against both TGFβ1 and TGFβ3 than against TGFβ2, which is consistent with their potential use as therapeutic agents for the treatment of TGFβ-related disorders, particularly those mediated by TGFβ3.

[0273] References Akhurst,RJand Hata,A.Targeting the TGFβ signaling pathway in disease.Nat.Rev.Drug Discov.2012;11:790-811. Akhurst,RJTargeting TGF-β signaling for therapeutic gain.Cold Spring Harbor Perspect.Biol.2017;3;9:a022301. Allanore, Y., Simms, R., and Distler. Systemic Sclerosis. Nat. Rev. Dis. Primers. 2015;1:15002. Baardsnes,J.,Hinck,C.S.,Hinck,A.P.,O’Connor-McCourt,M.D.TbetaR-II Discriminates the High- And Low-Affinity TGF-beta Isoforms via Two Hydrogen-Bonded Ion Pairs.Biochemistry.2009;48(10):2146-55. Boocock,G.R.,Morrison,J.A.,and Popovic,M.Mutations in SBDS are associated with Shwachman-Diamond syndrome.Nature Genetics.2003;23:97-101. Bragado,P.,Estrada,Y.,and Parikh,F.TGFβ2 dictates disseminated tumour cell fate in target organs through TGFβ-RIII and p38β / β signaling.Nat.Cell.Biol.2013;15:1351-1361. De Crescenzo,G.,Pham,P.L.,Durocher,Y.,Chao,H.and O’Connor-McCourt,M.D.Enhancement of the antagonistic potency of transforming growth factor-beta receptor extracellular domains by coiled coil-induced homo- and heterodimerization.J.Biol.Chem.2004;279(25),26013-26018. Desmouliere,A.,Geinoz,A.,Gabbiani,F.,and Gabbiani,G.Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.The Journal of Cell Biology.1993;122(1):103-11. Durocher,Y.,Perret,S.,and Kamen,A.High-level and High-Throughput Recombinant Protein Production by Transient Transfection of Suspension-Growing Human 293-EBNA1 Cells.Nucleic Acids Res.2002;30(2):E9. Gabrielli,A.,Avvedminento,E.V.,and Krieg,T.Scleroderma.New England Journal of Medicine.2009;360:1989-2003. Gourh,P.,Remmers,E.F.,and Satpathy,A.Transforming Growth Factor Beta 3 (TGFB3)-a Novel Systemic Sclerosis Susceptibility Locus Involved in Fibrosis and Th17 Cell Development Identified By Genome-Wide Association Study in African Americans from the Genome Research in African American Scleroderma Patients Consortium.Arthritis Rheumatol.2017;69 (suppl 10). Grutter,C.,Wilkinson,T.,Turner,R.,Podichetty,S.,Finch,D.,McCourt,M.,Loning,S.,Jermutus,L.,and Grutter,M.G.A Cytokine-Neutralizing Antibody as a Structural Mimetic of 2 Receptor Interactions.Proc.Natl.Acad.Sci.USA.2008;105(51):20251-6. Herbertz,S.,Sawyer,J.S.,and Stauber,A.J.Clinical development of galunisertib (LY2157299 monohydrate),a small molecule inhibitor of transforming growth factor-beta signaling pathway.Drug Design,Development and Therapy.2015;5:4479-4499. Hunzelmann,N.and Krieg,T.Scleroderma:from pathophysiology to novel therapeutic approaches.Experimental Dermatology.2010;19(5):393-400. Komai,I.D.and Okamura,T.Reevaluation of Pluripotent Cytokine TGF-β3 in Immunity.Int.J.Mol.Sci.2018;19:2261. Kissin,E.Y.,Merkel,P.A.,and Lafyatis,R.Myofibroblasts and hyalinized collagen as markers of skin disease in systemic sclerosis.Arthritis and Rheumatism.2006;54(11):3655-60. Lacouture,M.E.and Morris,J.C.Cutaneous keratoacanthomas / squamous cell carcinomas associated with neutralization of transforming growth factor β by the monoclonal antibody fresolimumab (GC1008).Cancer Immunol.Immunother.2015;64:437-446. Langer,J.C.,Henckaerts,E.,Orenstein,J.,and Snoeck,H.W.Quantitative trait analysis reveals transforming growth factor-beta2 as a positive regulator of early hematopoietic progenitor and stem cell function.J.Exp.Med.2004;199(1):5-14. Lafyatis,R.Transforming growth factor β--at the centre of systemic sclerosis.Nature reviews Rheumatology.2014;10(12):706-19. Leask,A.Targeting the TGFβ,endothelin-1 and CCN2 axis to combat fibrosis in scleroderma.Cell Signal.2008;20(8):1409-14. Li,M.O.,Wan,Y.Y.,Sanjabi,S.,Robertson,A.K.,and Flavell,R.A.Transforming growth factor-β regulation of immune responses.Annu.Rev.Immunol.2006;24,99-146. Martin,C.J.,Datta,A.,Littlefield,C.,Kalra,A.,Chapron,C.,Wawersik,S.,Dagbay,K.B.,Brueckner,C.T.,Nikiforov,A.,Danehy Jr.,F.T.,Streich Jr.,F.C.,Boston,C.,Simpson,A.,Jackson,J.W.,Lin,S.,Danek,N.,Faucette,R.R.,Raman,P.,Capili,A.D.,Buckler,A.,Carven,G.J.,and Schurpf,T.Selective Inhibition of TGFβ1 Activation Overcomes Primary Resistance to Checkpoint Blockade Therapy by Altering Tumor Immune Landscape.Sci.Transl.Med.2020;12(536):eaay8456. Mayes,M.D.,Lacey,J.V.,and Beebe-Dimmer,J.Prevalence,incidence,survival,and disease characteristics of systemic sclerosis in a large US population.Arthritis Rheum.2003;48:2246-2255. Meng,X.