TGF-β receptor extracellular domain fusion molecule and its use

Abstract

The present invention relates generally to polypeptides capable of binding and neutralizing transforming growth factor beta (TGF-β) ligands, and the use of these polypeptides to treat disorders associated with TGF-beta expression or activation (e.g., cancer and fibrotic diseases), and methods for making such molecules. [Solution] Polypeptides capable of binding and neutralizing transforming growth factor beta (TGF-β) ligands, and the use of these polypeptides to treat disorders associated with TGF-beta expression or activation (e.g., cancer and fibrotic diseases), and methods for making such molecules are provided.

Description

Technical Field

[0001] The present invention relates to TGF-β receptor extracellular domain fusion molecules and their use. More specifically, the present invention relates to TGF-β superfamily receptor extracellular domain fusion molecules and their use in neutralizing TGF-β ligands.

Background Art

[0002] TGF-β is part of a superfamily of more than 30 ligands that regulate several physiological processes including cell proliferation, migration and differentiation. Perturbation of their levels and / or signaling causes significant pathological effects. For example, TGF-β and activin ligands play important pathogenic roles in many diseases including cancer ((Hawinkels & Ten Dijke, 2011; Massague et al, 2000; Rodgarkia-Dara et al, 2006). In particular, TGF-β is considered an important regulator of tumor progression and is overexpressed in most tumor types. By inducing epithelial-mesenchymal transition in epithelial tumor cells, it promotes tumorigenesis and leads to aggressive metastasis (Thiery et al, 2009). TGF-β also promotes tumorigenesis by acting as a potent suppressor of the immune response in the tumor microenvironment (Li et al, 2006). Indeed, TGF-β is recognized as one of the most potent immunosuppressive factors present in the tumor microenvironment. TGF-β inhibits the differentiation, proliferation and survival of many immune cell types including dendritic cells, macrophages, NK cells, neutrophils, B cells and T cells. Thus, it regulates both innate and adaptive immunity (Santarpia et al, 2015; Yang et al, 2010). The importance of TGF-β in the tumor microenvironment is emphasized by evidence that in several tumor types (including melanoma, lung, pancreas, colorectal, liver and breast), elevated TGF-β ligand levels correlate with disease progression and recurrence, metastasis, and death. Therefore, significant efforts have been devoted to devising antitumor treatment approaches including TGF-β inhibition (Arteaga, 2006; Mourskaia et al, 2007; Wojtowicz-Praga, 2003).These approaches include the use of fusions of polypeptides based on the extracellular domain of the TGF-β receptor that bind or "trap" TGF-β ligands (see WO01 / 83525; WO2005 / 028517; WO2008 / 113185; WO2008 / 157367; WO2010 / 003118; WO2010 / 099219; WO2012 / 071649; WO2012 / 142515; WO2013 / 000234; US5693607; US2005 / 0203022; US2007 / 0244042; US8318135; US8658135; US8815247; US2015 / 0225483; and US2015 / 0056199).

[0003] One approach to developing therapeutics that inhibit TGF-β function has been to use antibodies or soluble decoy receptors (also referred to as ligand traps based on the extracellular domain (ECD) of the receptor) to bind and sequester the ligand, thereby blocking ligand access to cell surface receptors (Zwaagstra et al, 2012). In general, receptor ECD-based traps are a class of therapeutics that can sequester a broad range of ligands and can be optimized using protein engineering approaches (Economides et al, 2003; Holash et al, 2002; Jin et al, 2009).

[0004] Previously, a novel protein engineering design strategy was used to generate single-chain, bivalent TGF-β type II receptor extracellular domain (TβRII-ECD) traps that could potently neutralize members of the TGF-β superfamily of ligands by the avidity effect (Zwaagstra et al, 2012) [WO 2008 / 113185; WO 2010 / 031168]). In this case, bivalency was achieved through covalent linkage of two TβRII extracellular domains using a region of the intrinsically disordered region (IDR) adjacent to the structured ligand-binding domain of TβRII. An example of these single-chain bivalent traps, T22d35, showed ~100-fold higher TGF-β neutralizing ability than the monovalent, unmodified TβRII extracellular domain, but the bivalent trap did not neutralize the TGF-β2 isoform and had a relatively short circulating half-life.

[0005] It would be useful to provide TβRII-ECD-based traps with improved properties such as enhanced potency.

[0006] All patents, patent applications, and publications referred to throughout the application are set forth below.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0007]

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[0008] [Non-Patent Document 1] Arteaga CL (2006) Inhibition of TGFβeta signaling in cancer therapy. Curr Opin Genet Dev 16: 30-37 [Non-Patent Document 2] De Crescenzo G, Grothe S, Zwaagstra J, Tsang M, O'Connor-McCourt MD (2001) Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta ) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding. J Biol Chem 276: 29632-29643 [Non-Patent Document 3] Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30: E9 [Non-Patent Document 4] Economides AN, Carpenter LR, Rudge JS, Wong V, Koehler-Stec EM, Hartnett C, Pyles EA, Xu X, Daly TJ, Young MR, Fandl JP, Lee F, Carver S, McNay J, Bailey K, Ramakanth S, Hutabarat R, Huang TT, Radziejewski C, Yancopoulos GD, Stahl N (2003) Cytokine traps: multi-component, high-affinity blockers of cytokine action. Nat Med 9: 47-52 [Non-Patent Document 5] Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179: 125-142 [Non-Patent Document 6] Gajewski TF (2015) The Next Hurdle in Cancer Immunotherapy: Overcoming the Non-T-Cell-Inflamed Tumor Microenvironment. Semin Oncol 42: 663-671 [Non-Patent Document 7] Garberg P, Ball M, Borg N, Cecchelli R, Fenart L, Hurst RD, Lindmark T, Mabondzo A, Nilsson JE, Raub TJ, Stanimirovic D, Terasaki T, Oberg JO, Osterberg T (2005) In vitro models for the blood-brain barrier. Toxicol In Vitro 19: 299-334 [Non-Patent Document 8] Hahn T, Akporiaye ET (2006) Targeting transforming growth factor beta to enhance cancer immunotherapy. Curr Oncol 13: 141-143 [Non-Patent Document 9] Haqqani AS, Caram-Salas N, Ding W, Brunette E, Delaney CE, Baumann E, Boileau E, Stanimirovic D (2013) Multiplexed evaluation of serum and CSF pharmacokinetics of brain-targeting single-domain antibodies using a NanoLC-SRM-ILIS method. Mol Pharm 10: 1542-1556

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Brief Explanation of Drawings

[0009]

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[0010] The present invention provides a polypeptide construct with enhanced efficacy in inhibiting TGFβ.

[0011] The polypeptide construct of the present invention comprises a first region and a second region, wherein the first region comprises the first and / or second TGFβ receptor extracellular domain (ECD); the second region comprises the second constant domain (C H 2) and / or the third constant domain (C H 3). In a preferred non-limiting embodiment, the C-terminus of the first region is linked to the N-terminus of the second region. In a preferred non-limiting embodiment, the first region of the polypeptide construct comprises the first TβRII-ECD (ECD1) and / or the second TβRII-ECD (ECD2), where ECD1 and ECD2 are linked in tandem.

[0012] A first region containing a (TβRII-ECD)-(TβRII-ECD) doublet linked at its C-terminus to an antibody constant domain inhibits TGFβ activity with at least 600-fold higher potency than a corresponding construct having a single TβRII-ECD linked at its C-terminus to an antibody constant domain (i.e., in the absence of a second ECD, herein also referred to as a singlet). A polypeptide construct was provided.