-M.,Nikolic-Paterson,D.J.,and Lan,H.Y.TGF β:the master regulator of fibrosis.Nat.Rev.Nephrology.2016;12:325-338. Midgley,A.C.,Rogers,M.,Hallett,M.B.,Clayton,A.,Bowen,T.,and Phillips,A.O.Transforming growth factor-β1 (TGF-β1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts.The Journal of Biological Chemistry.2013;288(21):14824-38. Morris,J.C.,Tan,A.R.,Olencki,T.E.,Shapiro,G.I.,Dezube,B.J.,Reiss,M.,Hsu,F.J.,Berzofsky,J.A.,and Lawrence,D.P.Phase I Study of GC1008 (Fresolimumab):A Human Anti-Transforming Growth Factor-Beta (TGFβ) Monoclonal Antibody in Patients With Advanced Malignant Melanoma or Renal Cell Carcinoma.PLoS One.2014;9(3):e90353. Nanthakumar,D.B.,Hatley,R.J.D.,and Lemma,S.Dissecting fibrosis:therapeutic insights from the small-molecule toolbox.Nat.Rev.Drug Disc.2015;14:693-720. Prud’homme,G.J.Pathobiology of transforming growth factor beta in cancer,fibrosis and immunologic disease,and therapeutic considerations.Lab Invest.2007;87(11):1077-91. Rice,L.M.,Ziemek,J.,and Stratton,E.A.A longitudinal biomarker for the extent of skin disease in patients with diffuse cutaneous systemic sclerosis.Arthritis Rheumatol.2015;67:3004-3015. Rio,P.and Bueren,J.A.TGF-β:a master regulatory of the bone marrow failure puzzle in Fanconi anemia.Stem Cell Investig.2016;3:75 doi:10.21037 / sci.2016.09.17. Roberts,A.B.,Roche,N.S.,and Winokur,T.S.Role of transforming growth factor-beta in maintenance of function of cultured neonatal cardiac myocytes.Autocrine action and reversal of damaging effects of interleukin-1.J.Clin.Invest.1992;90(5):2056-2062. Rogers,Z.R.Shwachman-Diamond Syndrome.2018.UpToDate;https: / / www.uptodate.com / contents / shwachman-diamond-syndrome / print. Tatusova,T.A.and Madden,T.L. Blast 2 sequences - a new tool for comparing protein and nucleotide sequences.FEMS Microbiol Lett.1999;174:247-250. Todd,N.W.,Atamas,S.P.,Luzina,I.G.,and Galvin,J.R.Permanent alveolar collapse is the predominant mechanism in idiopathic pulmonary fibrosis.Expert Review of Respiratory Medicine.2015;9(4):411-8. Vannucchi,A.M.,Bianchi,L.,and Paoletti,F.A pathobiologic pathway linking thrombopoietin,GATA-1,and TGF-beta1 in the development of myelofibrosis.Blood.2005;105:3493-501. Varga,J.and Abraham,D.Systemic sclerosis:a prototypic multisystem fibrotic disorder.J.Clin.Invest.2007;117:557-567. Varga,J.and Pasche,B.Antitransforming growth factor-β therapy in fibrosis:recent progress and implications for systemic sclerosis.Current Opinion in Rheumatology.2008;20(6):720-8. Varga,J.and Whitfield,M.L.Transforming growth factor-beta in systemic sclerosis scleroderma.Frontiers in Bioscience :a journal and virtual library.2009;S1(1):226-35. Vincenti,F.,Fervenza,F.C.,Campbell,K.N.,Diaz,M.,Gesualdo,L.,Nelson,P.,Praga,M.,Radhakrishnan,J.,Sellin,L.,Singh,A.,Thornley-Brown,D.,Veronese,F.V.,Accomando,B.,Engstrand,S.,Ledbetter,S.,Lin,J.,Neylan,J.,Tumlin,J.,Focal Segmental Glomerulosclerosis Study Group.A Phase 2,Double-Blind,Placebo-Controlled,Randomized Study of Fresolimumab in Patients With Steroid-Resistant Primary Focal Segmental Glomerulosclerosis.Kidney Int.Rep.2017;2(5):800-810. Wang,J.C.,Chang,T.H.,and Goldberg,A.Quantitative analysis of growth factor production in the mechanism of fibrosis in agnogenic myeloid metaplasia.Exp.Hematol.2006;34(12):1617-23. Wang, H., Wang, B., Zhang, A., Hassounah, F., Seow, Y., Wood, M., Ma, F., Klein, JD, Price, SR, and Wang, XHExosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.Mol.Ther.2019;27(3):571-583. Zhang, H., Korono, DE, and O'Connor, KWStem cell defects and bone marrow failure in Fanconi anemia.Cell Stem Cell.2016;18:668-681.

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

[0275] The present invention will be described in detail with reference to its embodiments, which are provided not to limit the invention but to illustrate it. It is possible to construct other embodiments using the principles of the present invention, as well as other embodiments that fall within the scope defined by the spirit and appended claims.

Claims

1. A polypeptide construct useful for inhibiting the effects of transforming growth factor β (TGFβ) isoforms, wherein the construct is TGFβ binding region, and Multimerization domain Including; Here, the N-terminus of the polymerizing domain is bound to the C-terminus of the TGFβ binding region; The TGFβ binding region comprises, in the direction from the N-terminus to the C-terminus, 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; Here, the inhibitory efficacy of the polypeptide construct against both TGFβ1 isoform activity and TGFβ3 isoform activity is higher than that against TGFβ2 isoform activity; Here, the first linker and the second linker are used to determine the relative inhibitory efficacy of the polypeptide construct against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1). ５０ Polypeptide constructs selected such that the ratio is approximately 2.5:1 or less.

2. The relative inhibitory efficacy of the polypeptide construct against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The polypeptide construct according to claim 1, wherein the ratio is less than approximately 2.5:1, approximately 2.3:1 or less, approximately 2:1 or less, approximately 1.8:1 or less, approximately 1.5:1 or less, approximately 1.3:1 or less, approximately 1:1 or less, approximately 1:1 or less, approximately 0.8:1 or less, or approximately 0.5:1 or less.

3. The relative inhibitory efficacy of the polypeptide construct against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ A polypeptide construct according to claim 1 or 2, wherein the ratio is approximately 1:1 to approximately 2:

1.

4. The relative inhibitory efficacy of the polypeptide construct against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The polypeptide construct according to claim 3, wherein the ratio is approximately 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.

5. The relative inhibitory efficacy of the polypeptide construct against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The polypeptide construct according to claim 4, wherein the ratio is approximately 1.4:1 to approximately 1.6:

1.

6. The relative inhibitory efficacy of the polypeptide construct against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The polypeptide construct according to claim 5, wherein the ratio is approximately 1.4:1, approximately 1.5:1, or approximately 1.6:

1.

7. The polypeptide construct according to any one of claims 1 to 6, wherein the polypeptide construct inhibits both TGFβ1 isoform activity and TGFβ3 isoform activity with at least 20, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higher efficacy than TGFβ2 isoform activity.