[0013] The provided polypeptide construct contains a second TGFβ receptor extracellular domain tandemly linked to the first ECD, wherein the polypeptide construct linked to the antibody constant domain (i.e., the ECD doublet construct) exhibits at least 100, 200, 300, 400, 500, 600, 700, 800, or 900-fold higher TGFβ neutralization (inhibition) than a corresponding construct in which the antibody constant domain is absent (i.e., the ECD doublet construct, herein also referred to as a non-Fc fusion doublet).

[0014] In relation to TβRII-ECDs and their efficacy in inhibiting TGF-β activity, it has been found that surprisingly enhanced efficacy can be obtained by carefully selecting their composition. This occurs when certain TβRII-ECDs are tandemly linked and their C-terminus is linked to the N-terminus of the antibody constant domain (Fc). In the case of fusions and dimeric forms containing two such polypeptides cross-linked via a cysteine bridge between the constant domains of each polypeptide, the so-called "Fc fusion" resulting in having two TβRII-ECDs (ECD "doublet") in each of the two "arms" can exhibit inhibitory activity more than 600-fold higher for TGF-β1 and more than 20-fold higher for TGF-β3, as shown by the inhibition of TGF-β1- and -β3-induced IL-11 secretion by human non-small cell lung cancer (NSCLC) A549 cells, compared to an "Fc fusion" having one extracellular domain in each of the two "arms". The enhancement of efficacy is evident compared to any corresponding ones lacking the Fc region or lacking the second ECD (i.e., ECD "singlet"). The enhancement of efficacy is at least 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 600-fold higher for the Fc fusion doublet than for the Fc fusion singlet. The enhancement of efficacy is at least 100, 200, 300, 400, 500, 600, 700, 800, 900-fold and approximately 1000-fold higher for the Fc fusion doublet than for the non-Fc fusion doublet. The enhancement of efficacy is evident compared to corresponding ones lacking the Fc region (the Fc fusion doublet T22d35 is 972-fold and 243-fold superior to the non-Fc fusion doublet for TGF-β1 and TGF-β3, respectively) or lacking the second ECD (ECD "singlet"; the Fc fusion doublet T22d35-Fc is 615-fold and 24-fold superior to the non-Fc doublet for TGF-β1 and TGF-β3, respectively). More specifically, the Fc-doublet (T22d35-Fc) shows an enhancement of efficacy of at least 970-fold for TGF-β1 and at least 240-fold for TGF-β3 compared to the non-Fc fusion ECD doublet.Furthermore, the Fc-dimer (T22d35-Fc) shows an enhancement in potency that is at least 600-fold higher for TGF-β1 and at least 20-fold higher for TGF-β3 compared to the Fc-singleton (T2m-Fc).

[0015] In a general aspect, a polypeptide construct comprising at least two tandemly linked TβRII-ECDs (i.e., an ECD dimer), and at least the second constant domain (C H 2) and / or the third constant domain (C H 3) of an antibody heavy chain are provided, wherein the C-terminus of the extracellular domain is linked to the N-terminus of the antibody constant domain. In this form, the construct is a single-chain polypeptide. Thus, the antibody constant domain may comprise only the C H 2 domain, or may comprise the C H 2 domain and the C H 3 domain.

[0016] In an embodiment, the polypeptide is provided as a dimeric fusion polypeptide comprising two single-chain polypeptides and cross-linking means for covalently linking the chains.

[0017] In other embodiments, the two extracellular domains are generally identical with respect to their binding targets and / or their target species.

[0018] In a preferred embodiment, the first region comprises two TGFβ receptor extracellular domains (TGFβR-ECD or “TβR-ECD”). In a preferred embodiment, the TβR-ECD is a type II TGF-β receptor extracellular domain (TβRII-ECD). In a preferred embodiment, the TβR-ECD comprises SEQ ID NO: 1 and sequences substantially identical thereto.

[0019] The second region may comprise a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24, and sequences substantially identical thereto.

[0020] In a preferred embodiment, the second region of the polypeptide construct of the present invention further comprises C H 1. In embodiments, the construct is monofunctional.

[0021] The polypeptide construct of the present invention utilizes an antibody heavy chain of human origin. In a preferred embodiment, the antibody heavy chain is selected from the group consisting of human IgG1 (SEQ ID NO: 15) and IgG2 (SEQ ID NO: 24).

[0022] Accordingly, in another aspect, there is provided a polypeptide construct according to the present invention, the construct being a dimeric polypeptide; wherein the dimeric polypeptide comprises: (i) a first single-chain polypeptide comprising a second region comprising the second constant domain (C H 2) and the third constant domain (C H 3) of the antibody heavy chain, and a first region comprising two TGF-β receptor extracellular domains (TβRII-ECD), wherein the C-terminus of the first region is linked to the N-terminus of the second region, and (ii) a second single-chain polypeptide comprising a second region comprising the second constant domain (C H 2) and the third constant domain (C H 3) of the antibody heavy chain, and a first region comprising two tandemly linked TGF-β receptor extracellular domains (TβRII-ECD), wherein the C-terminus of the first region is linked to the N-terminus of the second region, and the first single-chain polypeptide is cross-linked to the second single-chain polypeptide.

[0023] There is also provided a nucleic acid molecule encoding any polypeptide construct of the present invention. There is also provided a vector comprising the nucleic acid molecule of the present invention.

[0024] There is also provided a composition comprising one or more independently selected polypeptide constructs of the present invention and a pharmaceutically acceptable carrier, diluent, or excipient.

[0025] Transgenic cell hosts comprising the nucleic acid molecules or vectors of the invention are also provided. The transgenic cell host may further comprise a second nucleic acid molecule or a second vector encoding a second polypeptide construct, where the second polypeptide construct may be the same as or different from the first polypeptide construct. When the two polypeptide constructs are different (heterodimer), a second nucleic acid molecule or a second vector is necessarily present, but when the constructs are identical (homodimer), it is not necessarily the case.

[0026] Use of the polypeptide constructs according to the invention for the treatment of medical conditions, diseases or disorders is also provided; wherein the medical condition, disease or disorder includes, but is not limited to, cancer, eye diseases, fibrotic diseases, or genetic disorders and immune disorders of connective tissue.

[0027] The peptide constructs of the invention comprise C H 2 and C H 3 or C H 2 only. For example, but not wishing to be limiting, the antibody heavy chain may be selected from the group consisting of human IgG1 and IgG2. In embodiments, the constant domain in the construct is itself C H 2, or is itself C H 3, or C H 2-C H 3. Suitably, the antibody heavy chain component provides disulfide bonds between the same or different single-chain polypeptide constructs. Similarly, suitably, the antibody heavy chain provides protein A-based isolation of the dimeric polypeptide produced by the host cell.

[0028] In embodiments, the receptor extracellular domain region comprises two independently selected extracellular domains linked tandemly, i.e., linearly. In some embodiments, the extracellular domains are identical or at least identical in sequence with respect to their target ligand.

[0029] The present invention also provides a nucleic acid molecule encoding the polypeptide construct described herein. Vectors containing the above nucleic acid molecules are also included in the present invention. The present invention also includes a transgenic cell host containing the nucleic acid molecule or vector described herein; the cell host may further contain a second nucleic acid molecule or a second vector encoding a second polypeptide construct different from the first polypeptide construct. The system used to produce the polypeptide of the present invention can be, in particular, a secretion system when dimerization via disulfide bridges is required, and thus the expression polynucleotide encodes a secretion signal that is cleaved by the host upon secretion into the culture medium.

[0030] Compositions comprising one or more independently selected polypeptide constructs described herein and a pharmaceutically acceptable carrier, diluent, or excipient are also included in the present invention.

[0031] These and other features of the present invention will be described below by way of example with reference to the accompanying drawings.

[0032] Additional aspects and advantages of the present invention will become apparent upon consideration of the following description. The detailed description and examples illustrate preferred embodiments of the present invention, but various changes and modifications within the scope of the present invention will be apparent to those skilled in the art, and are provided by way of example only.

Modes for Carrying Out the Invention

[0033] Detailed Description of the Invention Currently, polypeptide constructs are provided that bind to and neutralize all transforming growth factor beta (TGF-β1, β2, and β3) isoforms. These polypeptides utilize the TGF-β receptor extracellular domain to trap or sequester various TGFβ species, including TGF-β1 and TGF-β3, and to a lesser extent TGF-β2. The ability of the polypeptide constructs of the invention to neutralize TGF-β1 and -β3 is surprisingly much higher than that of related constructs, as demonstrated herein. For this reason, this construct is expected to be particularly useful as a pharmaceutical for the treatment of medical indications such as cancer, fibrotic diseases, and certain immune disorders.

[0034] This polypeptide construct contains two TβR-ECDs, such as TβRII-ECD, which are linked in tandem (C-terminus to N-terminus) and further includes an antibody constant domain comprising at least the second constant domain (C H 2) and / or the third constant domain (C H 3) of the antibody heavy chain. The antibody constant domain (Fc) is linked at its N-terminus to the C-terminus of the extracellular domain. Using the extracellular domain as a doublet and linking this doublet to the N-terminus of the antibody constant domain provides a "trap" with a neutralizing potency enhanced 615-fold against TGFβ1 and 24-fold against TGFβ3 compared to a construct having a single ECD linked to the N-terminus of the antibody constant domain.

[0035] As used herein, the term TβRII-ECD refers to the extracellular region of the TGF-β type II receptor that binds to the TGF-β ligand. In a preferred embodiment of this construct, the TGFβRII-extracellular domain is the extracellular domain of a TGFβR species that includes a sequence that forms a stable three-dimensional folded structure (i.e., TβRII-ECD): QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF (SEQ ID NO: 4).

[0036] In related forms that include a flexible native franking sequence, as shown below, the ECD can include the underlined structure: IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO: 1).

[0037] This sequence binds to TGF-β ligand isotypes designated TGF-β1 and TGF-β3. The binding affinity for TGF-β2 is low.

[0038] In the polypeptide constructs of the present invention, the two TβRII-ECDs can contain the same sequence. The two extracellular domains are linked in tandem, resulting in a linear polypeptide in which the C-terminus of one extracellular domain is linked to the N-terminus of the other extracellular domain.

[0039] The two extracellular domains can be linked by direct fusion so that no additional amino acid residues are introduced. Alternatively, the additional amino acid residues can form a linker that links the two receptor extracellular domains in tandem. In the protein constructs of the present invention, the first and second regions of the polypeptide constructs of the present invention are also linked. The term "linked" means that the two regions are covalently bonded. The chemical bond can be achieved by a chemical reaction or can be the recombinant expression product of two regions of a single polypeptide chain. In certain non-limiting examples, the C-terminus of the first region is directly linked to the N-terminus of the second region, i.e., there are no additional "linker" amino acids between the two regions. In the case where no linker is present, i.e., in the case of direct fusion of the two regions, the C-terminus of the complete extracellular domain and the antibody constant region C H 2-C HThere will be a direct connection between the N-terminus of 3. By fusing the Fc variants (SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24) to SEQ ID NO: 1 via the endogenous linker of SEQ ID NO: 8, this is part of the TβRII-ECD having SEQ ID NO: 1 (i.e., no additional "linker" amino acids are added), and it connects the aspartic acid at the last position of SEQ ID NO: 1 to glutamic acid, threonine, valine or the respective valines found in SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 24.

[0040] A common practice when producing fusion constructs is GGGGS or [G4S] nIt is to introduce a glycine - serine linker (GSL) such as (n is more than 1, 2, 3, 4 or 5, for example 10, 25 or 50) between the fusion components. As taught in the above paragraph, the polypeptide fusions of the present invention can be produced by direct ligation without using any additional amino acid sequences except those present in the Fc region and the receptor extracellular domain region. Thus, the use of foreign sequences as linkers can be avoided, providing advantages in terms of potential for unwanted immunogenicity and the addition of molecular weight. Entropic factors are also a potential hindrance for glycine and glycine - serine linkers, which are highly flexible and partially restricted upon target binding, causing an unfavorable loss of entropy in binding affinity. Thus, in embodiments of the present invention, only the flexible, intrinsically disordered N - terminal region of TGFβRII - ECD was used as a native linker. However, the specific amino acid composition and length of these native disordered linkers (e.g., SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8) precluded an accurate prediction of whether the resulting direct fusion construct would have the shape and preferred molecular interactions necessary for correct binding to their intended dimeric ligand. In other embodiments, the fusion polypeptide can include a flexible artificial GSL as exemplified in the construct of SEQ ID NO: 20, where the GS linker of SEQ ID NO: 21 is introduced between the aspartic acid (D) at the last position of TβRII - ECD having SEQ ID NO: 1 and the threonine (T) at the first position of the Fc region variant having SEQ ID NO: 15.

[0041] In embodiments, the first and second regions of the polypeptide construct are connected by a native intrinsically disordered polypeptide linker selected from the group consisting of SEQ ID NO: 8, 13, 16, 19, 25, and sequences substantially identical thereto. In other embodiments, the regions of the polypeptide construct are connected by a flexible linker selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22, and sequences substantially identical thereto.

[0042] In this embodiment, one region of the present polypeptide construct comprises first and second receptor extracellular domains tandemly linked by the native native-modified polypeptide linker of SEQ ID NO: 6, and comprises a TβRII-ECD doublet having the following amino acid sequence: PPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO: 5).

[0043] The construct also comprises a region comprising an antibody constant domain comprising at least the second constant domain (C H 2) and / or the third constant domain (C H 3) of the antibody heavy chain. Since the constant domain of the antibody is attached at its N-terminus to the C-terminus of the extracellular domain doublet, the orientation of the construct is TβRII-ECD (linked) TβRII-ECD (linked) C H 2-C H 3 as a single chain.

[0044] The antibody constant domain confers cross-linking between two of the present polypeptide constructs. This is achieved when the expressed polypeptide constructs are secreted from their expression hosts. Thus, production of the single-chain polypeptide provides a construct in dimeric form in which the two constructs are cross-linked via disulfide bonds comprising one or more cysteine residues within each antibody constant domain present in each construct.

[0045] The antibody constant domain present in the construct preferably is derived from an IgG constant region, particularly the constant domain of either IgG1 or IgG2.