8. The polypeptide construct according to any one of claims 1 to 7, wherein the first linker has a length of 33 amino acids or less.

9. The polypeptide construct according to any one of claims 1 to 8, wherein the second linker has a length of 10 amino acids or more.

10. A polypeptide construct according to any one of claims 1 to 9, wherein one or more of the first linker and the second linker include or consist of an IDR linker, an IDR linker variant, a hybrid linker, a hybrid linker variant, a cleavage linker, a cleavage linker variant, or an extension linker.

11. The polypeptide construct according to claim 10, wherein one of the first linker and the second linker includes or consists of a non-IDR linker.

12. The polypeptide construct according to any one of claims 1 to 10, wherein both the first linker portion and the second linker portion include or consist of an IDR linker, an IDR linker variant, a hybrid linker, a hybrid linker variant, a cleavage linker, a cleavage linker variant, or an extension linker.

13. The polypeptide construct according to any one of claims 1 to 12, wherein the first linker is 10 amino acid length or longer, 15 amino acid length or longer, or 18 amino acid length or longer.

14. The polypeptide construct according to any one of claims 1 to 13, wherein the first linker has a length of about 15 to 33 amino acids, or about 18 to about 30 amino acids.

15. The polypeptide construct according to any one of claims 1 to 14, wherein the first linker has a length of about 16, about 18, about 30, or about 32 amino acids.

16. The polypeptide construct according to claim 15, wherein the first linker has a length of 18 amino acids.

17. The polypeptide construct according to claim 15, wherein the first linker has a length of 16 amino acids.

18. The polypeptide construct according to claim 15, wherein the first linker has a length of 30 amino acids.

19. The polypeptide construct according to claim 15, wherein the first linker has a length of 32 amino acids.

20. The polypeptide construct according to any one of claims 1 to 19, wherein the second linker has a length of 35 amino acids or less, or a length of 10 to 34 amino acids.

21. The polypeptide construct according to any one of claims 1 to 20, wherein the second linker has a length of about 15 to about 35 amino acids.

22. The polypeptide construct according to any one of claims 1 to 21, wherein the second linker has a length of about 16, about 30, about 32, or about 34 amino acids.

23. The polypeptide construct according to claim 22, wherein the second linker has a length of 30 amino acids.

24. The polypeptide construct according to claim 22, wherein the second linker has a length of 16 amino acids.

25. The polypeptide construct according to claim 22, wherein the second linker has a length of 32 amino acids.

26. The polypeptide construct according to claim 22, wherein the second linker has a length of 34 amino acids.

27. A polypeptide construct according to any one of claims 1 to 26, wherein one or more of the first linker and the second linker comprises or consists of an amino acid sequence described in any one of Sequence IDs 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.

28. The polypeptide construct according to claim 27, wherein the first linker comprises or consists of an amino acid sequence described 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.

29. The polypeptide construct according to claim 27 or 28, wherein the second linker comprises or consists of an amino acid sequence described 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.

30. The polypeptide construct according to any one of claims 1 to 29, wherein the first linker comprises or consists of the amino acid sequence described in Sequence ID No.

12.

31. The polypeptide construct according to any one of claims 1 to 29, wherein the first linker comprises or consists of the amino acid sequence described in Sequence ID No.

8.

32. The polypeptide construct according to any one of claims 1 to 31, wherein the second linker comprises or consists of the amino acid sequence described in Sequence ID No.

11.

33. The polypeptide construct according to any one of claims 1 to 31, wherein the second linker comprises or consists of the amino acid sequence described in Sequence ID No.

9.

34. The first linker mentioned above, (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) Deletion of at least one C-terminal amino acid residue as compared to SEQ ID NO: 3, SEQ ID NO: 12, or SEQ ID NO: 8; (c) Deletion of at least one internal amino acid residue as compared to SEQ ID NO: 3, SEQ ID NO: 12, or SEQ ID NO: 8; or (d) A polypeptide construct according to any one of claims 1 to 29, having one or more substitutions in the amino acid sequence compared to SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 8, or comprising or consisting of any one of the amino acid sequences of (a) to (c).

35. The polypeptide construct according to claim 34, wherein the amino acid deletion is the deletion of the 16 amino acids of SEQ ID NO:

3.