[0046] The constructs provided are monofunctional in the sense that, other than acting as a structure in which a dimer of the polypeptide construct can be formed, the constant region itself has no specific activity. These minimal constant regions can be modified to provide several advantages by incorporating the corresponding hinge region and altering the cysteine residue composition as necessary. Thus, some or all of the cysteine residues involved in cross-linking the two Fc fragments, or the cysteine residues that are naturally used to cross-link between the heavy and light chains of a full-length antibody, can be substituted or deleted. One advantage of minimizing the number of cysteine residues is to reduce the incidence of disulfide bond scrambling that can promote aggregation. For example, these cysteine residues and their modifications are found in natural or non-natural linker sequences around the junction of the first and second regions of the polypeptide construct and are represented below: SEEYNTSNPDTHTCPPCPAPE (SEQ ID NO: 16), SEEYNTSNPDVEPKSSDKTHTCPPCPAPE (SEQ ID NO: 19), SEEYNTSNPDGGGSGGGSGGGTHTCPPCPAPE (SEQ ID NO: 22) incorporating variations of the human IgG1 hinge sequence; and SEEYNTSNPDERKCCVECPPCPAPP (SEQ ID NO: 13) and SEEYNTSNPDVECPPCPAPP (SEQ ID NO: 25) incorporating variations of the human IgG2 hinge sequence; and sequences substantially identical thereto.

[0047] Note that while the stability of the Fc homodimer can depend on the number of intermolecular disulfide bonds, not all of the naturally occurring inter-hinge disulfide bonds need to be formed for Fc homodimerization to occur.

[0048] In the present disclosure, an "antibody", also referred to in the art as an "immunoglobulin" (Ig), refers to a protein composed of heavy and light polypeptide chain pairs. As summarized herein, the structures of antibodies and each domain are well established and well known to those skilled in the art. When an antibody is correctly folded, each chain folds into a number of different globular domains that are folded and linked by more linear polypeptide sequences; the immunoglobulin light chain folds into variable (V L ) and constant (C L ) domains, and the heavy chain folds into variable (V H ) and three constant (C H 1, C H 2, C H 3) domains. When paired, the variable domains (V H and V L ) of the heavy and light chains and the interaction of the first constant domains (C L and C H 1) form a Fab (fragment, antigen-binding) containing a binding region (Fv); the interaction of the two heavy chains pairs the C H 2 domain and the C H 3 domain to form an Fc (fragment, crystallizability). The features described herein for the C H 2 and C H 3 domains also apply to the Fc.

[0049] In the present invention and its specific embodiments, polypeptide constructs that exhibit significantly enhanced potency include the following: T22d35-Fc: IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10) T22d35-Fc-IgG2-v2(CC): IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 23) T22d35-Fc-IgG1-v1(CC): IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 14) T22d35-Fc-IgG1-v2(SCC): IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD VEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17); and T22d35-Fc-IgG1-v3(GSL-CC): IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPDGGGSGGGSGGG THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20).

[0050] In certain embodiments, the polypeptide construct comprises a polypeptide of the invention that exhibits significantly enhanced potency. For example, a polypeptide that exhibits significantly enhanced potency may comprise SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23. In other certain embodiments, the polypeptide construct may be a homodimer that comprises a polypeptide that exhibits significantly enhanced potency; for example, where the polypeptide construct that exhibits significantly enhanced potency is SEQ ID NO: 14, the polypeptide construct is a homodimer that comprises two polypeptide constructs, each of which comprises SEQ ID NO: 14. Similarly, where the polypeptide construct that exhibits enhanced potency is SEQ ID NO: 10, 14, 17, 20, or 23, the homodimer of the invention likewise comprises two polypeptide constructs, each polypeptide of the homodimer comprising SEQ ID NO: 10, 14, 17, 20, or 23.

[0051] As described above, these single-chain polypeptide constructs dimerize upon secretion from the production host, resulting in a dimeric polypeptide construct that includes two single-chain polypeptides linked by a disulfide bond formed between the constant domains of the two single-chain polypeptides.

[0052] "Significantly enhanced potency" means that the effect or activity of this dimeric form of the polypeptide construct is greater than that of the corresponding construct when measured in an assay related to the evaluation of the biological activity of TGF-β. Appropriate means for making this determination are exemplified herein. For example, as shown by TGF-β-induced IL-11 release by A549 cells, the N-terminal Fc-fused T22d35 doublet neutralizes TGF-β and extends much better than the N-terminal Fc-fused T2m singlet (Figure 7).

[0053] This type of fusion construct has been observed to have advantages compared to several other versions of molecules based on the extracellular domain of the TβRII receptor, which include non-Fc fusion bivalent TGF-β receptor extracellular domain constructs (such as the T22d35 doublet) and constructs where a single receptor extracellular domain is fused to the N-terminus of the Fc region. In particular, the currently provided Fc fusion constructs have improved manufacturability due to the presence of the Fc region (e.g., production can be achieved using protein A chromatography). The Fc region also enables improvement of the circulating half-life. Importantly, this construct has substantially higher TGF-β neutralizing ability compared to the singlet fusion (T2m-Fc) and non-Fc fusion doublet extracellular domain (T22d35). The provided N-terminal fusion TGF-βECD doublet Fc construct (T22d35-Fc) shows advantages with respect to a significant improvement in TGF-β ligand neutralizing ability (as shown in Figure 7, for example, an improvement of over 970-fold in TGF-β1 neutralization compared to the non-Fc fusion doublet). Furthermore, they show improved manufacturability, as demonstrated by biophysical analysis showing a monomer content of over 99% (i.e., minimal presence of aggregates and absence of fragments of the purified N-terminal fusion T22d35-Fc construct) (as shown in Figure 6). Thus, an advantage of the present invention is the unexpectedly high potency of TGF-β ligand neutralization, including some neutralization of TGF-β2, which is not observed with the T2m-Fc (Fc singlet) or T22d35 (non-Fc fusion) constructs.

[0054] In certain embodiments, the second region of the polypeptide construct of the invention is selected from the group of N-terminal sequence variations as exemplified by SEQ ID NOs: 12, 15, 18, 24. These may differ in the length and number of cysteine residues retained from the hinge region as a means of modulating the degree of dimerization of the Fc region and thus can affect both efficacy and manufacturability. Thus, in embodiments, the polypeptide construct comprises variations of the constant domain, where at least one cysteine residue involved in cross-linking is deleted or substituted. Suitable substitutions include serine or alanine, and preferably substitutions with serine.

[0055] Substantially identical sequences may include one or more conservative amino acid mutations that further provide proper folding upon secretion into the culture medium. It is well known in the art that one or more conservative amino acid mutations relative to a reference sequence can result in a mutant peptide having substantially no change in physiological, chemical, physicochemical or functional properties compared to the reference sequence; in such cases, the reference sequence and the mutant sequence will be considered "substantially identical" polypeptides. Conservative amino acid substitutions are defined herein as substitutions of an amino acid residue with another amino acid residue having similar chemical properties (e.g., size, charge, or polarity). These conservative amino acid mutations can be made to the framework region while maintaining the overall structure of the constant domain; thus, the function of the Fc is maintained.

[0056] In certain non-limiting examples, the first region of the polypeptide construct of the invention may include the TGF-β receptor type II, such as the following: IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO: 1, also referred to herein as T2m). may include.

[0057] In a preferred embodiment, the polypeptide construct comprises a TβRII-ECD "doublet" in which the TβRII-ECD is linked in tandem with another TβRII-ECD, where the extracellular domains are, for example, as follows: IPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF -Linker- QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF SEEYNTSNPD (SEQ ID NO: 5, also referred to herein as T22d35), where in one non-limiting embodiment, the linker corresponds to SEQ ID NO: 6; and sequences that are substantially identical thereto, which may be the same or different TGF-β superfamily receptor extracellular domains such as etc. "Substantially identical" is as defined above.

[0058] The extracellular domain doublet can incorporate the same or different extracellular domains, both belonging to the TGFβ superfamily receptor family. In embodiments, the extracellular domains bind to the same target. In other embodiments, the extracellular domains are of the same receptor species. In other embodiments, the extracellular domains are identical and thus are homomers.

[0059] For example, the polypeptide construct of the present invention can have a TGF-β neutralizing potency selected from the group consisting of at least 900-fold and 200-fold more potent than the T22d35 doublet alone for TGF-β1 and TGF-β3, respectively. For example, in an IL-11 release assay, the T22d35 doublet construct is approximately 972-fold more potent in neutralizing TGF-β1 and approximately 243-fold more potent in neutralizing TGF-β3 compared to the non-Fc fusion T22d35 doublet alone.

[0060] In other embodiments, the construct is at least 600-fold and at least 20-fold more potent for neutralization of TGF-β1 and TGF-β3, respectively, than constructs in which the antibody constant domain is linked to a single external domain other than a doublet. The polypeptide construct of the present invention can have a neutralizing potency that is at least 615-fold and 24-fold better against TGF-β1 and TGF-β3, respectively, when compared to the potency of a construct in which the antibody constant domain is linked to a single external domain other than a doublet.

[0061] The neutralizing potency can be summarized as follows: The neutralizing potency of the Fc fusion doublet (ECD-ECD-Fc) is higher than that of the Fc fusion ECD monomer (ECD-Fc); that is, ECD-ECD-Fc, ECD-Fc are more potent than the non-Fc fusion doublet (ECD-ECD), and the non-Fc fusion doublet ECD is more potent than the non-Fc fusion singlet ECD; that is, ECD-ECD-Fc >> ECD-Fc > ECD-ECD >> ECD. With respect to formulation properties, the presence of the Fc protein enables purification by protein A and obviates the need to use a cleavable tag. In addition, placing a singlet or doublet ECD at the N-terminus of the Fc portion prevents problems of aggregation due to inappropriate pairing of cysteine residues in the hinge region of the Fc portion. Thus, fusion of the ECD singlet or doublet to the N-terminus of the Fc portion improves manufacturability compared to C-terminal fusion (N-terminal fusion has a higher proportion of monomeric species, fewer aggregates, and fewer fragments). In addition, an unexpectedly significant increase in the TGF-β neutralizing ability of all TGF-β isoforms of the N-terminal Fc fusion doublet ECD is observed compared to the N-terminal Fc fusion T2m singlet ECD.

[0062] In addition, when the polypeptide construct of the present invention comprises a TβRII-ECD that binds to TGF-β, the polypeptide construct can neutralize all three isoforms of TGF-β (i.e., TGF-β1, TGF-β2, and TGF-β3) to varying degrees.

[0063] The polypeptide construct of the present invention has a TGF-β neutralizing ability that is significantly higher (more than 20-fold) than that of the bivalent comparator polypeptides, namely, non-Fc fusion T22d35 (doublet only) and T2m-Fc (Fc fusion singlet), as evaluated in cell-based assays. Among a series of polypeptide constructs of the present invention, those containing two or more copies of TβRII-ECD fused to the N-terminus of the Fc constant region have higher potency than constructs containing only one copy, as evaluated in cell-based assays.

[0064] The polypeptide construct of the present invention is expressed as a single polypeptide chain. Once expressed, the polypeptide construct of the present invention forms a dimer, where the C H 2 and C H 3 domains interact to form a properly assembled Fc region such as occurs when the expression product is secreted into the culture medium.

[0065] The polypeptide construct of the present invention may also contain additional sequences that assist in the expression, detection, or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those skilled in the art may be used. For example, without wishing to be limiting, an antibody or fragment thereof may contain a targeting or signal sequence (e.g., but not limited to ompA), a detection / purification tag (e.g., but not limited to, c-Myc, His5, His6, or His8G), or a combination thereof. In other embodiments, the signal peptide may be MDWTWRILFLVAAATGTHA (SEQ ID NO: 11). In further embodiments, the additional sequence may be a biotin recognition site as described in [WO / 1995 / 04069] or [WO / 2004 / 076670]. Similarly, as known to those skilled in the art, a linker sequence may be used in combination with an additional sequence or tag, or may function as a detection / purification tag. Suitably, the constant region has a Protein A binding site (typically C H 2 and CH It includes those present near 3).

[0066] The present invention also encompasses nucleic acid sequences encoding the above-mentioned molecules. Considering the degeneracy of the genetic code, many nucleotide sequences will have the effect of encoding the desired polypeptide, as will be readily understood by those skilled in the art. The nucleic acid sequences can be codon-optimized for expression in various microorganisms. The present invention may also encompass vectors containing the above-mentioned nucleic acids, where the vectors typically include a promoter and a signal sequence operably linked to a polynucleotide encoding a construct for promoting its expression in a selected cell production host. The vectors can be the same or different, provided that both result in the secretion of the dimeric polypeptide construct.

[0067] Furthermore, the present invention encompasses cells, also referred to as transgenic cell hosts, containing the nucleic acids and / or vectors described herein. The host cells can contain a second nucleic acid and / or vector encoding a second polypeptide construct different from the first polypeptide construct. Co-expression of the first and second polypeptide constructs can result in the formation of heterodimers.

[0068] The present invention also encompasses compositions containing one or more polypeptide constructs described herein. The compositions can contain a single polypeptide construct as described above or a mixture of polypeptide constructs. The compositions can also contain one or more polypeptide constructs of the present invention linked to one or more cargo molecules. For example, without wishing to be limited by any means, the compositions can contain one or more polypeptide constructs of the present invention linked to a cytotoxic drug for generating an antibody-drug conjugate (ADC) according to the present invention.

[0069] The composition may also include a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier can be any suitable diluent, excipient, or carrier well-known in the art, must be compatible with the other components of the composition and the method of delivery of the composition, and must not be harmful to the recipient of the composition. The composition can be in any suitable form; for example, the composition can be provided in suspension form, powder form (e.g., limited to lyophilization or encapsulation), capsule or tablet form. For example, without wishing to be limiting, if the composition is provided in suspension form, the carrier can include water, physiological saline, a suitable buffer, or additives to improve solubility and / or stability; the reconstitution to produce the suspension is carried out with a suitable pH buffer to ensure the viability of the antibody or fragment thereof. The dry powder can also include additives to improve safety and / or a carrier to increase bulk / volume; for example, without wishing to be limiting, the dry powder composition can include sucrose or trehalose. In certain non-limiting examples, the composition can be formulated to deliver an antibody or fragment thereof to the gastrointestinal tract of a subject. Thus, the composition can include encapsulation, sustained release, or other suitable techniques for the delivery of the antibody or fragment thereof. Preparing a suitable composition containing the present compound would be within the capabilities of one of ordinary skill in the art.

[0070] The construct of the present invention can be used to treat diseases or disorders associated with overexpression or overactivation of ligands of the TGF-β superfamily. The disease or disorder can be selected from, but is not limited to, cancer, eye diseases, fibrotic diseases, or genetic disorders of connective tissue.

[0071] In the field of cancer treatment, it has recently been demonstrated that TGF-β is an important factor that inhibits the anti-tumor response induced by immunotherapies such as immune checkpoint inhibitors (ICI’s) (Hahn & Akporiaye, 2006). Specifically, the therapeutic response to ICI antibodies is mainly due to the reactivation of T cells localized in the tumor. Resistance to ICI antibodies depends on the presence of an immunosuppressive mechanism with a lack of T cells in the tumor microenvironment. Therefore, it is currently recognized that in order to induce a response in resistant patients, ICI antibodies need to be combined with agents that can activate T cells and induce the replenishment of the tumor, i.e., the reversal of the "non-T cell inflammatory" tumor phenotype. One literature pointed out that overcoming the microenvironment of non-T cell inflammatory tumors is the most important next hurdle in immuno-oncology (Gajewski, 2015).