36. The second linker mentioned above, (a) Deletion of at least one N-terminal amino acid residue, as compared to SEQ ID NO: 9 or SEQ ID NO: 11; (b) Deletion of at least one C-terminal amino acid residue, as compared to SEQ ID NO: 9 or SEQ ID NO: 11; (c) Deletion of at least one internal amino acid residue as compared to SEQ ID NO: 9 or SEQ ID NO: 11; or (d) A polypeptide construct according to any one of claims 1 to 35, having one or more substitutions in the amino acid sequence compared to SEQ ID NOs: 4, 9, or 11, or comprising or consisting of any one of the amino acid sequences of (a) to (c).

37. The polypeptide construct according to any one of claims 1 to 36, wherein the N-terminal region comprises or consists of an IDR linker, an IDR linker variant, a hybrid linker, a hybrid linker variant, a cleavage linker, a cleavage linker variant, or an elongation linker.

38. The polypeptide construct according to any one of claims 1 to 37, wherein the N-terminal region comprises or consists of the amino acid sequence described in Sequence 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.

39. A polypeptide construct according to any one of claims 1 to 38, wherein one or more of the first TGFβR-LBD and the second TGFβR-LBD include or consist of 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 to the amino acid sequence described in SEQ ID NO:

2.

40. A polypeptide construct according to any one of claims 1 to 39, wherein the first TGFβR-LBD and the second TGFβR-LBD are the same or substantially the same.

41. The polypeptide construct according to claim 40, wherein both the first TGFβR-LBD and the second TGFβR-LBD contain or consist of the amino acid sequence described in SEQ ID NO: 2, 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.

42. The polypeptide construct according to any one of claims 1 to 41, wherein the polymerizing domain enables dimerization of the polypeptide construct by the second polypeptide construct according to any one of claims 1 to 41 in a non-covalent manner.

43. The polypeptide construct according to any one of claims 1 to 41, wherein the polymerizing domain enables dimerization of the polypeptide construct by the second polypeptide construct according to any one of claims 1 to 41 in a covalent manner.

44. The polypeptide construct according to any one of claims 1 to 43, wherein the polymerizing domain comprises one or more constant regions of an antibody.

45. The aforementioned polymerizing domain is the second constant domain (C) of the antibody heavy chain. Ｈ 2) and / or a third constant domain (C Ｈ The polypeptide construct according to claim 44, comprising (3).

46. The polypeptide construct according to any one of claims 1 to 45, wherein the polymerizing domain includes the Fc region of an antibody heavy chain.

47. The polypeptide construct according to any one of claims 44 to 46, wherein the antibody is an IgG antibody.

48. The polypeptide construct according to claim 47, wherein the IgG antibody is optionally a human antibody, either an IgG1, IgG2, IgG3, or IgG4 antibody.

49. The polypeptide construct according to claim 48, wherein the polymerizing 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.

50. The polypeptide construct according to any one of claims 1 to 49, wherein the polymerizing domain comprises a cysteine ​​residue for crosslinking the polypeptide construct together with the second polypeptide construct according to any one of claims 1 to 49.

51. The polypeptide construct according to claim 50, wherein the polymerizing domain comprises at least two cysteine ​​residues for forming a disulfide crosslink together with the second polypeptide construct.

52. The polypeptide construct according to any one of claims 1 to 51, wherein the polymerizing domain is operated to reduce aggregation or to regulate the stability of the dimer or polymer of the polypeptide construct.

53. The polypeptide construct according to any one of claims 1 to 52, wherein the polymerizing domain comprises or consists of the amino acid sequence described in Sequence ID No.

49.

54. The polypeptide construct according to any one of claims 1 to 52, wherein the polymerizing domain comprises or consists of the amino acid sequence described in Sequence ID No.

50.

55. The polypeptide construct according to any one of claims 1 to 52, wherein the polymerizing domain comprises or consists of an amino acid sequence described in any one of Sequence IDs 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.

56. The polypeptide construct according to any one of claims 1 to 55, wherein the TGFβ-binding region includes or comprises an amino acid sequence described 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.

57. The polypeptide construct according to any one of claims 1 to 56, wherein the TGFβ-binding region comprises or consists of the amino acid sequence described in Sequence ID No.

27.