[0072] Using the proof-of-concept TGF-β trap, T22d35, it has been shown that blocking TGF-β effectively reverses the "non-T cell inflammatory" tumor phenotype (Zwaagstra et al, 2012). Thereby, anti-TGF-β molecules are positioned as a potential combination with synergistic effects with ICI and other immunotherapeutic agents. Supporting this, a 2014 study (Holtzhausen et al., ASCO poster presentation) examined the effect of a TGF-β blocker when combined with an anti-CTLA-4 antibody in a physiologically relevant transgenic melanoma model. This study demonstrated that anti-CTLA-4 antibody monotherapy could not suppress the progression of melanoma, but the combination of a TGF-β antagonist and an anti-CTLA-4 antibody significantly and synergistically suppressed both the growth of primary melanoma tumors and melanoma metastasis. These observations correlate with a significant increase in effector T cells in melanoma tissue.

[0073] In this specification, it is shown that the polypeptide having the basic structure of T22d35-Fc significantly reduces tumor growth in the syngeneic mouse MC-38 colon cancer model. Therefore, this positions the anti-TGF-β molecule for use in potential synergistic combinations with other immunotherapeutic agents.

[0074] The constructs of the present invention may be useful for the treatment of fibrotic diseases, including but not limited to those that affect any organ of the body, including the kidney, lung, liver, heart, skin, and eye. These diseases include but are not limited to chronic obstructive pulmonary disease (COPD), glomerulonephritis, liver fibrosis, post-infarct cardiac fibrosis, restenosis, systemic sclerosis, fibrosis due to ophthalmic surgery, and scarring.

[0075] Genetic disorders of connective tissue may also be treated, including but not limited to Marfan syndrome (MFS) and osteogenesis imperfecta (OI).

[0076] The present invention is further illustrated by the following examples. However, it should be understood that these examples are for illustrative purposes only and should not be used in any way to limit the scope of the present invention.

Examples

[0077] Materials and Methods Production and Purification Transient CHO Expression Various TβRII-ECD fusion mutants (such as T2m-Fc and T22d35-Fc) are each composed of the heavy chain Fc region and contain the signal sequence MDWTWRILFLVAAATGTHA (SEQ ID NO: 11) at their N-terminus. The DNA coding regions of the constructs were prepared synthetically (Biobasic Inc. or Genescript USA Inc.) and cloned into the HindIII (5'-end) and BamH1 (3'-end) sites of the pTT5 mammalian expression plasmid vector (Durocher et al, 2002). The fusion proteins were produced by transient transfection of Chinese hamster ovary (CHO) cells using the heavy chain T2m or T22d35 fused to IgG heavy chain (T2m-HC and T22d35-HC constructs respectively). Briefly, T2m-HC or T22d35-HC plasmid DNA was transfected into 2.5 L and 4.6 L cultures of CHO-3E7 cells in FreeStyle F17 medium (Invitrogen) containing 4 mM glutamine and 0.1% Kolliphor p-188 (Sigma) and maintained at 37°C. Transfection conditions: DNA (80% plasmid construct, 15% AKT plasmid, 5% GFP plasmid): PEIpro (ratio = 1:2.5): PEI (polyethyleneimine)pro (polyplex) (ratio = 1:2.5). Twenty-four hours after transfection, 10% tryptone N1 feed (TekniScience Inc.) and 0.5 mM valproic acid (VPA, Sigma) were added, the temperature was shifted to 32°C to promote production and secretion of the fusion protein, and after maintaining for 15 days post-transfection, the cells were harvested. The cell viability at final harvest was 89.6%.

[0078] Stable pool CHO expression CHO cells were transfected with a vector expressing the target gene encoding various Fc-fusion TβRII-ECD proteins BRI / rcTAA cell pool was generated. On the day following transfection, the cells were centrifuged at 250 rpm for 5 minutes and seeded in selection medium (PowerCHO2 medium supplemented with 50 μM methionine sulfoximine) at a density of 0.5×10 6 cells / mL. The selection medium was changed every 2 - 3 days for 14 - 18 days and seeded at 0.5×10 6 cells / mL. The cell number and viability were measured using the above-mentioned Cedex Innovatis automated cell counter Cedex Analyzer. The cell number and viability were measured using the above-mentioned Cedex Analyzer manufactured by Cedex Innovatis. When the cell viability exceeded 95%, the pool was seeded at 0.2×10 6 cells / mL into 125 or 250 mL Erlenmeyer flasks. In fed-batch culture, the CHO BRI / rcTA cell pool was seeded as described above. On the 3rd day after inoculation, when the cell density reached 3.5 - 4.5×10 6 cells / mL, the expression of the recombinant protein was induced by adding 2 μg / mL of cumate. The MSX concentration was adjusted to 125 μM, and after adding F12.7 feed (Irvine Scientific), the temperature was shifted to 32°C. Every 2 - 3 days, 5% (v:v) F12.7 was supplied to the culture, and samples were collected for the measurement of the concentrations of the recombinant protein (pA-HPLC) and glucose (VITROS 350, Orthoclinical Diagnostics, USA). Glucose was added to maintain a minimum concentration of 17 mM.

[0079] Purification The supernatant recovered from CHO cells was filtered (0.2 μm) and loaded onto a Protein A MabSelect Sure column (GE Healthcare). The column was washed with 2 column volumes of PBS, and the protein was eluted with 3 column volumes of 0.1 M sodium citrate pH 3.6. To maximize the yield, the flow-through was reloaded onto the Protein A column and eluted as described above. The eluted fractions were neutralized with 1 M Tris, and the fractions containing the fusion protein were pooled. Subsequently, the formulation buffer (Ca 2+Mg-free 2+ Loaded onto a Hi-load Superdex S200 26 / 80 size exclusion chromatography (SEC) column (GE Healthcare) equilibrated with DPBS (minus Ca 2+ 2+ , minus Mg 2+ 2+ ). The protein was eluted using 1 column volume of formulation buffer, collected in consecutive fractions, and detected at 280 nm UV absorbance. Subsequently, the main peak SEC fractions containing the fusion protein were pooled and concentrated. The integrity of the Prot-A and SEC-purified fusion proteins in the pooled fractions was further analyzed by UPLC-SEC and SDS-PAGE (4-15% polyacrylamide) under reducing and non-reducing conditions (SYPRO Ruby staining). For UPLC-SEC, 2-10 μg of protein in DPBS (Hyclone, minus Ca

[0080] Cell line Human A549 non-small cell lung cancer cells were purchased from ATCC (Cat# CCL-185, Cedarlane, Burlington, ON). The cells were cultured in Dulbecco's modified Eagle's medium (MEM) supplemented with 5% fetal bovine serum (FBS). MC-38 mouse colorectal adenocarcinoma cells were purchased from Kerafast (Cat# ENH204, Boston, MA) and cultured in Dulbecco's modified MEM supplemented with 2 mM L-glutamine and 10% fetal bovine serum. Both cell lines were maintained at 37 °C in a humidified atmosphere with 5% CO2.