58. The polypeptide construct according to any one of claims 1 to 56, wherein the TGFβ-binding region comprises or consists of the amino acid sequence described in Sequence ID No.

29.

59. The polypeptide construct according to any one of claims 1 to 56, wherein the TGFβ-binding region comprises or consists of the amino acid sequence described in SEQ ID NO:

32.

60. The polypeptide construct according to any one of claims 1 to 56, wherein the TGFβ-binding region comprises or consists of the amino acid sequence described in Sequence ID No.

40.

61. The polypeptide construct according to any one of claims 1 to 56, wherein the TGFβ-binding region comprises or consists of the amino acid sequence described in Sequence ID No.

41.

62. The polypeptide construct according to any one of claims 1 to 61, wherein the polypeptide construct comprises or consists of an amino acid sequence described in any one of Sequence IDs 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.

63. The polypeptide construct according to any one of claims 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence described in Sequence 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.

64. The polypeptide construct according to any one of claims 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence described in Sequence 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.

65. The polypeptide construct according to any one of claims 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence described in Sequence 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.

66. The polypeptide construct according to any one of claims 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence described in Sequence 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.

67. The polypeptide construct according to any one of claims 1 to 61, wherein the polypeptide construct comprises or consists of the amino acid sequence described in Sequence 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.

68. The polypeptide construct according to any one of claims 1 to 67, wherein the polypeptide construct further comprises an amino acid sequence suitable for the expression, detection and / or purification of the TGFβ binder.

69. The polypeptide construct according to claim 68, wherein the polypeptide construct further comprises a signal peptide having the sequence described in Sequence ID No. 104, or a sequence substantially identical thereto.

70. The polypeptide construct according to any one of claims 1 to 69, wherein the polypeptide construct is a dimer polypeptide comprising the first and second polypeptide constructs according to any one of claims 1 to 69, wherein the polypeptide construct is linked between each of the polymerizing domains by at least one disulfide crosslink.

71. The polypeptide construct according to claim 70, wherein the first and second polypeptide constructs comprise the same or substantially the same amino acid sequence.

72. The polypeptide construct according to claim 70, wherein the first and second polypeptide constructs comprise different amino acid sequences.

73. The polypeptide construct according to claim 72, wherein the first and second polypeptide constructs comprise the same or substantially the same polymerizing domain and different TGFβ-binding regions.

74. A polypeptide construct according to any one of claims 70 to 73, wherein the first polypeptide and / or the second polypeptide construct further comprises a site for conjugation.

75. The polypeptide construct according to claim 74, wherein the first polypeptide and / or the second polypeptide construct is conjugated with a targeting agent, a therapeutic portion, a detectable portion, or a diagnostic portion.

76. The polypeptide construct according to claim 75, wherein the targeting agent, the therapeutic portion, the detectable portion, or the diagnostic portion comprises an antibody or its antigen-binding fragment, a binder having affinity for another member of the TGFβ family or another therapeutic target, a radiotherapy agent, a contrast agent, a fluorescent portion, a cytotoxic agent, a cell division inhibitor, a nanoparticle-based carrier, a drug conjugated to a polymer, a nanocarrier, a contrast agent, a stabilizer, a drug, a nanocarrier, or a dendrimer.

77. 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 polypeptide construct containing the Fc region of human IgG1.

78. A nucleic acid molecule encoding a polypeptide construct according to any one of claims 1 to 77.

79. The nucleic acid molecule according to claim 78, wherein the nucleic acid molecule encodes the polypeptide construct in a form that can be secreted by a selected expression host.

80. A nucleic acid molecule encoding at least one polypeptide having an amino acid sequence described in any one of Sequence IDs 81-103 and 105, or a sequence substantially identical thereto.

81. A nucleic acid molecule having a sequence described in any one of sequence numbers 106 to 109, or a sequence substantially identical thereto.

82. The nucleic acid molecule according to claim 80, further comprising at its 5' end the sequence described in SEQ ID NO: 110 or SEQ ID NO: 111, or a sequence substantially identical thereto.

83. A vector comprising a nucleic acid molecule according to any one of claims 78 to 82.

84. A cell host comprising a nucleic acid molecule according to any one of claims 78 to 82 or a vector according to claim 83.