[0081] TGF-β-induced A549 cell IL-11 release assay Human A549 lung cancer cells were seeded in 96-well plates (5×10 3(Cells / Well). The next day, 10 pM TGF-β in complete medium was added to the cells after incubation at RT for 30 min in the absence or presence of a series of dilutions of the TGF-β trap fusion protein. After 21 h of incubation, the conditioned medium was harvested and added to an MSD streptavidin gold plate coated with 2 μg / mL biotinylated mouse anti-human IL-11 antibody (MAB618, R&D Systems, Minneapolis, MN). After 18 h (4 °C), the plate was washed with PBS containing 0.02 Tween20, 2 μg / mL goat anti-human IL-11 antibody conjugated with SULFO tag (AF-218-NA, R&D Systems Minneapolis, MN) was added, and the plate was incubated at room temperature for 1 h. After the final wash, the plate was read on a MESO QuickPlex SQ120 machine (Meso Scale Diagnostics, Gaithersburg, MD). The IL-11 readout was expressed as the percent IL-11 release compared to control cells treated with TGF-β alone. Graphpad Prism (4-PL algorithm ((log(inhibitor) VS. response - variable slope (4 parameters)) was used and the IC 50 was calculated (automatic outlier option was used if necessary).

[0082] In vitro evaluation of the syngeneic mouse colon cancer MC-38 subcutaneous mouse model Female C57BL / 6-Elite mice (5 - 7 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). Thirteen C57BL / 6 mice were subcutaneously injected with 3 × 10 5 MC-38 cells on the right flank on day 0. When the tumors reached a volume of 50 - 100 mm 3 (day 5), the animals were divided into two cohorts and treatment was initiated: · Cohort 1 (7 animals): Isotype control (CTL IgG; BioxCell InVivo MAb Rat IgG2b, anti-KLH; clone LTF-2, Cat# BE0090); 200 μg in 100 μL phosphate-buffered saline (PBS), intraperitoneal (i.p.), on days 5, 7, 9, and 11. · Cohort 2 (6 animals): T22d35-Fc, 5 mg / kg in 100 μL PBS, i.p., on days 5, 9, 12, and 16. Tumors were measured twice weekly using digital calipers for up to 15 days after treatment began. Tumor volume was calculated from these measurements using the modified ellipsoid formula described above (Tvol = π / 6 × (length × width × width)) (Tomayko et al., 1986).

[0083] Results and Discussion Fusion Construct Design To generate the desired TGF-β trap, the TβRII-ECD singlet (named T2m) was fused to another such singlet, thereby forming an external domain doublet (named T22d35) linked to the N-terminus of the heavy chain of the human (h) IgG2 Fc region and the human IgG1 Fc region. Figure 1 shows a schematic diagram (Figure 1A) and amino acid sequence (Figure 1B) of T2m and T22d35. These modules were fused to the N-terminus of the heavy chain of the IgG Fc region using several linker variations (Figure 2E) to generate T2m-Fc (Figure 2B) and T22d35-Fc variants (Figure 2C) fusions (Figure 2A). The sequences of these fusions are shown in Figure 2D. Also, variants of T22d35-Fc were designed to explore the number of cysteine residues in the hinge region of the Fc domain, different IgG isotypes (human IgG1 vs IgG2), and various lengths and properties as linkers between T22d35 and the N-terminus of the Fc domain (Figure 2E, 2F, and 2G). These variations aim to investigate and ultimately optimize the functional and manufacturability characteristics of the T22d35-Fc design.

[0084] Expression and Purification Purification of Transient CHO Material Each fusion protein construct was transiently expressed in CHO-3E7 cells (see Table 1), after which the conditioned medium was harvested and purified using a Protein A affinity column, followed by size exclusion chromatography (SEC). The SEC elution profiles of T2m-Fc (Figure 3A) and T22d35-Fc (Figure 4A) showed that these fusion proteins were relatively pure and aggregate-free. Fractions 6 - 11 (T2m-Fc) and 7 - 10 (T22d35-Fc) were pooled and concentrated to 5.6 mg / ml (T2m-Fc) and 6.03 mg / ml (T22d35-Fc). The final yields of T2m-Fc and T22d35-Fc were 267 mg and 168, respectively. The final products (shown as SEC-pooled fractions) were shown to have a purity >99% by UPLC-SEC (Figures 3B and 4B). SDS-PAGE evaluation (Figures 3C and 4C, Sypro RUBY staining) showed bands for T2m-Fc and T22d35-Fc of ~60 kDa and ~90 kDa under reducing conditions, although bands of approximately 90 kDa and 150 kDa were detectable, and under non-reducing conditions, showed fully assembled high-purity T2m-Fc and T22d35-Fc fusion proteins, respectively. An overview of the details of production and purification is described in Table 1. Collectively, these results demonstrate the good manufacturability of the T2m-Fc and T22d35-Fc fusion proteins.

[0085]

Table 1

[0086] Purification of stable CHO pool material N-terminal and C-terminal Fc-fused T22d35 variants were used in CHO to compare some of their expression levels and biological properties BRI / rcTAIt was stably expressed in cells. The coding regions of each variant were ligated into a mammalian cell expression plasmid, and after transfection, an enriched pool of cells stably expressing each variant was selected. The main difference between the variants can be found in the amino acid sequence containing the linker region that separates the T22d35 doublet from the Fc domain (in the case of N-terminal fusion). However, in the case of C-terminal Fc fusion, the difference between each variant is at the amino terminus at the tip of the protein (Table 2).

[0087]

Table 2

[0088] The fusion protein was purified by protein A affinity using 100 mM citric acid (pH 3.6) as the elution buffer. Samples of the eluted fusion protein were neutralized with 1 M HEPES and then buffer-exchanged into DPBS using a Zeba spin column (Table 3), and some integrity of the purified fusion protein was evaluated by SDS-PAGE. Purification of each variant was performed similarly. Many of the properties were very similar among the variants, but some differences became apparent due to the possibility of aggregation indicating improper folding. Protein aggregation may indicate a decrease in the stability of the three-dimensional structure, and as a result, the activity, potency, or potential may be reduced. Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) was used to determine the purity of the N- and C-terminal Fc fusion variants. This method can accurately measure the proportion of the complete monomeric species and the presence of impurities such as aggregates and / or degradation products. As shown in Figure 6, significant differences can be observed between the T22d35 variant expressed as an N-terminal Fc fusion and the T22d35 variant expressed as a C-terminal fusion. In particular, the proportion of the complete monomer (Figure 6A) was approximately 99% for all five N-terminal fusion mutants (SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23), but was significantly lower for the three C-terminal fusions (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28). This significant decrease in the proportion of the complete monomer is due to the accumulation of an increase in high molecular weight aggregates observed in all C-terminal Fc fusions (Figure 6B), as well as the accumulation of an increase in low molecular weight fragments in two of the three C-terminal Fc fusions. In addition, evaluation of the titer of individual 500 mL products and the average titer of the N-terminal Fc fusion T22d35 product and the C-terminal Fc fusion T22d35 product indicates that the N-terminal Fc fusion T22d35 variant can be produced in higher yields compared to the C-terminal fusion (Table 4). Overall, these results indicate that there are significant and unexpected advantages to expressing the T22d35 doublet at the N-terminus of a moiety such as the Fc portion of an immunoglobulin. Summarizing these data, it can be seen that the manufacturability of the N-terminal Fc fusion T22d35 protein is improved.

[0089]

Table 3

[0090]

Table 4

[0091] Functional in vitro evaluation As shown in FIGS. 7A / B / C, the TGF-β neutralizing ability of the T2m-Fc and T22d35-Fc fusion proteins was compared to the non-Fc fusion T22d35 single-chain doublet trap using the A549 cell IL-11 release assay. This data shows that for all TGF-β isotypes, the potency of T22d35-Fc is superior to that of T2m-Fc and the non-Fc fusion T22d35 single-chain trap, and the calculated IC 50 is 0.003348 and 0.003908 nM, respectively (Table 5). These values represent potencies that are at least 970-fold and at least 240-fold superior to T22d35 (IC 50 is 3.253 and 0.9491 nM, respectively), and 615-fold and 24-fold higher than T2m-Fc (IC 50are 2.059 and 0.0943 nM, respectively. In addition, T22d35-Fc neutralizes TGF-β2, although to a much lesser extent than TGF-β1 and -β3. In contrast, no neutralization of TGF-β2 is observed for either T2m-Fc or the T22d35 single-chain trap. The neutralizing potency of the T22d35-Fc trap is similar for TGF-1β and -β3, but it should be noted that the T2m-Fc variant showed a ~22-fold higher neutralizing potency for TGF-β3 compared to TGF-β1 (2.059 nM and 0.0943 nM, respectively). Evaluation of additional N-terminal Fc-fused T22d35 fusions [T22d35-Fc-IgG2-v2(CC), T22d35-Fc-IgG1-v1(CC), T22d35-Fc-IgG1-v2(SCC), and T22d35-Fc-IgG1-v3(GSL-CC)] (Figure 8, Table 6) indicated that all of these fusions exhibited comparable TGF-β1 neutralizing potencies, which were very similar to the potency of T22d35-Fc. Additional evaluation of the T22d35-Fc-IgG1-v1(CC) variant (Figure 9) confirmed that, in line with the T22d35-Fc variant, the neutralizing potencies for TGF-β1 and -β3 were very similar (IC 50 = 0.003327 nM and 0.003251 nM, respectively), although this potency was much lower for TGF-β2 (IC 50 = 17.33 nM).

[0092]

Table 5

[0093]

Table 6

[0094] Functional in vivo evaluation The T22d35-Fc fusion protein (SEQ ID NO: 10) was evaluated in vivo using a syngeneic MC-38 mouse colon cancer model (Figure 9). The tumor growth of animals treated with the T22d35-Fc fusion was compared to the tumor growth of animals treated with control IgG (CTL IgG). As shown in Figure 9, no significant difference in tumor growth was observed until day 11 after treatment, but a significant decrease in tumor growth was confirmed in the tumor volume of animals treated with T22d35-Fc on day 15 when compared to CTL IgG (two-way ANOVA). This data indicates that administration of T22d35-Fc significantly causes suppression of MC-38 growth compared to the group treated with CTL IgG, suggesting that blocking TGF-β1 in vivo can inhibit tumor growth in this syngeneic model of colorectal cancer.

[0095]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Claims

**Claim 1** A polypeptide construct useful for inhibiting the effects of transforming growth factor beta (TGF-β) isotypes, said construct comprising: a first region comprising a first TGF-β receptor extracellular domain (TβR-ECD) tandemly linked to a second TβR-ECD, wherein the C-terminus of the first TβR-ECD is linked to the N-terminus of the second TβR-ECD, wherein the first TβR-ECD and the second TβR-ECD each consist of the amino acid sequence of SEQ ID NO: 1, and The second constant domain (C Ｈ 2) and / or the third constant domain (C Ｈ 3) of the heavy chain of the antibody, comprising a second region comprising, wherein the C-terminus of the first region is bound to the N-terminus of the second region, a polypeptide construct. **Claim 2** The polypeptide construct of claim 1, wherein the polypeptide construct inhibits TGF-β activity with at least 20-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 600-fold higher potency than a corresponding construct having a single TβR-ECD. **Claim 3** The polypeptide construct of claim 1, wherein the polypeptide construct inhibits TGF-β activity with at least 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, or 900-fold higher potency than a non-Fc fused TβR-ECD doublet. **Claim 4** The polypeptide construct of claim 1, wherein the second region comprises the Fc region of an antibody heavy chain. **Claim 5** The polypeptide construct of claim 1, wherein the C-terminus of the first TβR-ECD directly fuses to the N-terminus of the second TβR-ECD. **Claim 6** wherein the second region comprises the C Ｈ 2 and C Ｈ 3 domains, the polypeptide construct according to any one of claims 1 to 5. **Claim 7** The polypeptide construct of claim 6, wherein the IgG antibody is an IgG1 or IgG2 antibody. **Claim 8** The polypeptide construct according to any one of claims 1 to 5, wherein the second region comprises a hinge region containing a cysteine residue for cross-linking the polypeptide construct to another construct. **Claim 9** The polypeptide construct according to claim 8, wherein the second region has an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 24, or a variation is further introduced in the amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 24, wherein at least one cysteine residue in the hinge region, but not all cysteine residues, is deleted or substituted with a serine or alanine residue.

10. The polypeptide construct according to claim 8, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 17, and SEQ ID NO: 20, wherein a variation is further introduced in which at least one cysteine residue in the hinge region, but not all cysteine residues, is deleted or substituted with a serine or alanine residue.

11. A homodimer polypeptide construct useful for inhibiting the effects of a transforming growth factor beta (TGF-β) isoform, comprising a first single-chain polypeptide and a second single-chain polypeptide linked between their respective hinge regions by at least one disulfide bridge, wherein the first single-chain polypeptide and the second single-chain polypeptide each comprise a first region and a second region; wherein each of the first regions comprises a first TβRII-ECD and a second TβRII-ECD; Here, each of the second regions includes the second constant domain (C Ｈ 2) and the third constant domain (C Ｈ 3) of the antibody heavy chain, and the heavy chain variable region of a predetermined antibody; wherein each of the second regions comprises a hinge region containing at least one cysteine residue to enable the formation of at least one disulfide bridge between the first single-chain polypeptide and the second single-chain polypeptide; wherein the C-terminus of the first region of the first single-chain polypeptide is linked to the N-terminus of the second region of the first single-chain polypeptide; wherein the C-terminus of the first region of the second single-chain polypeptide is linked to the N-terminus of the second region of the second single-chain polypeptide; and Here, each of the first single-chain polypeptide and the second single-chain polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 17, and SEQ ID NO: 20, and in the hinge region, at least one cysteine residue, but not all cysteine residues, has been deleted or substituted with a serine or alanine residue, and a homodimer polypeptide construct having a sequence into which a further variation has been introduced.

12. A nucleic acid molecule encoding the polypeptide construct according to any one of Claims 1 to 5, 10, or 11.

13. An expression vector containing the nucleic acid molecule according to Claim 12.

14. A composition for inhibiting TGF-β activity, comprising an effective amount of the polypeptide construct according to any one of Claims 1 to 5, 10, or 11, and a pharmaceutically acceptable carrier or diluent or excipient.

15. A transgenic cell host for producing the polypeptide construct according to any one of Claims 1 to 5, 10, or 11, comprising the nucleic acid molecule according to Claim 12 or the expression vector according to Claim 13.

16. A method for producing a homodimer polypeptide construct, comprising recovering the homodimer polypeptide construct according to Claim 11 from a pretreated medium by culturing and growing the host, wherein the host is a transgenic cell host containing a nucleic acid molecule encoding the homodimer polypeptide construct.

17. Use of the polypeptide construct according to any one of Claims 1 to 5, 10, or 11 in the manufacture of a pharmaceutical composition for the treatment of cancer.

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