85. A first polypeptide construct according to any one of claims 1 to 77, and A second polypeptide construct according to any one of claims 1 to 77 A TGFβ binder containing; Here, the first polypeptide construct and the second polypeptide construct are bound together by their respective polymerizing domains. Here, the inhibitory effect of the TGFβ binder on both TGFβ1 isoform activity and TGFβ3 isoform activity is higher than that on TGFβ2 isoform activity; Here, the first linker and the second linker are selected such that the relative inhibitory potency of the TGFβ binder against the TGFβ3 isoform activity compared to the TGFβ1 isoform activity (IC ５０ ratio) is about 2.5:1 or less, a TGFβ binder.

86. The relative inhibitory efficacy of the TGFβ binder against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The TGFβ binder according to claim 85, wherein the ratio is less than approximately 2.5:1, approximately 2.3:1 or less, approximately 2:1 or less, approximately 1.8:1 or less, approximately 1.5:1 or less, approximately 1.3:1 or less, approximately 1:1 or less, approximately 1:1 or less, approximately 0.8:1 or less, or approximately 0.5:1 or less.

87. The relative inhibitory efficacy of the TGFβ binder against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The TGFβ binder according to claim 85 or 86, wherein the ratio is approximately 1:1 to approximately 2:

1.

88. The relative inhibitory efficacy of the TGFβ binder against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The TGFβ binder according to claim 87, wherein the ratio is approximately 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.

89. The relative inhibitory efficacy of the TGFβ binder against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The TGFβ binder according to claim 88, wherein the ratio is approximately 1.4:1 to approximately 1.6:

1.

90. The relative inhibitory efficacy of the TGFβ binder against TGFβ3 isoform activity compared to TGFβ1 isoform activity (TGFβ3: IC2 for TGFβ1) ５０ The TGFβ binder according to claim 89, wherein the ratio is approximately 1.4:1, approximately 1.5:1, or approximately 1.6:

1.

91. The TGFβ binder according to any one of claims 85 to 90, wherein the TGFβ binder inhibits both TGFβ1 isoform activity and TGFβ3 isoform activity with at least 20, 100, 200, 300, 400, 500, 600, 700, 800, or 900 times higher efficacy than TGFβ2 isoform activity.

92. The TGFβ binder according to any one of claims 85 to 91, wherein the TGFβ binder is a dimer, and the first polypeptide construct and the second polypeptide construct are linked between their respective polymerization domains by at least one disulfide crosslink.

93. The TGFβ binder according to claim 92, wherein the TGFβ binder is a homodimer, and the first polypeptide construct and the second polypeptide construct are the same or substantially the same.

94. The TGFβ binder according to claim 93, wherein the first polypeptide construct and the second polypeptide construct contain or consist of a sequence described in any one of SEQ ID NOs: 81, 84, 87, or 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.

95. The TGFβ binder according to claim 93 or 94, wherein the first polypeptide construct and the second polypeptide construct contain or consist of the sequence described in Sequence ID No.

87.

96. The TGFβ binder according to claim 93 or 94, wherein the first polypeptide construct and the second polypeptide construct contain or consist of the sequence described in Sequence ID No.

96.

97. The TGFβ binder according to claim 93, wherein the first polypeptide construct and the second polypeptide construct contain or consist of the sequence described in Sequence 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.

98. The TGFβ binder according to claim 97, wherein the first polypeptide construct and the second polypeptide construct contain or consist of the sequence described in Sequence ID No.

95.

99. The TGFβ binder according to claim 92, wherein the TGFβ binder is a heterodimer, and the first polypeptide construct and the second polypeptide construct contain different amino acid sequences.

100. The TGFβ binding agent according to claim 99, wherein the first polypeptide construct and the second polypeptide construct comprise the same or substantially the same polymerizing domain and different TGFβ binding regions.

101. The TGFβ binding agent according to claim 100, wherein the different TGFβ binding regions include or consist of an amino acid sequence described in any one of SEQ ID NOs: 27, 29, 87, and 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.

102. The TGFβ binding agent according to claim 100, wherein the different TGFβ binding regions include or consist of the amino acid sequence described in Sequence 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.

103. 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 polypeptide construct comprising the first Fc region of human IgG1, and 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 polypeptide construct containing the second Fc region of human IgG1. A TGFβ binder containing; Herein, the first polypeptide construct and the second polypeptide construct are linked together via the first and second Fc regions of human IgG1 in a TGFβ-binding agent.

104. The inhibitory efficacy of the TGFβ binder against both TGFβ1 and TGFβ3 isoform activity is higher than that against TGFβ2 isoform activity; the relative inhibitory efficacy of the TGFβ binder against TGFβ3 isoform activity compared to TGFβ1 isoform activity (IC2) ５０ The TGFβ binder according to claim 103, wherein the ratio is approximately 2.5:1 or less.

105. A TGFβ binder which is a homodimer of a polypeptide construct according to any one of claims 1 to 77.

106. A pharmaceutical composition comprising a polypeptide construct according to any one of claims 1 to 77 or a TGFβ binder according to any one of claims 85 to 105 and a pharmaceutically acceptable carrier, diluent, or excipient.

107. The pharmaceutical composition according to claim 106, wherein the composition comprises the polypeptide construct according to claim 64 or 67, the TGFβ binder according to claim 95 or 96, or a combination thereof.

108. The pharmaceutical composition according to claim 106, wherein the composition comprises the polypeptide construct according to claim 66 or the TGFβ binder according to claim 98, or a combination thereof.

109. The pharmaceutical composition according to claim 106, wherein the composition comprises the polypeptide construct according to claim 66 or the TGFβ binder according to claim 103, or a combination thereof.

110. The pharmaceutical composition according to any one of claims 106 to 109, wherein the composition is formulated for administration by injection or infusion.

111. The pharmaceutical composition according to claim 110, wherein the composition is formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

112. A method for producing a polypeptide construct according to any one of claims 1 to 77 or a TGFβ binder according to any one of claims 85 to 105, comprising expressing the first polypeptide construct and / or the second polypeptide construct in a cell.

113. The method according to claim 112, further comprising culturing the cells and isolating and / or purifying the polypeptide construct or the TGFβ binder expressed in the cells.

114. The method according to claim 113, wherein the polypeptide construct and / or the TGFβ binder is secreted by the cells, and the polypeptide construct and / or the TGFβ binder is obtained from the culture medium in which the cells are cultured.

115. A method for treating or preventing a TGFβ-related disease or condition in a subject requiring treatment, comprising administering to the subject a polypeptide construct according to any one of claims 1 to 77 or a TGFβ-binding agent according to any one of claims 85 to 105, such that the TGFβ-related disease or condition is treated or prevented in the subject.

116. The method according to claim 115, wherein the subject is a mammal.

117. The method according to claim 116, wherein the mammal is a human.

118. The method according to any one of claims 115 to 117, wherein the subject is suffering from or suspected to be suffering from a disease or condition mediated by TGFβ1 and / or TGFβ3.

119. The method according to any one of claims 115 to 118, wherein the subject is suffering from or suspected to be suffering from a disease or condition mediated by TGFβ3.

120. A method for treating or preventing a disease or condition mediated by TGFβ1 and / or TGFβ3 in a subject, wherein the disease or condition mediated by TGFβ1 and / or TGFβ3 is treated or prevented in the subject. A method comprising administering to the subject a polypeptide construct according to any one of claims 1 to 77 or a TGFβ binder according to any one of claims 85 to 105.

121. The method according to claim 120, wherein the disease is mediated by TGFβ3.

122. The method according to any one of claims 115 to 121, wherein the disease or condition is characterized by overexpression or overactivation of TGFβ1 and / or TGFβ3.

123. The method according to any one of claims 115 to 122, wherein the disease or condition is fibrosis.

124. The method according to claim 123, wherein the fibrosis is pulmonary fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, renal fibrosis, myelofibrosis, systemic sclerosis, cutaneous fibrosis, cardiac fibrosis, myelofibrosis, fibroproliferative disease, or connective tissue disorder.

125. The method according to any one of claims 115 to 122, wherein the disease or condition is a bone marrow failure disease.

126. The method according to claim 125, wherein the disease or condition is Schwachmann-Bodian-Diamond syndrome or Fanconi anemia.

127. A method for producing a polypeptide construct according to any one of claims 1 to 77 or a TGFβ binder according to any one of claims 85 to 105, comprising: culturing a host cell according to claim 84 under conditions suitable for protein expression; and collecting a polypeptide construct according to any one of claims 1 to 77 or a TGFβ binder according to any one of claims 85 to 105.

128. A polypeptide construct or TGFβ binder produced by the method of claim 127.

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